| SOU 1997:132 | 225 |
Annex A: Avilamycin
A.1 Introduction
Avilamycin is a mixture of oligosaccharides of the orthosomycin group, that are produced by 3TREPTOMYCES VIRIDOCHROMOGENES. Other members of this
group include curamycin and everninomycins (Wolf, 1973). Avilamycin is mainly active against
A.2 Mode of action and resistance mechanisms
Avilamycin acts on the bacterial ribosome, by inhibiting the binding of
No data has been published on mechanisms of resistance, but it seems logical to assume that structural changes in the ribosomal 30 S subunit or ribosomal protection could confer resistance to avilamycin in naturally susceptible bacteria. In view of the ease and speed at which resistance to some other
Full
| 226 | Annex A | SOU 1997:132 |
The figures in this report is only available in the printed version
Figure A.I. Tentative sketch of avilamycin and everninomycins
| SOU 1997:132 | Annex A | 227 |
possible
A.3 Development of resistance
Unfortunately, only two publications concerning bacterial susceptibility to avilamycin have been found. This is hardly enough to illustrate the prevalence of avilamycin resistance, but the data from these studies are compiled in table A.I.
Table A.I. Reported prevalence of avilamycin resistance
| "ACTERIAL | 3OURCE | .O /F | 9EAR S | 2ESISTANCE | 2EFERENCE | #OUNTRY |
| SPECIES | OF | ISOLATES | IN | |||
| ISOLATES | ||||||
| #LOSTRIDIUM | various | 95 | 1991 | 0 | Devriese ET AL, | Belgium |
| PERFRINGENS | 1993 | |||||
| 3TAPHYLOCOCCUS | swine | 71 | 0 | DANMAP, | Denmark | |
| HYICUS | 1997 | |||||
| 3TAPHYLOCOCCUS | cattle | 211 | 0 | DANMAP, | Denmark | |
| AUREUS | 1997 | |||||
| #OAGULASE | cattle | 371 | 0 | DANMAP, | Denmark | |
| NEGATIVE | 1997 | |||||
| STAPHYLOCOCCI | ||||||
| %NTEROCOCCUS | swine | 225 | 1 | DANMAP, | Denmark | |
| FAECALIS | 1997 | |||||
| %NTEROCOCCUS | swine | 58 | 2 | DANMAP, | Denmark | |
| FAECIUM | 1997 | |||||
| %NTEROCOCCUS | poultry | 54 | 69 | DANMAP, | Denmark | |
| FAECIUM | 1997 | |||||
| %NTEROCOCCUS | cattle | 13 | 0 | DANMAP, | Denmark | |
| FAECIUM | 1997 |
| 228 | Annex A | SOU 1997:132 |
In Denmark, 84 % of the annual consumption of avilamycin in feed is used
in poultry, which is reflected in the differences in resistance to this compound in % FAECIUM from cattle swine and poultry, respectively. The
consumption of avilamycin increased
coinciding with its introduction into poultry feed. Unfortunately, there is no data available on avilamycin resistance in %NTEROCOCCUS FAECIUM isolates
collected before this increase in the consumption. As no data has been published on the development of resistance, it is not possible to evaluate the risk of this. An adequate amount of this type of data is essential in risk evaluation and should be made available as soon as possible.
A.4 Acquisition of resistance
No published information has been found about genes conveying resistance to avilamycin, transfer of avilamycin resistance, or bacterial hosts for resistance genes. If no such investigations have been undertaken, they should be planned immediately.
A.5 Impact of resistance on animal and human health
As avilamycin is not yet used for therapy in humans or animals, resistance would not be expected to cause clinical problems unless
A.6 Other effects on the microflora
A.6.1 Salmonella
Hinton (1988) investigated the effect of
| SOU 1997:132 | Annex A | 229 |
were infected with 3ALMONELLA Kedougou in the feed for two weeks, at concentrations from 1.6 to 176 bacterial cells per g feed. All birds in the same replicate experiment received the same dose of organisms. Samples for bacterial culture were taken on day 7 and 14 after the introduction of infected feed. The author concluded that no evidence was obtained to suggest that avilamycin, at concentrations of 2.5 or 10 ppm in the feed, favoured colonisation of the intestinal tract in chickens with 3 Kedougou when they were challenged with this organism in the feed.
This is the only published study on the effect of avilamycin on intestinal salmonellae. One single study, no matter how well performed, is hardly enough to form the basis for any definite conclusions, especially when this study did not result in any clear evidence as to whether avilamycin presents a risk in this aspect or not.
A.6.2 Other enteric pathogens
No publications on the effects of avilamycin on other enteric phogens have been found.
A.7 Effects on specific animal diseases
Avilamycin at growth promoting levels has been shown to reduce the amount of #LOSTRIDIUM PERFRINGENS in the intestinal tract of chickens (Elwinger ET AL ,
1993; Elwinger ET AL , 1995) and may thus be used prophylactically against necrotic enteritis in poultry. Kyriakis (1989) investigated the effect of avilamycin at 40 or 80 ppm in the control of
A.8 Toxicological aspects
No information about possible toxic effects either on the target species or on humans has been found. Such effects may be totally absent or, as it is a
| 230 | Annex A | SOU 1997:132 |
comparatively new compound, reports on allergy and other side effects may not yet have appeared.
One publication (Magnussen ET AL , 1991) has been found concerning residues. The results in this study indicate that avilamycin fed to swine at a concentration of 60 ppm gives rise to small but measurable residues in tissues. At zero withdrawal time the residue levels were 0.14 ppm in muscle, 0.66 ppm in liver, 0.34 ppm in kidney and 0.55 ppm in fat. There is no available information to suggest that this would present a risk for the consumer.
A.9 Environmental effects
Source separated municipal solid waste and agricultural waste can be utilised for biogas production. Substances with an antimicrobial effect against anaerobic bacteria could disturb this process. In studies on manure from pigs and poultry fed avilamycin, Sutton (1989) reported efficient operation of experimental and large mesophilic digesters. The presence of avilamycin appeared to alter the metabolism of the microflora, increasing the efficiency to degrade volatile solids.
No other publications on possible environmental effects of avilamycin have been found. As the compound is produced by a soil microbe, it would be expected to be microbially degraded in soil.
A.10 Summary comments
Use of avilamycin in poultry appears to have caused an increase in avilamycin resistance in % FAECIUM from this animal species. This resistance
might have appeared after only a few years of avilamycin use. Avilamycin is closely related to everninomycins, and
| SOU 1997:132 | Annex A | 231 |
References
#HOPRA ) (ODGSON *
#ORMICAN M ' AND *ONES 2 . , 1996. Emerging resistance to antimicrobial agents in
$EVRIESE , ! $AUBE ' (OMMEZ * AND (AESEBROUCK & , 1993. In vitro susceptibility of #LOSTRIDIUM PERFRINGENS isolated from farm animals to growthenhancing antibiotics. *OURNAL OF !PPLIED "ACTERIOLOGY
%LWINGER + %NGSTR¶M " "ERNDTSON % &OSSUM / AND 4EGL¶F " , 1993. Effect ov Avotan (avoparcin) and Maxus (avilamycin) on the caecal growth of #LOSTRIDIUM PERFRINGENS and the occurrence of necrotic enteritis in broilers (report). Swedish
University of Agricultural Sciences, Uppsala.
%LWINGER + %NGSTR¶M " "ERNDTSON % &OSSUM / AND 7ALDENSTEDT , , 1995.
Effect of Maxus (avilamycin), Avotan (avoparcin), Monteban (narasin) and Elancoban
broilers (report). Swedish University of Agricultural Sciences, Uppsala.
(INTON
+YRIAKIS 3 # , 1989. The effects of avilamycin in the control of
.ICAS 4 ) :ECKEL
3UTTON ! , .YE * # 0ATTERSON * ! +ELLY $ 4 AND &URUMOTO %LKIN % * , 1989.
Effects of avilamycin in swine and poultry wastes on methane production in anaerobic digesters. "IOLOGICAL 7ASTES
5RBAN #
* * , 1996. Comparative
| 232 | Annex A | SOU 1997:132 |
7OLF (, 1973. Avilamycin, an inhibitor of the 30 S ribosomal subunits function. &%"3 ,ETTERS
| SOU 1997:132 | 233 |
Annex B: Bacitracin
B.1 Introduction
Bacitracin is a complex mixture of cyclic polypeptides produced by "ACILLUS SUBTILIS and "ACILLUS LICHENIFORMIS. The compound has bactericidal effect on
Bacitracin is poorly absorbed from the gastrointestinal tract (Donoso ET AL , 1970; Froyshov ET AL , 1986), as well as from skin and mucosal surfaces. Absorbed bacitracin is excreted by glomerular filtration (Prescott and Baggot, 1993).
The substance is used in human therapy, mostly for topical treatment of superficial infections of the skin and mucosal surfaces. However, its effectiveness against
Recommended dosages for prophylaxis and therapy in poultry are in the range between 50 and 200 ppm, and for swine around 250 ppm (Prescott and Baggot, 1993). The corresponding dosages permitted for growth promotion are between 5 and 100 ppm for poultry, and between 5 and 80 ppm for swine. Growth promoting dosages for calves, lambs and kids are in the range of 5- 80 ppm and for fur animals
Bacitracin is widely used for laboratory purposes, in selective media for bacterial culture and in cell culture media. It is also used experimentally as a protease inhibitor (Fukuda ET AL , 1995).
| 234 | Annex B | SOU 1997:132 |
The figures in this report is only available in the printed version
Figure B.I. Tentative sketch of bacitracin
B.2 Mode of action and resistance mechanisms
Bacitracin inhibits the formation of bacterial cell wall peptidoglycan by complexing directly with the lipid isoprenyl pyrophosphate (IPP) carrier, inhibiting the dephosphorylation reaction that is required for its regeneration. This leads to accumulation of phospholipids inside the cell and inhibition of cell wall formation. Proposed resistance mechanisms include active efflux, increased production of IPP kinase, suppression of autolytic systems, reduced membrane permeability and suppressed exopolysaccharide secretion (see below).
No information about
It has been claimed that bacitracin "cures" resistance against other antimicrobials and that its use as a feed additive would therefore be purely beneficial from a resistance point of view (Walton, 1978; Gedek, 1981; Walton, 1984; Walton and Wheeler, 1987). However, due to weaknesses in study design in relation to the specific question to be answered, as well as
| SOU 1997:132 | Annex B | 235 |
inconsistent results, the only conclusion that can be drawn from these studies is that bacitracin does not seem to induce resistance to any of the other, unrelated, antimicrobials tested. However, as resistance to bacitracin was not determined or characterised, the results provided little information about the potential for
B.3 Development of resistance
B.3.1 Prospective studies
Only two studies that deal specifically with development of bacitracin resistance over time have been found (Linton ET AL , 1985; Kaukas ET AL , 1988). These include one experimental study and one field study.
In the study by Kaukas ET AL (Kaukas ET AL , 1988), groups of 10 chickens were fed different antibiotics, with one group serving as a
enterococci from
bacitracin as high as 47% in the medicated group, while in the
In the publication by Linton ET AL (Linton ET AL , 1985), the first part is a field survey comparing five commercial premises and a total of nine batches of broiler chickens. One farm, where one batch of birds was reared, used feed with bacitracin at the recommended level for growth promotion. No
| 236 | Annex B | SOU 1997:132 |
coccidiostats were used was provided. Cloacal swabs were taken at the beginning and at the end of the rearing period and isolated enterococci were tested for antimicrobial susceptibility. The percentage of strains resistant to bacitracin, among the strains isolated from birds fed bacitracin, increased from 87% to 100% during the rearing period. However, the enterococci were not identified to the species level and no information is provided on how many birds were sampled in each group in this experiment. This, taken together with the fact that there was no
The second part of the same publication describes an experimental study, where enterococci were also tested for their susceptibility to bacitracin. Bacitracin was not a factor of the experiment but one of the four groups of chickens was a
B.3.2
Data from different studies on prevalence of resistance to bacitracin have been compiled in table B.I. Unfortunately, most studies do not report MIC values and there seems to be some confusion about what should be the breakpoint value for different bacteria. Further, the number of strains included are often inadequate for an estimate of the prevalence of resistance. Bearing this in mind, it is noteworthy that the proportion of enterococcal isolates with presumably acquired resistance shows a range from
| SOU 1997:132 | Annex B | 237 |
Table B.I. Resistance to bacitracin in various bacterial species, reported in different studies
| Bacterial species | Source | No. Of | Year | Resis- | Reference | Country |
| of | iso- | tance | ||||
| isolates | lates | in % | ||||
| * | ||||||
| Clostridium | cattle | 32 | 1991 | 9 | Devriese et al, 1993Belgium | |
| perfringens | ||||||
| C. perfringens | poultry | 31 | 1992 | 6 | Devriese et al, 1993Belgium | |
| C. perfringens | poultry | 80 | >50 | Benno et al, 1988 | Japan | |
| C. perfringens | swine | 32 | 1992 | 0 | Devriese et al, 1993Belgium | |
| Clostridium spp. | cattle | * | 9 | Dutta and Devriese, Belgium | ||
| 1984 | ||||||
| Clostridium spp. | poultry | * | 6 | Dutta and Devriese, Belgium | ||
| 1984 | ||||||
| Clostridium spp. | swine | * | 1 | Dutta and Devriese, Belgium | ||
| 1984 | ||||||
| Enterococcus | poultry | 23 | 1980 | 21 | Dutta and Devriese, Belgium | |
| faecalis subsp | 1982 | |||||
| liquefaciens | ||||||
| E. faecalis | poultry | 8 | 1980 | 62 | Dutta and Devriese, Belgium | |
| 1982 | ||||||
| E. faecalis | poultry | 60 | 1977 | 17 | Barnes et al, 1978 | UK |
| E. faecalis | swine | 225 | 3 | DANMAP, 1997 | Denmark | |
| Enterococcus | poultry | 15 | 1980 | 67 | Dutta and Devriese, Belgium | |
| faecium | 1982 | |||||
| E.faecium | cattle | 13 | 8 | DANMAP, 1997 | Denmark | |
| E.faecium | poultry | 54 | 41 | DANMAP, 1997 | Denmark | |
| E.faecium | poultry | 13 | 1977 | 77 | Barnes et al, 1978 | UK |
| E.faecium | swine | 58 | 31 | DANMAP, 1997 | Denmark | |
| Enterococcus spp. humans | 9 | 1992- | 11 | Everett et al, 1995 | USA | |
| 1993 | ||||||
| Streptococcus | humans | 50 | 1992- | 2 | Everett et al, 1995 | USA |
| spp. | 1993 | |||||
| Staphylococcus | cattle | 211 | 0 | DANMAP, 1997 | Denmark | |
| aureus | ||||||
| S. aureus | various | 324 | <1% | Devriese, 1980 | Belgium | |
| S. aureus, | humans | 106 | 1989 | 2 | Maple et al, 1989 | various |
| methicillin | ||||||
| resistant | ||||||
| S. hyicus | swine | 71 | 0 | DANMAP, 1997 | Denmark | |
| Coagulase | humans | 119 | 17 | Everett et al, 1995 | USA | |
| positive | ||||||
| staphylococci | ||||||
| Coagulase | cattle | 371 | 0 | DANMAP, 1997 | Denmark | |
| negative | ||||||
| staphylococci | ||||||
| Coagulase | humans | 261 | 6 | Everett et al, 1995 | USA | |
| negative | ||||||
| staphylococci | ||||||
* total number of strains for cattle, poultry and swine = 192
| 238 | Annex B | SOU 1997:132 |
B.4 Acquisition of resistance
Published information on bacitracin resistance is sparse, but some hitherto identified mechanisms of resistance to bacitracin are shown in Table B.II.
Table B.II. Mechanisms of bacitracin resistance
| Bacterial species | Gene | Mechanism | Reference |
| Bacillus | bcr | active efflux | Podlesek et al, 1995 |
| licheniformis | |||
| plasmid pXV62, | bac A | production of IPP | Cain et al, 1993 |
| original bacterial | kinase | ||
| source not given | |||
| Enterococcus spp. | not identified | suppressed autolytic | Krogstad and |
| system | Pargwette, 1980* | ||
| various gram- | not identified | reduced membrane | Mukherjee et al, |
| positive and gram- | permeability | 1989 | |
| negative bacteria | |||
| various gram | not identified | suppressed | Pollock et al, 1994 |
| negative bacteria | exopolysaccharide | ||
| secretion |
*this is the mechanism proposed although not proven in the article
Podlesek and
binding transport system in the cell membrane. The proposed action of this transport system is active efflux of the bacitracin molecule. Another resistance gene, located on plasmid pXV62, named BACA (Cain ET AL , 1993),
proposedly encodes a phosphokinase involved in IPP metabolism. A similar enzyme has been characterised in 3TAPHYLOCOCCUS AUREUS (Sandermann Jr and
Strominger, 1971), although its possible effects on the bacitracin susceptibility of this bacterium was not investigated. Resistance to bacitracin due to altered cell membrane permeability has been reported (Mukherjee ET AL , 1989). Resistance due to suppressed autolytic enzyme systems has also been suggested (Krogstad and Pargwette, 1980). Suppression of autolytic enzymes makes the bacterium resistant to substances that inhibit peptidoglycan synthesis (Tomasz ET AL , 1970; Krogstad and Pargwette, 1980).
Other mechanisms for reduced bacitracin susceptibility that have been suggested include increased production of the carrier IPP, which may competitively overcome the inhibitory effect of bacitracin on peptidoglycan synthesis, and cessation of exopolysaccharide synthesis (Pollock ET AL , 1994). The excretion of polysaccharides require the same carrier IPP that is needed for the synthesis of peptidoglycan and a halt in this excretion will leave more IPP available for cell wall synthesis. This will also occur in the
| SOU 1997:132 | Annex B | 239 |
absence of essential components required for exopolysaccharide synthesis, e.g. in an environment depleted of certain sugars (Pollock ET AL , 1994).
Transfer of bacitracin resistance seems to have attracted little attention from researchers. Transduction between strains of 3TREPTOCOCCUS PYOGENES
has been shown to occur (Stuart and Ferretti, 1978). However, this study did not investigate what gene(s) and mechanisms were involved in the observed resistance. No other studies, concerning transfer or
B.5 Effects on specific animal diseases
Some reports indicate that bacitracin, even at concentrations used for growth promotion, may prevent necrotic enteritis in poultry (Wicker ET AL , 1977; Prescott ET AL , 1978; Stutz ET AL , 1983). Stutz and
5.5, 16.5, or 55 ppm of bacitracin significantly reduced the number of #LOSTRIDIUM PERFRINGENS organisms in the ileal contents of chicks (p<0.05).
Prescott and
Some authors have investigated the possible effects of orally administered bacitracin on the immune response to certain forms of challenge (Harmon ET AL , 1973; Wasinska, 1980). Harmon and
Wasinska (1980) investigated the antibody response and resistance to infection after vaccinating pigs against erysipelas, colibacillosis and swine
| 240 | Annex B | SOU 1997:132 |
fever. In addition,
B.6 Impact of resistance on animal and human health
Increased resistance to bacitracin in clostridia and enterococci could lead to therapeutic failures when bacitracin is used for the treatment of infections with these organisms in animals and humans. Without further information about the extent of the therapeutic use of bacitracin, the impact on human and animal health of such incidents is impossible to assess.
B.7 Other effects on the microflora
B.7.1 Effects on salmonella colonisation
Only a few published studies investigating the association between salmonella colonisation of the gut and bacitracin in the feed have been found (Nurmi and Rantala, 1974; Smith and Tucker, 1975; Smith and Tucker, 1980; Latour and Barnum, 1981; Humbert ET AL , 1991; Manning ET AL , 1994). All use poultry as the experimental animal species and
birds. All animals were kept on used litter, and challenged with 106 organisms of 3ALMONELLA Enteritidis. The concentration of bacitracin used
was considerably higher than what is used for growth promotion, and the selective culture technique used appears to be based on visual examination only. This makes the results difficult to evaluate.
In the study by Humbert and
| SOU 1997:132 | Annex B | 241 |
with
and without further confirmation.
Nurmi and Rantala (1974) compared the
treatment with CE. The results indicate a decrease in the amount and prevalence of 3ALMONELLA shedding in birds receiving bacitracin, as compared
to
Smith and Tucker reported two studies including bacitracin (Smith and Tucker, 1975; Smith and Tucker, 1980). In the first (Smith and Tucker, 1975) birds were fed bacitracin at concentrations of either 10 or 100 ppm and inoculated with 3. Typhimurium. Faecal samples were compared with samples from similarly infected
In another study by the same authors (Smith and Tucker, 1980) chickens were challenged with five different salmonella serovars (3 Heidelberg 3 Infantis 3 Oranienburg, 3 Senftenberg and 3 Typhimurium). The percentage of
| 242 | Annex B | SOU 1997:132 |
identification by visual examination only. As in the study by Humbert and
Latour and Barnum (1981) used ducks as experimental animals. Two concentrations of
Bailey and
No studies investigating the effect of bacitracin on the infectious dose necessary to achieve establishment of salmonella colonisation have been found, nor any studies investigating
B.7.2 Other enteric pathogens
No studies on the possible effects on colonisation by other enteric pathogens have been found.
B.8 Toxicological aspects
Bacitracin is highly nephrotoxic when administered parenterally (1993), but as it is poorly absorbed from the gut, no adverse effects would be expected after oral administration.
B.8.1 Adverse effects in ruminants
Bacitracin is used as a feed additive in calves, lambs and kids. Adult cattle, however, react adversely to bacitracin in the feed. Sudden milk drops, in
| SOU 1997:132 | Annex B | 243 |
herds fed concentrate feed contaminated with bacitracin at the feed mill, have been reported (Woodger, 1979). Higher mortality and decreased effectiveness of antimicrobial therapy was noticed in calves fed 50 ppm bacitracin in the milkreplacement in a study by Jonson and Jacobsson (1973). It is discussed whether resistant bacteria or immunosuppression in the medicated animals were the cause, but no investigations to determine the cause were included in the article. In sheep, biotransformation of plant toxins, that occurs in the rumen of some
B.8.2 Allergy
Allergic reactions to bacitracin are frequently reported (Katz and Fisher, 1987; Grandinetti and Fowler, 1990; Knowles and Shear, 1995). Both anaphylaxis, eczema, urticaria and delayed reactions may be seen (Katz and Fisher, 1987). Most reports concern patients treated with bacitracin ointment. It has been stated that bacitracin is the topical agent most commonly implicated in anaphylactic reactions (Katz and Fisher, 1987). Considering this, it is somewhat surprising that no reports have been found on allergic reactions in people who come in contact with bacitracin professionally, such as farmers, hospital personnel and people working in the pharmaceutical industry.
B.9 Environmental effects
Like for other AFA, if bacitracin reduces the amount of feed consumed per kg weight gain in the target animal, it would also be expected to reduce the amount of nitrogen output per kg weight gain.
Very little of ingested bacitracin is absorbed and most is excreted unmetabolised in the faeces (Donoso ET AL , 1970; Froyshov ET AL , 1986). Gavalchin and Katz (1994) studied the degradation of bacitracin in sandy loam from a
| 244 | Annex B | SOU 1997:132 |
Similar results have been obtained earlier by Jagnow (1978) reporting half life in soil of 22,5 days at 20 °C and 12 days at 30°C .
Vogtmann and
B.10 Summary comments
Bacitracin has a bactericidal effect mainly on
Data available on colonisation by enteric pathogens in animals fed bacitracin is too inconsistent and too scarce to form the basis of any firm conclusions about the effects of bacitracin.
Bacitracin administered at growth promoting concentrations has prophylactic and therapeutic effects on necrotic enteritis in poultry.
Allergic reactions to bacitracin are documented in humans undergoing bacitracin treatment. People who are exposed to the substance on a daily basis may be at risk of being sensitised.
Bacitracin is degraded in soil. The environmental degradation appears to be inversely related to soil temperature.
In conclusion, available information is to scarce for an assessment of the possible risks of bacitracin usage to human and animal health. Bacitracin usage does not appear to represent any substantial danger to the environment.
| SOU 1997:132 | Annex B | 245 |
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| SOU 1997:132 | Annex B | 247 |
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resistance in enterococci consequent upon feeding growth promoters active against
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.URMI % AND 2ANTALA
3ALMONELLA INFANTIS in the intestine of broiler chickens. 2ESEARCH IN 6ETERINARY 3CIENCE
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2 ( + , 1994. Enteric eradication of
| 248 | Annex B | SOU 1997:132 |
0ODLESEK : #OMINO ! (ERZOG 6ELIKONJA " :GUR "ERTOK $ +OMEL 2 AND
'RABNAR
976.
0OLLOCK 4 * 4HORNE , 9AMAZAKI -
1994. Mechanism of bacitracin resistance in
0RESCOTT * & AND "AGGOT * $ EDS , 1993. !NTIMICROBIAL THERAPY IN VETERINARY MEDICINE 2nd ed. Iowa State University Press, Ames. pp. 612.
0RESCOTT * & 3IVENDRA 2 AND "ARNUM $ ! , 1978. The use of bacitracin in the
prevention and treatment of
1UINLAN ' * AND 'UTTERIDGE *
damage DNA and carbohydrate in the presence of iron and copper salts. &REE 2ADICALS 2ESEARCH #OMMUNATIONS
3ANDERMANN *R ( AND 3TROMINGER * , , 1971.
.ATIONAL !CADEMY OF 3CIENCES 53!
3MITH 7 ( AND 4UCKER * & , 1975. The effect of feeding diets containing permitted antibiotics on the faecal excretion of 3ALMONELLA TYPHIMURIUM by experimentally infected chickens. *OURNAL OF (YGIENE
3MITH 7 ( AND 4UCKER * & , 1980. Further observations on the effect of feeding diets
containing avoparcin, bacitracin and sodium arsenilate on the colonization of the alimentary tract of poultry by salmonella organisms. *OURNAL OF (YGIENE 137-
150.
3TUART * ' AND &ERRETTI * * , 1978. Genetic analysis of antibiotic resistance in
3TREPTOCOCCUS PYOGENES. *OURNAL OF "ACTERIOLOGY
3TUTZ - 7 *OHNSON 3 , AND *UDITH & 2 , 1983. Effects of diet and bacitracin on growth, feed efficiency, and populations of #LOSTRIDIUM PERFRINGENS in the intestine of broiler chicks. 0OULTRY 3CIENCE
4OMASZ ! !LBINO ! AND :ANATI % , 1970. Multiple antibiotic resistance in a bacterium with suppressed autolytic system. .ATURE
7ACHENHEIM $ % "LYTHE , , AND #RAIG !
on in vitro ovine ruminal biotransformation of the hepatotoxic pyrrolizidine alkaloid jacobine. !PPLIED AND %NVIRONMENTAL
7ALTON * 2 , 1978. The effect of zinc bacitracin on the susceptibility of selected gram negative and gram positive bacteria to therapeutic antibiotics. :ENTRALBLATT F¼R 6ETERIN¤RMEDIZINE 2EIHE "
| SOU 1997:132 | Annex B | 249 |
7ALTON * 2 , 1984. The effect of dietary zinc bacitracin on the resistance status of intestinal %SCHERICHIA COLI and enterococci from broiler chickens. :ENTRALBLATT F¼R 6ETERIN¤RMEDIZINE 2EIHE "
7ALTON * 2 AND 7HEELER * % , 1987. Loss of resistance to the tetracyclines from porcine %SCHERICHIA COLI in contact with dietary bacitracin methylene disalicylate.
:ENTRALBLATT F¼R 6ETERIN¤RMEDIZINE 2EIHE "
7ASINSKA " , 1980. Effect of prolonged feeding of mixtures supplemented with Zn-
bactracin and ronidazole on specific immunity in pigs after their vaccination against swine fever, erysipelas and colibacillosis. )N PROCEEDINGS OF TH )063 #ONGRESS.
Copenhagen. p. 178.
7ICKER $ , )SGRIGG 7 . AND 4RAMMEL * ( , 1977. The control and prevention of necrotic enteritis in broilers with zinc bacitracin. 0OULTRY 3CIENCE
6OGTMANN ( /BRIST 7 (AUSER + 0FIRTER ( 0 AND !UGSTBURGER & , 1978.
Composting and plant growth: use of chicken manure containing antibiotics.
#OMPOST 3CIENCE ,AND 5TILIZATION
7OODGER ' * ! , 1979. Antibiotic contamination of compound cattle feed. 6ETERINARY 2ECORD 173.
SOU 1997:132
Annex C: Flavomycin
C.1 Introduction
Flavomycin, also known as flavophospholipol, bambermycins or moenomycin, is a
complexes of very similar components. It is produced by a group of
3TREPTOMYCES SPP, including 3 BAMBERGENSIS, 3 GHANAENSIS, 3 GEYSIRENSIS and 3 EDERENSIS . At the moment it is used for growth promoting purposes
only and is not included in any therapeutic drug either in human or veterinary medicine. Flavomycin inhibits cell wall synthesis, mainly in
Concentrations used for growth promotion are between 1 and 20 ppm for swine,
Flavomycin is not absorbed to any great extent after oral administration. When parenterally administrated it is excreted in the urine at a very slow rate and has, therefore, a prolonged activity in blood. The antimicrobial activity is reduced by serum and is optimal at pH 5.0 - 6.5. (Huber, 1979)
C.2 Mode of action and resistance mechanisms
Flavomycin exerts its effect by inhibiting bacterial cell wall synthesis. The transglycosylation reaction necessary for peptidoglycan synthesis, that is catalysed by the
No reports on mechanisms for resistance to flavomycin have been found. This is somewhat surprising, since investigations on resistance mechanisms and resistance genes might determine whether
Flavomycin is reportedly mainly active against
However, several publications indicate that the compound is sometimes active against
and Moeller, 1977a; Witte, 1996). Unfortunatley, pure flavomycin for
| SOU 1997:132 | Annex C | 251 |
laboratory use is not commercially available. Thus, no experiments could be performed to clarify this issue.
The figures in this report is only available in the printed version
Figure C.I. Tentative sketch of flavomycin
C.3 Development of resistance
C.3.1 Prospective studies
Unfortunately, and somewhat surprising, most studies where antimicrobial resistance patterns are determined in faecal bacteria, before and after the supplementation of feed with flavomycin, do not include any investigations on susceptibility to flavomycin itself in these bacteria.
A study by Dealy and Moeller (1977a) shows a significant (p<0.001) increase in the percentage of
| 252 | Annex C | SOU 1997:132 |
Table C.I. Prevalence of resistance to flavomycin in various bacterial species
| Bacterial species | Source | No. Of | Year | Resistance | Reference | Country |
| of | iso- | in % | ||||
| isolates | lates | |||||
| Clostridium | various | * | 0 | Dutta and | Belgium | |
| botulinum | Devriese, | |||||
| 1984 | ||||||
| Enterococcus | poultry | 23 | 1980 | 0 | Dutta and | Belgium |
| faecalis subsp | Devriese, | |||||
| liquefaciens | 1982 | |||||
| E. faecalis | poultry | 8 | 1980 | 0 | Dutta and | Belgium |
| Devriese, | ||||||
| 1982 | ||||||
| E. faecalis | poultry | 54 | 1995- | 0 | DANMAP, | Denmark |
| products | 1996 | 1997 | ||||
| E. faecalis | swine | 38 | 1986- | 0 | Devriese | Belgium |
| 1995 | and | |||||
| Haeseb- | ||||||
| rouck, 1996 | ||||||
| E. faecalis | swine | 225 | 0 | DANMAP, | Denmark | |
| 1997 | ||||||
| E. faecalis | pork | 38 | 6 | DANMAP, | Denmark | |
| 1997 | ||||||
| E. faecalis | beef | 21 | 19 | DANMAP, | Denmark | |
| 1997 | ||||||
| Enterococcus spp. | swine | 21 | 1986- | 71 | Devriese | Belgium |
| 1995 | and | |||||
| Haeseb- | ||||||
| rouck, 1996 | ||||||
| Coagulase negative | cattle | 371 | 0 | DANMAP, | Denmark | |
| staphylococci | 1997 | |||||
| Staphylococcus | swine | 71 | 0 | DANMAP, | Denmark | |
| hyicus | 1997 | |||||
| Staphylococcus | cattle | 211 | 0 | DANMAP, | Denmark | |
| aureus | 1997 | |||||
| S. aureus | various | 792 | 1970- | 0 | Devriese, | Belgium |
| 1980 | 1980 | |||||
| Streptococcus spp. | swine | 19 | 1986- | 21 | Devriese | Belgium |
| 1995 | and | |||||
| Haeseb- | ||||||
| rouck, 1996 |
* total number of strains: C.perfringens = 142, C. sporogenes = 6, C. botulinum = 3
| SOU 1997:132 | Annex C | 253 |
C.3.2
Data on prevalence of resistance to flavomycin from various studies are compiled in table C.I.
Some bacterial species are reported to be naturally resistant to flavomycin, including #LOSTRIDIUM PERFRINGENS, #LOSTRIDIUM SPOROGENES and
%NTEROCOCCUS FAECIUM (Dutta and Devriese, 1984). However, susceptible strains of % FAECIUM have been reported (DANMAP, 1997). These
discrepancies could be due to methodological differences regarding breakpoints etc.
C.4 Acquisition of resistance
No published investigations on resistance genes, transfer of resistance or resistance determinants in different bacterial hosts have been found.
C.4.1 Influence on resistance against other antimicrobials
George and Fagerberg (1984) investigated the effect of flavomycin on
A similar investigation, with similar conclusions was conducted by Sepulchre (1979). This study also includes IN VIVO experiments on effects of flavomycin in the feed on resistance in enteric microflora in pigs. However, the decrease in resistance observed in bacteria isolated from medicated pigs was also seen in the
Another study, by Pohl (1975), also showed decreased transfer frequency of some, but not all, R plasmids between % COLI strains in the presence of flavomycin.
| 254 | Annex C | SOU 1997:132 |
Brophy (1988) conducted an IN VIVO study on the effect of
Corpet (1984) used a mouse model to study changes in chlortetracycline resistance of faecal % COLI after supplementing the drinking water with flavomycin. The percentage of
The proposed reason for flavomycin’s limited action against
C.5 Effects on specific animal diseases
The fact that all # PERFRINGENS seem to be
(1984) fed flavomycin at 55 ppm to chickens whithout noticing any increase of # PERFRINGENS in ileal contents. Brenes et al (1989) conducted a similar
experiment with similar results. However, 55 ppm is more than twice as much as the maximum dose used for growth promotion in chickens and would therefore not quite correspond to the
C.6 Impact of resistance on animal and human health
No
| SOU 1997:132 | Annex C | 255 |
Several other
C.7 Other effects on the microflora
C.7.1 Salmonella
A few studies on the effects of
Moeller, 1976; Dealy and Moeller, 1977b; George ET AL , 1982; Humbert ET AL , 1991).
Dealy and Moeller (1976; 1977b) investigated the shedding of 3ALMONELLA Typhimurium in experimentally infected calves and pigs. Animals given flavomycin at growth promoting levels were compared to
animals. The results indicate that the use of flavomycin reduced the duration and prevalence of 3ALMONELLA shedding in both pigs and calves. However, the
experimental groups are rather small and, oddly enough, the challenge strain of 3. Typhimurium used in the trials was susceptible to flavomycin and did
not develop resistance during the experimental period. Since there are no reports on the regular susceptibility of 3ALMONELLA spp. to flavomycin, it
cannot be determined whether this is unusual or not. If the infecting strain is sensitive to flavomycin, feeding this drug would be expected to reduce shedding. This might be regarded as therapy and not growth promotion, though.
In the study by Humbert and
| 256 | Annex C | SOU 1997:132 |
given as to the design of this study, regarding culturing methods, presentation of the results and length of study period (see annex B).
George and
Smith and Tucker (1975) also compared
C.7.2 Other enteric pathogens
No studies regarding the effects of
C.8 Toxicological aspects
The toxicity of flavomycin is very low, even after intravenous administration (Huber, 1979), and is only absorbed from the gut in small quantities (Sambeth ET AL , 1974).Therefore, no toxic effects would be expected in either animals or humans due to the usage of flavomycin in animal feed.
C.8.1 Allergy
Frese and Blobel (1973) studied the antigenicity of flavomycin in rabbits and found that neither oral administration nor subcutaneous injection of the substance produced any antibody responses or anaphylactic reactions. This is the only published report on flavomycin in association with allergy. It is hard to say whether data obtained from rabbit experiments are applicable to people exposed to the substance.
| SOU 1997:132 | Annex C | 257 |
C.9 Environmental effects
Like other AFA, if flavomycin reduces the amount of feed consumed per kg weight gain in the target animal, it would also be expected to reduce the amount of nitrogen output per kg weight gain.
Flavomycin shows a high degree of stability through the procedure of pelleting feed (Waals, 1973), which would suggest heat stability. The substance is degraded in soil and manure, but at a slow rate (Jagnow, 1978). According to Jagnow (1978), it takes 17 weeks for complete aerobic degradation of 10 ppm flavomycin in fresh manure, and 2 weeks in a soil mixture. Galvachin and Katz (1994) found that at 20°C or more, flavomycin, in a
C.10 Summary comments
Flavomycin appears to be a very attractive substance for therapy, as it is fairly atoxic and has good pharmacokinetic properties. However, if its use for growth promoting purposes causes increased resistance among animal bacteria, both flavomycin and related substances may be rendered useless for therapy in animal and human medicine. In general, very little information about flavomycin is available. As the substance has been in use for more than 20 years it is remarkable that so few investigations have been published on resistance in various bacterial species, various animal species and in different geographical regions. For substances not used in therapy, such investigations are essental, since resistance will not be noticed in clinical practice.
| 258 | Annex C | SOU 1997:132 |
References
Brenes, A., Trevino, J., Centeno, C. and Yuste, P., 1989. Influence of peas (Pisum sativum) as a dietary ingredient and flavomycin supplementation on the performance and intestinal microflora of broiler chicks. Brittish Poultry Science
Brophy, P.O., 1988. Antibiotics on the farm: the UCD Lyons experience. Irish Veterinary Journal.
Corpet, D.E., 1984. The effect of bambermycin, carbadox, chlortetracycline and olaquindox on antibiotic resistance in intestinal coliforms: a new animal model.
Annales de Microbiologie. 135
DANMAP, 1997. Consumption of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in Denmark. No. 1. DANMAP, Copenhagen.
Dealy, J. and Moeller, M.W., 1976. Influence of bambermycins on Salmonella infection and antibiotic resistance in swine. Journal of Animal Science.
Dealy, J. and Moeller, M.W., 1977a. Effect of bambermycins on Escherichia coli and antibiotic resistance in calves. Journal of Animal Science.
Dealy, J. and Moeller, M.W., 1977b. Influence of bambermycins on Salmonella infection and antibiotic resistance in calves. Journal of Animal Science.
Devriese, L.A., 1980. Sensitivity of staphylococci from farm animals to antibacterial agents used for growth promotion and therapy - a ten year study. Annales de Recherches Veterinaires.
Devriese, L.A., Daube, G., Hommez, J. and Haesebrouck, F., 1993. In vitro susceptibility of Clostridium perfringens isolated from farm animals to growthenhancing antibiotics. Journal of Applied Bacteriology.
Devriese, L.A. and Haesebrouck, F., 1996. Susceptibility of enterococci and intestinal streptococci from pigs to the growth enhancing antibiotics flavomycin and avoparcin. In proceedings of: 14th IPVS Congress
Dutta, G.N. and Devriese, L.A., 1982. Susceptibility of fecal streptococci of poultry origin to nine
Dutta, G.N. and Devriese, L.A., 1984. Observations on the in vitro sensitivity and resistance of Gram positive intestinal bacteria of farm animals to growth promoting antimicrobial agents. Journal of Applied Bacteriology.
Frese, E. and Blobel, H., 1973. Testing the antigenicity of Flavomycin. Zentrablatt fur Veterinarmedizin.
| SOU 1997:132 | Annex C | 259 |
Gavalchin, J. and Katz, S.E., 1994. The persistence of
Journal of the Association of Official Analytical Chemists.
George, B.A. and Fagerberg, D.J., 1984. Effect of bambermycins, in vitro, on plasmidmediated antimicrobial resistance. American Journal of Veterinary Research.
George, B.A., Fagerberg, D.J., Quarles, C.L., Fenton, J.M. and McKinley, G.A., 1982. Effect of bambermycins on quantity, prevalence, duration, and antimicrobial resistance of Salmonella typhimurium in experimentally infected broiler chickens.
American Journal of Veterinary Research.
Huber, G., 1979. Moenomycin and related
Huber, G. and Nesemann, G., 1968. Moenomycin, an inhibitor of cell wall synthesis.
Biochemical and Biophysical Research Communications.
Hudd, D.L., 1983. The addition of antibiotics to feedingstuffs. In : Pharmacological basis of large animal medicine. Eds Lees, J.A.B.P., Yoxall, A.T. Balckwell Scientific Publications, Oxford, UK.
Humbert, F., Lalande, F., l’Hospitalier, R., Salvat, G. and Bennejean, G., 1991. Effect of four antibiotic additives on the Salmonella contamination of chicks protected by an adult caecal flora. Avian Pathology.
Jagnow, G., 1978. Microbial degradation of the feed antibiotics
Meyers, E., Miraglia, G.J., Smith, D.A., Basch, H.I., Pansy, F.E., Trejo, W.H. and Donovick, R., 1968. Biological characterization of prasinomycin, a phosphoruscontaining antibiotic. Applied Microbiology.
Pohl, P., Laub Kupersztejn, R., Thomas, J. and van Robaeys, G., 1975. Effets de la flavomycine et de quelques agents antiparasitaires sur une souche colibacillaire hébergeant divers
Sambeth, W., Bauer, F., Dost, G. and Nesemann, G., 1974. Evaluation of the saferty of flavomycin in poultry. In proceedings of: XV World's Poultry Congress August 11- 16, New Orleans. pp.
Sepulchre, M., 1979. Flavomycine: inhibition de l’antibioresistance chez les enterobacteries. Bulletin Mensuel de la Societé Vétérinaire Pratique de France.
Smith, W.H. and Tucker, J.F., 1975. The effect of feeding diets containing permitted antibiotics on the faecal excretion of Salmonella typhimurium by experimentally infected chickens. Journal of Hygiene.
| 260 | Annex C | SOU 1997:132 |
Stutz, M.W. and Lawton, G.C., 1984. Effects of diet and antimicrobials on growth, feed efficiency, intestinal Clostridium perfringens, and ileal weight of broiler chicks.
Poultry Science.
Waals, P.v.d., 1973. [Stability of drugs in feeds. Effect of processing on a number of antibiotics and coccidiostats when these are incorporated in pelleted feeds: a snapshot]. Tijdschrift voor Diergeneeskunde.
van Heijenoort, Y., Leduc, M., Singer, H. and van Heijenoort, J., 1987. Effects of moenomycin on Escherichia coli.
Witte, W., 1996. Impact of antibiotic use in animal feeding on resistance of bacterial pathogens in humans. In proceedings of: Antibiotic resistance: origins, evolution, selection and spread. Whiley, Chichester (Ciba Foundation Symposium 207), July
| SOU 1997:132 | 261 |
Annex D: Ardacin and avoparcin
D.1 Introduction
Ardacin and avoparcin are glycopeptide antibiotics. The glycopeptides are
large molecules produced by a variety of bacterial genera including
3TREPTOMYCES, !CTINOPLANES .OCARDIA and +IBDELOSPORANGIUM. Avoparcin and ardacin are both compounds of two or more substances with similar molecular structure.
Chemically, the glycopeptides all have a common core and differ in the four side chains (figure D.I). The basic peptide structure possesses a nucleus of seven amino acid residues and five amino acids. Sugars and amino sugars, linked to the core structure, are mainly located on the outside of the molecule. They do not markedly affect the antimicrobial activity, but give the substances different pharmacokinetic properties (Reynolds, 1990).
Glycopeptides are active against
staphylococci, streptococci, enterococci, corynebacteria, clostridia and ,ISTERIA spp. Presently known glycopeptides are not active against gram-
negative bacteria because the antibiotic molecules are unable to pass through the outer membrane and hence cannot reach their target (Reynolds, 1990).
Many glycopeptides, including avoparcin, are poorly or not at all absorbed from the gastrointestinal tract (Zulalian ET AL , 1979; Hudd ET AL , 1983).
Avoparcin is, in accordance with Directive 97/6/EC, presently not approved in the EU. Similarly, ardacin has been approved for growth promotion in annex II of Directive 70/524/EEC according to Directive 94/77/EEC, but there are indications that the authorisation will not be prolonged.
Vancomycin and teicoplanin (formerly teichomycin) are well known substances that are used therapeutically in human medicine for the treatment of severe infections caused by
D.2 Mode of action and resistance mechanisms
The bacterial cell wall is composed of a three dimensional web of crosslinked peptidoglycans. Precursors for this web are transported to the outer surface of the cell membrane on a lipid carrier. The assembly of precursors by cross linking takes place on the outer surface of the cell membrane. At this step, glycopeptides inhibit cell wall formation by binding to peptide stems of the precursor ending with
| 262 | Annex D | SOU 1997:132 |
The figures in this report is only available in the printed version
Figure D.I. Tentative sketch of ardacin, avoparcin, vancomycin and teicoplanin
| SOU 1997:132 | Annex D | 263 |
The figures in this report is only available in the printed version
Figure D.I continued
| 264 | Annex D | SOU 1997:132 |
binding is the result of five hydrogen interactions between
assumed to be basically the same for all substances within the group (Arthur ET AL , 1996).
Bacteria producing peptidoglycan precursors not ending in
resistance to high levels of vancomycin and teicoplanin seen in most ,ACTOBACILLUS spp is due to the production of precursors ending in
lactate
Enterococci belonging to the %NTEROCOCCUS GALLINARIUM group are resistant to low levels of vancomycin but sensitive to teicoplanin (VanC phenotype). These enterococci produce peptidoglycan precursors where
In enterococci with acquired resistance, two main phenotypes have been described. Both these resistance phenotypes (VanA and VanB, see D.4) are due to the production of peptidoglycan precursors ending in
This will allow the bacteria to grow in presence of the antibiotic. Recently, a phenotype designated VanD has been described in a strain of % FAECIUM
(Perichon ET AL , 1997). The described strain was constitutively resistant to vancomycin and to low levels of teicoplanin. Apparently, this phenotype is also the result of the production of peptidoglycan precursors ending in D- lactate.
D.3 Development of resistance
Experimental studies
In a study by Walton (1978), the effects of avoparcin on resistance in faecal streptococci (enterococci) and staphylococci was investigated. Avoparcin was fed to chickens at dosages of 10 and 100 ppm and birds on a nonsupplemented feed were used as control group. In all groups, there was a wide variability in the total viable count of bacteria as well as in the proportion of resistant strains.
| SOU 1997:132 | Annex D | 265 |
between avoparcin and other antibiotics tested (vancomycin, among others). The results are not presented in a form that makes it possible to substantiate this claim. No species identification of resistant isolates was performed.
Kaukas and
group. This increase could be associated with an increase in the proportion of % FAECIUM in the enterococcal population of the treated birds. The proportion
of strains resistant to avoparcin (defined as having a minimum inhibitory concentration > 4µg/ml) in % FAECIUM and % FAECALIS was higher in the
control group than in the avoparcin group. The authors commented that avoparcin resistance was an overall common finding also in the other experimental groups
Cohort studies
An association between the use of avoparcin and prevalence of vancomycin resistant enterococci (VRE) in animals has been reported (Aarestrup, 1995; Klare ET AL , 1995a; Kruse, 1995; van den Bogaard ET AL , 1996). In studies from USA where avoparcin has never been used, and from Sweden where avoparcin has not been used for 10 years, no VRE were found in samples from animals when selective techniques were used (Coque ET AL , 1996; Greko, 1996). Thus, in the absence of avoparcin, the prevalence of VRE in animals is, at most, very low.
Bager and
Denmark in a retrospective cohort study. The relative risk for occurrence of high level vancomycin resistance in % FAECIUM was 3.3
herds exposed to avoparcin. The corresponding figure for poultry flocks was 2.9
Taken together, there is strong evidence of a causal relationship between avoparcin use and occurrence of high level vancomycin resistance.
Point prevalence studies
Shortly after the introduction of avoparcin, no glycopeptide resistance was found among 15 strains of % FAECIUM isolated on
(Dutta and Devriese, 1982), nor was resistance found in other enterococcal species. In table D.I, results concerning prevalence of glycopeptide resistance from Denmark and Sweden are presented. The results in these studies were
| 266 | Annex D | SOU 1997:132 |
obtained without the use of antibiotic containing media in the course of monitoring studies.
Studies using media favouring resistant isolates indicate that enterococci with high level resistance to glycopeptides are widespread among animals,
including pets and horses (Bates ET AL , 1994; Klare ET AL , 1995b; Devriese ET AL , 1996).
The lack of earlier data on VRE in animals precludes conclusions on whether the resistance trait was present in animal populations at the time of introduction of avoparcin in animal husbandry.
Table D.I Prevalence of glycopeptide resistance
| "ACTERIAL SPECIES | !NIMAL | .O OF | 9EAR | 2ESISTANCE | 2EFERENCE | #OUNTRY |
| SOURCE | ISOLATES | IN | ||||
| OR | ||||||
| SAMPLES | ||||||
| % FAECIUM | cattle | 13 | 0 | DANMAP, | Denmark | |
| 1997 | ||||||
| % FAECIUM | poultry | 54 | 59 | DANMAP, | Denmark | |
| 1997 | ||||||
| % FAECIUM | swine | 58 | 20 | DANMAP, | Denmark | |
| 1997 | ||||||
| % FAECALIS | cattle | 35 | 0 | DANMAP, | Denmark | |
| 1997 | ||||||
| % FAECALIS | poultry | 225 | 29 | DANMAP, | Denmark | |
| 1997 | ||||||
| %NTEROCOCCUS spp. swine | 46 | 1995 | 0 | Greko, | Sweden | |
| 1996 | ||||||
| %NTEROCOCCUS spp. poultry | 60 | 1995 | 0 | Greko, | Sweden | |
| 1996 | ||||||
| %NTEROCOCCUS spp. swine | 218 | 1996 | 0 | Greko, | Sweden | |
| 1997 | ||||||
| %NTEROCOCCUS spp. poultry | 207 | 1996 | 0 | Greko, | Sweden | |
| 1997 | ||||||
D.4 Resistance genes and gene transfer
Resistance to high levels of vancomycin, teicoplanin, avoparcin and, presumably, ardacin (the VanA phenotype) in enterococci is mediated by a cluster of genes designated the
| SOU 1997:132 | Annex D | 267 |
AL , 1996). The recently described VanD phenotype is encoded by a gene cluster designated VAND (Perichon ET AL , 1997).
As shown in table D.II, there is a high degree of similarity between the main mechanisms mediating the VanA and VanB phenotypes. The explanation for the difference in teicoplanin susceptibility between these phenotypes is likely to be derived from differences between the regulatory products
Table D.II. Genes encoded for by Tn (the
| 'ENES IN CLUSTER | 0RODUCT TYPE OF | &UNCTION OF RESPECTIVE | !MINO ACID | |
| RESPECTIVE GENE | GENE | IDENTITY | ||
| BETWEEN | ||||
| HOMOLOGOUS | ||||
| 6AN! | 6AN" | PRODUCTS | ||
| VANH | VANHB | dehydrogenase | Formation of |
67 |
| from pyruvate | ||||
| VANA | VANB | ligase | Binding between |
76 |
| and |
||||
| VANX | VANXB | dipeptidase | Hydrolysis of |
71 |
| Ala | ||||
| VANY | VANYB | carboxypeptidase | Hydrolysis of terminal | 30 |
| VANZ | - | unknown | Confers teicoplanin | - |
| resistance by unknown | ||||
| mechanism | ||||
| VANR | VANRB | Initiation of | 34 | |
| transcription of vanH, | ||||
| vanX and vanA/B | ||||
| VANS | VANSB | Regulation of VANR | 23 | |
| - | VANW | Unknown function | - | |
| ORF1 | Open reading frame, | |||
| transposase | ||||
| ORF2 | Open reading frame, | |||
| resolvase | ||||
Production of VanH, VanA and VanX (VanHAX), encoded for by the genes in the operon VANHAX, is normally inducible. This means that they are
| 268 | Annex D | SOU 1997:132 |
only produced in the presence of a suitable inducer such as a glycopeptide. Synthesis of VanHAX is thought to be activated (induced) by the phosphorylated form of VanR (the product of the gene VANR). Recent evidence indicate that VanS, encoded for by the VANS gene, controls the effect of VanR negatively by dephosphorylation in the absence of glycopeptides. VanS is likely to have a domain acting as a membrane associated sensor. Elimination of the VanS gene results in
Conflicting data about the inducing effects of other cell wall active antibiotics such as moenomycin (flavomycin), bacitracin, daptomycin, penicillin, cephalotin have been presented (Allen and Hobbs, 1995). Recent evidence indicate that apart from glycopeptides, only flavophospholipol
(moenomycin/flavomycin) has the capacity to act as an inducer (Baptista ET AL , 1996).
The regulatory system for production of VanHB, VanB and VanXB in strains with
Both the VANA and VANB gene clusters are generally located on plasmids and/or transposons (Arthur and Courvalin, 1993). High level resistance to
glycopeptides mediated by the
cluster is mostly associated with the conjugative transposon Tn and/or
3TAPHYLOCOCCUS AUREUS, and to various streptococci (Leclercq ET AL , 1989).
Transfer frequencies were
Resistance to glycopeptides mediated by the
| SOU 1997:132 | Annex D | 269 |
gene cluster has been found in % FAECALIS, % FAECIUM and, recently, in 3 BOVIS (Arthur and Courvalin, 1993; Poyart ET AL , 1997). As mentioned, the
Selection of mutants expressing the
AL , 1993; Green ET AL , 1995). Recently, transfer experiments with strains expressing VANB constitutively were reported (Hayden ET AL , 1997). The resulting transconjugants were either of constitutive or of inducible type. The use of a
VRE harbouring the VANA gene cluster, have been isolated from humans, both in hospitals and community, from swine, rabbits, dogs, cats, horses
chickens, turkeys, pheasants, ducks, foods of animal origin and sewage (Bates ET AL , 1994; Torres ET AL , 1994; Klare ET AL , 1995b; Chadwick ET AL ,
1996; Devriese ET AL , 1996; DANMAP, 1997). A polyclonal nature of the VRE strains has been demonstrated (Klare ET AL , 1995b). As shown in table D.II, the VANA gene cluster consists of 7 gene components. It is extremely unlikely that such a complicated gene should have developed separately in so many different host populations. Its occurrence therefore suggests an interspecies spread.
Human VRE have successfully been used to colonise mice experimentally (Whitman ET AL , 1996). This indicates that at least certain enterococcal strains can colonise, or transiently inhabit, a variety of hosts. A report on occupational exposure provides further evidence (van den Bogaard ET AL , 1997). The prevalence of VRE in turkeys, turkey farmers, turkey slaughterers and urban residents was found to be 50%, 39%, 20% and 14% respectively. Further investigations showed that VRE isolated from one of the farmers and his turkeys could not be differentiated by phenotypic or genotypic (pulsedfield gel electrophoresis) methods. Investigations of the
The question of ”identity” of genes has been a matter of debate in relation to the possible effects of the use of avoparcin in animal husbandry. The VANA
| 270 | Annex D | SOU 1997:132 |
gene cluster contains 9 genes (7 VAN and two transposition genes). Between those genes are intergenic,
and sequencing of the genes and/or their intergenic regions (Jensen, 1996; Haaheim ET AL , 1997; Kirk ET AL , 1997).
In the Norwegian study (Haaheim ET AL , 1997), PCR for the VANA and VANB genes combined with restriction fragment analysis of a long PCR covering the entire gene cluster and sequencing of the intergenic
Kirk and
supermarkets. By PCR, three intergenic regions of the
VANX, VANY and VANZ was also determined. In the chicken isolates, all three investigated genes were amplified as well as the three intergenic regions. Evidence of a not previously described insertion sequence in various locations of the intergenic region between VANX and VANY was found in some of the chicken isolates. The gene sequences of the strains from humans obviously differed from those of other described gene clusters from human strains (Arthur ET AL , 1993; Handwerger ET AL , 1995), as the VANY region as well as the intergenic regions
by Mackinnon and
124 clinical isolates, indicating the occasional finding of such variations. The fact that all the gene clusters from human strains in the study by Kirk and coworkers (1997) apparently had a deletion and/or insertion in the VANY region seems to indicate a horizontal spread of the gene within the hospital.
Therefore, it is questionable whether the investigated strains can be deemed representative for human strains in general. The authors conclusion was that the results indicate that the infections with VRE in humans may not be caused by VRE from chickens. Considering the indications of a horizontal spread of the gene cluster among the human strains in this particular material and the variation between the genes of the chicken strains, indicating a
| SOU 1997:132 | Annex D | 271 |
multiple origin, this study does not seem suitable for any general conclusions about the relation between human and animal strains.
Another report, cited above, of comparisons between the
The focus of a Danish study, reported by Jensen (1996), was slightly different. In order to investigate the degree of variation within the VANA gene cluster, isolates from different animals and humans from a wider geographic range were investigated. Similar to Kirk and
As mentioned, transfer of the
antibiotics was also present, the two traits were transferred EN BLOC (Leclercq ET AL , 1989). Conjugal
glycopeptides, erythromycin and chloramphenicol, from % FAECALIS to 3 AUREUS on the skin of hairless obese mice was demonstrated in an experiment
by Noble and
| 272 | Annex D | SOU 1997:132 |
The localisation of, for instance, MLS resistance determinants on mobile gene elements together with glycopeptide resistance means that selective pressures other that glycopeptides (e.g. macrolides) can result in increased or maintained resistance levels, and vice versa.
D.5 Effects on specific animal diseases
Glycopeptides are not used for therapy of animal diseases, although avoparcin can be used to prevent necrotic enteritis in chickens. Using a model with experimental infections, Prescott (1979) showed that inclusion of avoparcin at 20 ppm in the feed prevented necrotic enteritis, but 10 ppm was only marginally effective. Elwinger and
ppm of
additives.
D.6 Impact of resistance on animal and human health
Glycopeptides are used in human medicine for the treatment of infections (especially nosocomial) with multiresistant enterococci and staphylococci. Glycopeptides are also used to treat certain intestinal infections. Enterococci have a remarkable capacity for acquiring resistance to antimicrobial substances (Murray, 1990; Leclerq, 1997). In many instances, vancomycin is the sole drug with activity against these infectious agents.
Infections with bacteria resistant to vancomycin and/or teicoplanin have been reported with increasing frequency in human medicine. VRE are emerging as a significant cause of hospital acquired infections (HICPAC, 1995; Woodford ET AL , 1995a). Infections with VRE are associated with increased mortality (Linden ET AL , 1996). A further threat is the possible spread of vancomycin resistance genes from their enterococcal hosts to
multiresistant staphylococci. Although transfer of the VANA gene cluster from enterococci to 3TAPHYLOCOCCUS AUREUS on the skin of mice in an IN VIVO model
has been reported (Noble ET AL , 1992), no such resistance has yet been reported in clinical isolates of staphylococci.
Multiple factors predispose a person to infection with VRE, but colonisation precedes most infections (Edmond ET AL , 1995). In Europe, a community reservoir has been demonstrated. The reported rates of VRE carriage in
| SOU 1997:132 | Annex D | 273 |
According to the findings of Whitman (1996), administration of glycopeptides seems to be an important factor in establishing a persistent infection. Van der Auwera (1996) studied the effects of administration of oral glycopeptides to healthy volunteers. Before exposure, no VRE were recovered from the faecal samples whereas after exposure, 64% of the volunteers gave VRE positive samples. It is not clear whether these findings were due to a
The occurrence of VRE in food of animal origin has been demonstrated (Bates ET AL , 1994; Aarestrup, 1995; Klare ET AL , 1995a; Wegener ET AL , 1997). Once
Numerous reports are available on the spread of VRE between patients and hospital staff (for a review see Woodford ET AL , 1995a). Clearly, introduction into the ward from a community reservoir or through contaminated food may lead to an increased number of colonised patients, and consequently to an increased number of infections.
D.7 Other effects on the microflora
D.7.1 Salmonella
Of all AFA, avoparcin is probably the best studied regarding the possible influence on salmonella colonisation. Almost half of the studies discussed in chapter 4 include avoparcin. On the other hand, no studies have been found on the effect of ardacin on salmonella colonisation. All studies use chickens as experimental animals.
| 274 | Annex D | SOU 1997:132 |
Already in 1978, Smith and Tucker (1978) demonstrated an increase in the prevalence and duration of salmonella shedding among chickens fed 10 ppm
avoparcin, as compared to that of nonmedicated birds, both after direct and indirect experimental infection with 3ALMONELLA Typhimurium. The animals
were either directly infected with 03 ml of a nalidixic
In another, similarly designed, study by the same authors (Smith and Tucker, 1980), the earlier results were confirmed. In this study, 5 different serovars were used (3. Typhimurium, 3. Heidelberg, 3. Oranienburg, 3. Infantis and 3. Senftenberg). Three different poultry breeds and 3 different commercial diets were also tested. All experiments gave similar results; feeding avoparcin at a concentration of 10 ppm led to a higher prevalence and a longer duration of salmonella shedding. Different inoculation dosages were also tested. It was demonstrated that, in the medicated animals, the inoculation dose required to achieve colonisation was
In his thesis from 1981, Leuchtenberger (1981) compared the salmonella excretion of experimentally infected broilers treated with avoparcin, virginiamycin or tylosin with nonmedicated controls. In a series of experiments, 2 different concentrations of antibiotic (15 and 25 ppm avoparcin), 2 types of housing (wired cages and floor housing), 2 methods of experimental infection (direct oral inoculation or mixed in the feed) and 2 different infectious doses (103 or 104 organisms of 3 Typhimurium) were tried in various combinations. Most experiments were performed in duplicate, with groups of
| SOU 1997:132 | Annex D | 275 |
of infection, as well as the frequency of dosing with infectious organisms, and on the housing system.
It is rather surprising that so many of the trials yielded significant differences between treated and
In 1981 Gustafson and
avoparcin, virginiamycin or no antimicrobial were compared after receiving 3Typhimurium via the drinking water (GustafsonET AL, 1981). The authors
tried to achieve a level of infection that corresponds to the level of natural infection and the administration of the salmonellae was distributed over several days. The results indicate that the feeding of avoparcin led to a larger proportion of animals shedding salmonella as compared to controls during the first 3 weeks after infection. This proportion of positive birds then decreased to become lower than that in the control group at about 4 weeks post infection. However, in the samples taken from the caeca after slaughtering the birds at the end of the trial, the proportion of salmonellapositive birds was higher in the
Another study by Gustafson and
Linton and
| 276 | Annex D | SOU 1997:132 |
infected via contaminated food, and all animals, except for one control group in one experiment, also received monensin. This makes the results difficult to evaluate, but in the first study the authors stated that there was no statistical difference between the medicated group and the control group (Linton ET AL , 1985) and in the other that the results were inconclusive (Hinton ET AL , 1986).
In 1989, Barrow (1989) demonstrated a
Humbert and
In conclusion, avoparcin increases the prevalence of salmonella shedding among experimentally infected chickens, as demonstrated by a
D.7.2 Other enteric pathogens
No publications have been found on the influence of either avoparcin or ardacin on the intestinal colonisation of target animals with other zoonotic pathogens.
D.8 Toxicological aspects
Glycopeptides are poorly absorbed after oral ingestion (Prescott and Baggot, 1993).
In an experiment where radiolabelled avoparcin was fed to chickens for 7 consecutive days at the dose of 1 mg/kg body weight, virtually the entire dose was retrieved in
| SOU 1997:132 | Annex D | 277 |
(Zulalian ET AL , 1979). Residues in all tissues were less than 0.05 ppm. Based on these findings, the authors concluded that essentially no avoparcin was absorbed from the gastrointestinal tract of the animals. No residues are therefore to be expected. Maximum residue levels (MRL) have not been established.
Experimental feeding of broiler chickens with 15 ppm ardacin for 30 days resulted in liver residues of up to 50 µg/kg after a 7 day withdrawal period (Gottschall ET AL , 1995). Ardacin is not biotransformed to any large extent (Gottschall ET AL , 1995). No MRL has been established.
No information on toxicity to target species,
Occupationally derived contact dermatitis after contact with avoparcin has been reported (Barriga ET AL , 1992).
D.9 Environmental effects
As for other AFA, if feeding avoparcin or ardacin leads to a reduction in the amount of feed consumed per kg weight gain, this would be expected to lead to a reduction in nitrogen output.
Like other naturally produced antibiotics, avoparcin and ardacin would be expected to be microbially degraded in soil, but no reports relating to the environmental fate of either ardacin or avoparcin have been found. Regarding avoparcin, that has been used for over 20 years in some countries, it is remarkable that such information is not publicly available.
D.10 Summary comments
Increased glycopeptide resistance is a human health problem. Avoparcin has been shown to select for glycopeptide resistance among animal bacteria, and there is no reason to believe that ardacin would be any different in this respect. Numerous reports indicate that transfer of glycopeptide resistance between animal and human microflora can and does occur.
Avoparcin has also been shown to affect salmonella colonisation. The lowering of the infectious dose necessary for colonisation that is seen in
The available information on toxicological and environmental aspects is too scarce to form the basis of any assessment.
| 278 | Annex D | SOU 1997:132 |
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of four antibiotic additives on the Salmonella contamination of chicks protected by an adult caecal flora. !VIAN 0ATHOLOGY
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+AUKAS ! (INTON - AND ,INTON ! ( , 1988. The effect of
+IRK - #HEN ( 9 (ILL 2 #ASEWELL - 7 AND "EIGHTON $ , 1997. Novel insertion sequences from
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mediated high level glycopeptide resistance isolated from animal
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,ECLERCQ 2 $ERLOT % 7EBER - $UVAL * AND #OURVALIN 0 , 1989. Transferable vancomycin and teicoplanin resistance in %NTEROCOCCUS FAECIUM. !NTIMICROBIAL !GENTS AND #HEMOTHERAPY
,ECLERQ 2 , 1997. Enterococci acquire new kinds of resistance. #LINICAL )NFECTIOUS $ISEASES SUPPL
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,INDEN P + 0ASCULLE ! 7
vancomycin resistant Enterococcus faecium of vancomycin susceptible Enterococcus faecium. #LINICAL )NFECTIOUS $ISEASES
,INTON ! ( !L #HALABY : ! - AND (INTON - ( , 1985. Natural subclinical
salmonella infection in chickens: a potential model for testing the effects of various procedures on salmonella shedding. 6ETERINARY 2ECORD
characterization of IS , an insertion
.OBLE 7 # 6IRANI : AND #REE 2 ' , 1992.
0ANAGEA 3 AND #HADWICK 0 2 , 1996. Heat tolerance of vancomycin resistant
%NTEROCOCCUS FAECIUM. *OURNAL OF #LINICAL 0ATHOLOGY
0ERICHON " 2EYNOLDS 0 AND #OURVALIN 0 , 1997. VanD type
%NTEROCOCCUS FAECIUM BM 4339. !NTIMICROBIAL !GENTS AND #HEMOTHERAPY
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0OWER % ' - !BDULLA 9 ( 4ALSANIA ( ' 3PICE 7 !ATHITHAN 3 AND &RENCH ' , , 1995. VANA genes in
0OYART # 0IERRE # 1UESNE ' 0RON " "ERCHE 0 AND 4RIEU #UOT 0 , 1997. Emergence of vancomycin resistance in the genus 3TREPTOCOCCUS: characterisation of a VANB transferable determinant in 3TREPTOCOCCUS BOVIS. !NTIMICROBIAL !GENTS AND #HEMOTHERAPY
0RESCOTT * & , 1979. The prevention of experimentally induced necrotic enteritis in chickens by avoparcin. !VIAN $ISESES
0RESCOTT * & AND "AGGOT * $ EDS , 1993. !NTIMICROBIAL THERAPY IN VETERINARY MEDICINE 2nd ed. Iowa State University Press, Ames. pp. 612.
1UINTILIANI 2 * AND #OURVALIN 0 , 1994. Conjugal transfer of the vancomycin
resistance determinant VANB between enterococci is associated with the movement of large elements from chromosome to chromosome.
1UINTILIANI 2 * AND #OURVALIN 0 , 1996. Charachterization of Tn , a composite
transposon flanked by the IS and IS
2EYNOLDS 0 , 1990. Glycopeptide antibiotics: Pharmacokinetics, spectrum of activity, resistance patterns. (OSPITAL &ORMULARY
3MITH ( 7 AND 4UCKER * & , 1978. The effect of antimicrobial feed additives on the colonization of the alimentary tract of chickens by 3ALMONELLA TYPHIMURIUM.
*OURNAL OF (YGIENE
3MITH 7 ( AND 4UCKER * & , 1980. Further observations on the effect of feeding diets
containing avoparcin, bacitracin and sodium arsenilate on the colonization of the alimentary tract of poultry by salmonella organisms. *OURNAL OF (YGIENE 137-
150.
4ORRES # 2EGUERA * ! 3ANMARTIN M * 0EREZ $IAZ * # AND "AQUERO & , 1994.
7ALTON * 2 , 1978. The effect of dietary avoparcin on the antibiotic resistance patterns of enteric and pharyngeal bacteria isolated from broiler chickens. :ENTRALBLATT F¼R 6ETERI¤RMEDIZIN 2HEIE "
VAN DEN "OGAARD ! ,ONDON . $RIESSEN # AND 3TOBBERINGH % , 1996. Prevalence
of resistant faecal bacteria in turkeys, turkey farmers and turkey slaughterers. )N PROCEEDINGS OF TH )#!!# New Orleans.
VAN DEN "OGAARD ! * *ENSEN , " AND 3TOBBERING % % , 1997. An identical VRE isolated from a turkey and a farmer (letter). SUBMITTED.
| SOU 1997:132 | Annex D | 283 |
6AN DER !UWERA 0 0ENSART . +ORTEN 6
Influence of oral glycopeptides on the faecal flora of human volunteers: selection of highly
1136.
7EGENER ( #
7HITMAN - # 0ITSAKIS 0 ' $E*ESUS % /SBORNE ! * ,EVINSON - % AND
*OHNSON # # , 1996. Gastrointestinal tract colonisation with
%NTEROCOCCUS FAECIUM in an animal model. !NTIMICROBIAL !GENTS AND #HEMOTHERAPY
7OODFORD . *OHNSON ! 0
perspectives on glycopeptide resistance. #LINICAL
7OODFORD . *ONES " "ACCUS : ,UDLAM ( AND "ROWN $ , 1995b. Linkage of
vancomycin and high level gentamicin resistance genes on the same plasmid in a clinical isolate of %NTEROCOCCUS FAECALIS. *OURNAL OF !NTIMICROBIAL #HEMOTHERAPY
:ULALIAN * ,EE ! ( 'ARCES 4 "ERGER ( /RLOSKI % * AND %GGERT 2 ' , 1979. A
study of the excretion and tissue distribution in cattle and chickens fed
114.
| 284 | Annex D | SOU 1997:132 |
| SOU 1997:132 | 285 |
Annex E: Spiramycin, tylosin and virginiamycin
E.1 Introduction
Spiramycin and tylosin belong to the macrolide group of antibiotics, while virginiamycin belongs to the streptogramins. Macrolides and streptogramins are not chemically related but they are often grouped together because they show similar antibacterial spectra and are functionally related in their mode of action. A third class, the lincosamides is also often included for the same reason, leading to the acronym MLS (macrolides, lincosamides and streptogramins). They all inhibit protein synthesis and bind to the 50S subunit of the bacterial ribosome. They are mainly active against grampositive aerobic bacteria, various
E.1.1 Macrolides (tylosin and spiramycin)
Macrolides are derived from products from 3TREPTOMYCES spp. and are characterised by a macrocyclic lactone ring attached to one or more sugar moieties and can, according to the ring structure, be divided into
In veterinary medicine, the
Important clinical applications for those and related substances are swine dysentery (3ERPULINA HYODYSENTERIAE) and mycoplasmosis. They are also
| 286 | Annex E | SOU 1997:132 |
The figures in this report is only available in the printed version
Figure E.I. Tentative sketch of erythromycin, spiramycin and tylosin
| SOU 1997:132 | Annex E | 287 |
The figures in this report is only available in the printed version
Figure E.I. Continued; virginiamycin
therapeutic purposes, tilmicosin, has lately been introduced into the veterinary field, mainly for use against infections with !CTINOBACILLUS PLEUROPNEUMONIAE in swine, 0ASTEURELLA spp in cattle and mycoplasmosis in
various animal species (Prescott and Baggot, 1993). Therapeutic dosages of
The different macrolides differ to some extent with respect to their pharmacokinetic properties. Common characteristics of the group are high intracellular concentrations and a wide distribution in tissues (Prescott and
| 288 | Annex E | SOU 1997:132 |
Baggot, 1993), properties which together with their activity against important pathogens make them a valuable group for therapeutic purposes in both veterinary and human medicine.
E.1.2 Streptogramins (virginiamycin)
Streptogramins are natural cyclic peptides including substances such as
pristinamycins and virginiamycins (fig E.I). They are produced by a variety of 3TREPTOMYCES spp. Streptogramins consist of two components; A (also
called M) and B (also called S), each having bacteriostatic activity and acting synergistically.
Streptogramins have been sparingly used in human medicine, although there are geographic variations. Pristinamycin and virginiamycin are the most well known substances, but a new derivative,
In animals, virginiamycin is mainly used as a feed additive for growth promotion. Examples of prophylactic or therapeutic applications are swine dysentery and clostridial infections such as necrotic enteritis in poultry.
Virginiamycin is permitted for growth promotion in poultry, calves and swine at concentrations between 5 and 50 ppm for poultry,
There are few data available on the pharmacokinetics of streptogramins. Orally administered virginiamycin is not absorbed from the gut (Prescott and Baggot, 1993).
E.2 Mode of action and resistance mechanisms
The MLS antibiotics inhibit protein synthesis by binding to the 50 S subunit of the bacterial ribosome. By binding to, or near, the peptidyl transferase centre on the ribosome, macrolides prevent the elongation of the peptide being synthesised. Intrinsic resistance to macrolides, lincosamides and streptogramin B (MLSB) in gram negative bacteria is due to low permeability of the outer membrane (Leclercq and Courvalin, 1991b).
| SOU 1997:132 | Annex E | 289 |
The most common mechanism of acquired resistance to MLS antibacterials is alteration of the ribosomal target. Inactivation of the drug and active efflux from the bacterial cell have also been described.
Concerning streptogramin A, the mechanisms of intrinsic resistance are unclear. Described mechanisms of acquired resistance include enzymatic inactivation and active efflux.
Depending on the exact mechanism, a variety of resistance phenotypes are found, many of which are
positive cocci and clostridia. Regarding macrolide and streptogramin resistance in #AMPYLOBACTER spp. and 3ERPULINA spp., little is known about
the mechanisms and the genes conferring resistance.
Further information on resistance mechanisms and corresponding genes are found in E.4.
Table E.I. Examples of phenotypic and genotypic expression of some resistance genes (R=resistant, S=sensitive)
| 2ESISTANCE GENE | 0HENOTYPE | 'ENOTYPE | |||||||||||
| %R | 4Y | 3P | 3G" 3G! 3G!" | %R | 4Y | 3P | 3G" 3G! 3G!" | ||||||
| ERM | |||||||||||||
| R | R | R | R | S | S | R | R | R | R | S | S | ||
| staphylococci | R | S | S | S | S | S | R | R | R | R | S | S | |
| streptococci2 | R | R | S | S | R | R | R | R | S | S | |||
| % FAECALIS | R | R | R | R | R | R | R | R | |||||
| ERE | R | S | S | S | S | S | R | S | S | S | S | S | |
| SBH | S | S | S | R | S | S | S | S | S | R | S | S | |
| MSR | R | S | S | R | S | S | R | S | S | S | S | S | |
| SAT! | S | S | S | S | R | S | S | S | S | S | R | S | |
1 Er=erythromycin, Ty=tylosin, Sp=spiramycin, SgB= streptogramin B component, SgA=
streptogramin A component, SgAB= streptogramin components A+B
2 3 SANGUIS and 3 PYOGENES
E.3 Development of resistance
Most studies regarding development of resistance to macrolides and streptogramins have been investigating the association between clinical therapy and development of resistance. Thus, the dosages used are usually higher than what is permitted for growth promotion. Nonetheless, the results indicate that resistance develops in bacteria exposed to these substances. Moreover, as discussed in chapter 4, the concentrations used for growth
| 290 | Annex E | SOU 1997:132 |
promotion are well above the MIC values for common intestinal bacteria. Where relevant information is lacking about the situation when growth promoting dosages are used, results from studies employing therapeutic dosages have been cited.
E.3.1 Prospective studies
Experimental studies
Several studies by the research group of Linton (Linton ET AL , 1985; Hinton ET AL , 1986; Kaukas ET AL , 1987; Kaukas ET AL , 1988) illustrate the influence of macrolides and streptogramins (tylosin, virginiamycin) at growth promotion dosages on the prevalence of resistance in enterococci. In two of these studies (Hinton ET AL , 1986; Kaukas ET AL , 1987), the control groups were given a diet containing growth promoters (including virginiamycin) and/or a coccidiostat with antibacterial effect. A gradual increase in macrolide (tylosin) resistance in the control groups was noted in both these studies. This is in contrast to the studies where the control groups were provided feed without antibacterial agents (Linton ET AL , 1985; Kaukas ET AL , 1988). Considering the potential of virginiamycin to select for
In one of the studies (Linton ET AL , 1985) the enterococci were not identified to the species level, which makes the results hard to evaluate. Moreover, part of the study was conducted on commercial farms and it is not known whether any coccidiostats or other antimicrobial substances were used during the study period. In the other studies, the isolation frequencies of different species of enterococci on different sampling occasions were compared. Again, a marked difference can be observed between the two studies where the bacterial flora of the control groups was exposed to antibacterial agents and the one where it was not. The normal changes in composition of the enterococcal flora during the first weeks of life of chickens not exposed to antimicrobial agents was further investigated in another study (Kaukas ET AL , 1986). The results show the same trend in number of isolates of different enterococcal species as was observed in the unexposed control group in the study from 1988 (Kaukas ET AL , 1988). Thus, the confounding effect of growth promoters and anticoccidials in the other
control groups is further substantiated. All animals fed antimicrobial agents seem to have had a larger proportion of % FAECIUM in their intestinal flora
than animals not receiving any antibacterials.
| SOU 1997:132 | Annex E | 291 |
In the following, only the study from 1988, where the control group did not receive any antimicrobials will be discussed in detail. In this study (Kaukas ET AL , 1988), small groups of chickens were given feed containing either avoparcin, nitrovin, virginiamycin or zinc bacitracin during the first three weeks of life. The feed of the control group, as mentioned earlier, contained no additives. Each study group consisted of ten chickens and the experiment was performed in duplicate. The incidence of resistance to therapeutic antibiotics, expressed as Antibiotic Resistance Index (ARI) was significantly higher in all groups receiving antibacterials, as compared to the control group
(p=0.003). This increase was associated with an increase in the proportion of % FAECIUM in the enterococcal population of the treated birds. The incidence
of resistance to streptogramins in bacterial isolates from the group receiving
virginiamycin and from the control group differed significantly (p<0.001) for both % FAECALIS (46 and 25% respectively) and % FAECIUM (88 and 26% respectively). Thus, in addition to the selection of the species % FAECIUM by
virginiamycin, a specific selection for resistance traits was also observed. In a study from 1983 (Christie ET AL , 1983) an increase of MLS resistance
over time in gram positive cocci isolated from pigs fed 100 ppm tylosin was observed. Despite weaknesses in study design, sample size and methodology, and the fact that therapeutic dosages were used, this study nevertheless implies that tylosin in pig feed affects development of resistance in both staphylococci and enterococci from these animals. This study has been much criticised. Among other things, it appears that due to a feed mixing error the control group also received some tylosin early in the study (Anonymous, 1985). However, the influence of this error on the results would have been expected to be seen as a decrease in the difference between the two groups. Thus, it ought not to have caused any overestimation of the difference.
Field studies
The influence of virginiamycin on the prevalence of resistant enterococci within flocks of turkeys has been reported by Thal and
Different turkeys from the same flock were found to harbour the same type of
streptogramins and ampicillin increased with the age of the flock. In a German study (Hummel and Witte, 1981) macrolide resistant
staphylococci could be isolated from pigs in herds exposed to tylosin via the feed. Moreover, such resistant strains were also isolated from people who worked directly with the animals but not from their family members. All staphylococci from pigs in
| 292 | Annex E | SOU 1997:132 |
directly on the bacteria of people handling the feed and the animals. The failure to demonstrate a spread of the resistant strains or the genes mediating resistance to
E.3.2 Retrospective studies
The development of tylosin resistance in 3ERPULINA HYODYSENTERIAE was reported by Molnar (1996) in an investigation from Hungary. The proportion of strains classified as resistant to tylosin in this investigation increased from 11% during
data on consumption is presented.
The prevalence of resistance to macrolides in #LOSTRIDIUM PERFRINGENS isolated from cattle, swine and poultry was investigated in two studies from Belgium (Dutta and Devriese, 1984; Devriese ET AL , 1993). In the material included in the first study, collected between 1979 and 1982, the prevalence of resistance ranged from 1% to 21% depending on animal source. In the second study from
E.3.3
Enterococci
With the exception of % FAECIUM, enterococci are naturally resistant to the A component of streptogramins (Leclercq and Courvalin, 1991b). Acquired resistance in enterococci is, according to present knowledge, of the MLSB type, encoded for by a number of
| SOU 1997:132 | Annex E | 293 |
Table E.II. Resistance in enterococci to MLSB antibacterials as reported in different studies
| Bacterial species | Animal | No. of | Year | Resistance | Reference | Country |
| source | isolates | in % 1 | ||||
| Enterococcus | poultry | 23 | 1980 | 83 | Dutta and | Belgium |
| faecalis subsp. | Devriese, | |||||
| Liquefaciens | 1982 | |||||
| E.faecalis | poultry | 8 | 1980 | 50 | Dutta and | Belgium |
| Devriese, | ||||||
| 1982 | ||||||
| E.faecalis | swine | 225 | 91 | DANMAP, | Denmark | |
| 1997 | ||||||
| Enterococcus | poultry | 15 | 1980 | 67 | Dutta and | Belgium |
| faecium | Devriese, | |||||
| 1982 | ||||||
| E.faecium | poultry | 54 | 65 | DANMAP, | Denmark | |
| 1997 | ||||||
| E. faecium | cattle | 13 | 38 | DANMAP, | Denmark | |
| 1997 | ||||||
| E. faecium | swine | 58 | 91 | DANMAP, | Denmark | |
| 1997 | ||||||
| Enterococcus spp. | cattle | 34 | 26 | Rollins et al., | USA | |
| 1985 | ||||||
| Enterococcus spp. | swine | 72 | 79 | Rollins et al., | USA | |
| 1985 | ||||||
| Enterococcus spp. pork | 50 | 1995 | 15 | Quednau et | Denmark | |
| al., 1996 | ||||||
| Enterococcus spp. pork | 50 | 1995 | 2 | Quednau et | Sweden | |
| al., 1996 | ||||||
| Enterococcus spp. swine | 1996 | 33 | Greko, 1997 | Sweden | ||
| Enterococcus spp. poultry | 55 | Dutta and | Belgium | |||
| Devriese, | ||||||
| 1984 | ||||||
| Enterococcus spp. | poultry | 93 | 67 | Rollins et al., | USA | |
| 1985 | ||||||
| Enterococcus spp. poultry | 50 | 1995 | 27 | Quednau et | Denmark | |
| meat | al., 1996 | |||||
| Enterococcus spp. poultry | 50 | 1995 | 10 | Quednau et | Sweden | |
| meat | al., 1996 | |||||
| Enterococcus spp. poultry | 207 | 1996 | 17 | Greko, 1996 | Sweden | |
1When several macrolides have been investigated, the figures for erythromycin have been used.
| 294 | Annex E | SOU 1997:132 |
Staphylococci
Data on the prevalence of macrolide resistance in animal staphylococci from various studies are presented in table E.III. A majority of these strains have been isolated from pathological lesions or from skin. As can be seen in the table, there seems to be a marked difference in the prevalence of macrolideresistance between staphylococci from different animal species. The majority of the staphylococcal strains reported as resistant in table E.III expressed the resistance constitutively.
Table E.III. Resistance in 3TAPHYLOCOCCUS AUREUS to erythromycin as reported in different studies
| Bacterial | Animal | n of | Year | Resistan | Reference | Country |
| species | source | isolates | ce in % | |||
| S.aureus | cattle | 517 | Devriese, 1980 | Belgium | ||
| S.aureus | cattle | 211 | 1 | DANMAP, 1997 | Denmark | |
| S.aureus | cattle | 183 | 1995 | 1 | Nilsson, 1996 | Sweden |
| S.aureus | poultry | 399 | Devriese, 1980 | Belgium | ||
| S.aureus | swine | 124 | Devriese, 1980 | Belgium | ||
Campylobacter
# JEJUNI is associated mainly with poultry and # COLI with swine. The species most commonly isolated from humans is # JEJUNI. The incidence of macrolide resistance in # COLI is much higher than in # JEJUNI. (Wang ET AL ,
1984; Burridge ET AL , 1986; Lacey, 1988;
DANMAP, 1997). It has been implied that the widespread resistance to macrolides in # COLI may be linked to the use of macrolides (Davies ET AL ,
1996; Moore ET AL , 1996). One of the few studies made on MICs of tylosin for field isolates of # COLI (Ryden R, cit. by Burridge ET AL , 1986) showed that the prevalence of tylosin resistant # COLI among nontreated pigs, or pigs
given antibacterials other than tylosin, was 55%, and in pigs receiving therapeutic doses of
originating from poultry are rarely resistant to macrolides. The use of macrolides is presumably higher in swine as compared to poultry as tylosin is approved for growth promoting purposes only in the former species. In view of the zoonotic character of campylobacteriosis, it is remarkable that no further investigations on the possible relation between use of macrolides as growth promoters and selection of resistant strains of campylobacters have been made.
| SOU 1997:132 | Annex E | 295 |
Table E.IV. Prevalence of erythromycin resistance in campylobacter in various studies
| Bacterial | Source | n of | Year | Resistance | Reference | Country |
| species | isolates | in % | ||||
| C. coli | swine | 99 | 55 | DANMAP, 1997 | Denmark | |
| C. coli | poultry | 91 | 12 | Netherlands | ||
| al., 1994 | ||||||
| C. coli | sewage | 32 | 1995 | 25 | Koenraad et al., | Netherlands |
| 1995 | ||||||
| C. coli | human | 58 | 19 | Reina et al., 1992 | Spain | |
| C. coli | human | 801 | 1984 | 70 | Wang et al., | USA |
| and swine | 1984 | |||||
| C. jejuni | poultry | 55 | 4 | DANMAP, 1997 | Denmark | |
| C. jejuni | poultry | 177 | 2 | Netherlands | ||
| al., 1994 | ||||||
| C. jejuni | sewage | 121 | 1995 | 9 | Koenraad et al., | Netherlands |
| 1995 | ||||||
| C. jejuni | human | 614 | 2 | Reina et al., 1992 | Spain | |
| C.jejuni | human | 982 | 1984 | 3 | Wang et al., | USA |
| and swine | 1984 | |||||
| Campylo- | Poultry | 59 | 5 | Cabrita et al., | Portugal | |
| bacter spp. | 1992 | |||||
| Campylo- | Swine | 65 | 26 | Cabrita et al., | Portugal | |
| bacter spp. | 1992 |
111 isolates from humans, 69 isolates from swine
293 isolates from humans, 5 isolates from swine
Serpulina
During the 1970s, tylosin was regarded as the drug of choice in the treatment
of swine dysentery. This is no longer the case, as resistance to tylosin now seems to be widespread in strains of 3ERPULINA HYODYSENTERIAE (Smith ET AL ,
1991 see also E.3.2; Prescott and Baggot, 1993; Fellström ET AL , 1996; Molnar, 1996). A high proportion of virginiamycin resistance in this group of bacteria has also been reported from other countries in recent years (Ronne and Szancer, 1990).
E.4 Resistance genes and acquisition of resistance
Various genes conferring the different types of resistance mentioned above (E.2) have been identified (table E.IV). These genes, often of a highly mobile type, can be transferred between bacteria, within the same species and between different species. They are often carried on ”jumping genes” (transposons) which means that they can copy themselves from, for instance,
| 296 | Annex E | SOU 1997:132 |
the chromosome to a plasmid or from one plasmid to another plasmid. Further, they may be carried on
Table E.IV. Examples of resistance to MLS antibacterials by different mechanisms and genes. (Data compiled from Arthur ET AL , 1987; Brisson
Noel ET AL , 1988; Leclercq and Courvalin, 1991a; Leclercq and Courvalin, 1991b; Eady ET AL , 1993; Mullany ET AL , 1995; Tauch ET AL , 1995;
Weisblum, 1995a; Allignet ET AL , 1996)
| Phenotype | Mechanism | Gene | Described | Example of species |
| localisation | ||||
| MLSB | target | ermA | Tn554 | S. aureus, coagulase |
| modification | negative staphylococci | |||
| ermB, | Tn551, pAM77, | S. aureus, S. intermedius, S. | ||
| ermAM | Tn917, Tn1545, | hyicus, S. pneumoniae, | ||
| plus various other | Streptococcus spp., E. | |||
| plasmids and | faecalis, Lactobacillus spp. | |||
| transposons | ||||
| ErmC | pE194, plM13, | S. aureus, coagulase | ||
| pE5, pNE131 | negative staphylococci, S. | |||
| hyicus, Bacillus subtilis | ||||
| ermCX | Tn5432 | Corynebacterium xerosis | ||
| ermD | chromosome | B. licheniformis | ||
| ermBC | pIP1527 | E. coli | ||
| ermF | pBF4 | Bacteroides spp. | ||
| ErmE | chromosome | Streptomyces erythreus | ||
| ermP, | Cl. perfringens | |||
| ermQ | ||||
| ermZ | Tn5398 | Cl. difficile | ||
| ermJ | Bacillus anthracis | |||
| ermM | S. epidermidis | |||
| drug | ereA | pIP1100 | E. coli | |
| inactivation | ||||
| ereB | pIP1527 | E. coli | ||
| L | linA | pIP855 | S. haemolyticus | |
| linA’ | pIP856 | S. aureus | ||
| SB | sbh | pIP524 | S. aureus | |
| vgb | pIP680 | S. aureus | ||
| SA | satA | pAT424 | E. faecium | |
| vat | pIP680, IS257 | S. aureus | ||
| vatB | pIP1156 | S. aureus | ||
| active efflux | erpA | S. epidermidis | ||
| msrA | S. epidermidis | |||
| SA | vga | plasmids | S. aureus, S. epidermidis |
| SOU 1997:132 | Annex E | 297 |
E.4.1 Target modification
substantial sequence diversity but studies on their evolutionary relationship suggest that they are of ancient presence in at least 3TREPTOMYCES spp, grampositive cocci and "ACILLUS LICHENIFORMIS (Arthur ET AL , 1987). However,
transfer of ERM genes occurs under natural conditions to bacteria which are
phylogenetically remote from the above mentioned bacteria, such as %SCHERICHIA COLI (Brisson Noel ET AL , 1988). This transfer is believed to be a
recent event although the genes are already disseminated in enterobacteria. Transfer of
studies.
pIP811 together with other resistance determinants, between among others
,ISTERIA MONOCYTOGENES and % FAECALIS.
4N1545, harbouring ERMB, APHA3’ (kanamycin resistance) and TETM (tetracycline resistance) between % FAECALIS and , MONOCYTOGENES
McConnell and
Inducible or constitutive expression
Expression of ERM genes can be inducible or constitutive. When constitutively expressed, the methylase encoded for by the
The type of expression is related to the class of ERM gene but depends on a regulatory gene sequence upstream from the methylase gene sequence itself (the messenger). The mechanisms of resistance by target modification and induction of this resistance has been described by Weisblum (Weisblum, 1985; Weisblum, 1995a; Weisblum, 1995b; see figures).
| 298 | Annex E | SOU 1997:132 |
The figures in this report is only available in the printed version
Figure E.II. Induction of the ermC gene (Adapted from Weisblum, 1995a)
The entire gene consists of a leader peptide and a series of inverted complementary repeat sequences that can redistribute and assume alternative double stranded conformations, followed by the methylase gene sequence. In the presence of a macrolide the ribosomes are inhibited ("stalled"). Activation of the messenger depends on the degree to which ribosomes, during translation of the leader peptide, are inhibited by the macrolide and thereby disrupt the secondary structure in a certain part of the control region. The result of inverted repeat sequence redistribution is the unmasking of a ribosome binding site for synthesis of the methylase. The inductive capability of different macrolides is dependent on their capability of stalling the ribosome at the right step in the translation of the leader peptide. This varies between different ERM genes and between different bacterial species, but
| SOU 1997:132 | Annex E | 299 |
Enterococci and streptococci
In enterococci and streptococci (formerly group D streptococci), MLSB resistance can be expressed either inducibly or constitutively. However, in some cases all MLSB antibiotics can act as inducers which explains the diversity of resistance phenotypes coded for by similar genes. Thus, in the case of MLSB resistance, phenotypic characteristics do not necessarily provide a basis for conclusions on genotype. Reports from Japan and France have shown a trend from predominantly inducible resistance to predominantly constitutive resistance in staphylococci whereas in British and German reports inducible type still dominate (Jenssen ET AL , 1987). A noninducing macrolide or streptogramin would be expected to select for constitutively expressed resistance, since mutation to the constitutive form would be the only way bacteria harbouring inducible ERM genes could
survive exposure to a
Investigations of Swedish isolates from humans of 3TREPTOCOCCUS PYOGENES
revealed that the resistance determinant was present on an
(Schalen ET AL , 1995). In an Italian study on the same species, in addition to the MLSB type mediated by
an
and meningitis in piglets is today more commonly associated with bronchopneumonia in weaners and fatteners. In a study from USA (Stuart ET AL , 1992) a substantial increase in resistance to the MLS antibacterials in 3 SUIS was demonstrated when compared to a similar study undertaken previously. Further, the study showed that resistant isolates were capable of transmitting resistance by conjugation possibly mediated by a transposon similar to 4N916. Wasteson and
determined by ERMC and/or ERMB genes and furthermore, that the resistance genes could be transmitted from 3 SUIS to % FAECALIS
Enterococci are known to frequently exchange resistance genes, not only with other enterococci but also with other bacterial genera. Transfer of resistance genes between enterococci and staphylococci appears to take place in nature, maybe mediated by broad host range plasmids (Bonafede ET AL , 1997).
| 300 | Annex E | SOU 1997:132 |
Staphylococci
In staphylococci harbouring ERMC or ERMA genes, inducible resistance is triggered by 14- and
resulting transconjugants can be either of constitutive or of inducible type (Hayden ET AL , 1997).
Eady and
3TAPHYLOCOCCUS INTERMEDIUS from dogs.
Table E.II. Number of staphylococcal strains from various sources harbouring
| Gene | Swine | Dogs | Humans |
| ermA | 0 | 1 | 14 |
| ermB | 7 | 11 | 0 |
| ermC | 21 | 1 | 89 |
| msrA | 2 | 1 | 70 |
Fourteen of the human isolates, but none of the isolates from pigs, and only one isolate from a dog harboured ERMA. The ERMB gene was only found in animal isolates. The ERMC gene was the most common among human and pig isolates, while this gene was only found in one of the dog isolates. The
| SOU 1997:132 | Annex E | 301 |
MSRA gene was only found in 2 pig isolates and one dog isolate, but in 70 human isolates. Several isolates included in the study harboured more than one of the resistance determinants studied. All of the pig strains, half of the dog strains and 13 of the human strains expressed MLSB resistance constitutively. This illustrates the fact that expression of resistance genes cannot be used to predict type of resistance determinant, and that some ERM genes circulate in both human and animal populations. Since pigs, as opposed to dogs, are not exposed to
Westh and
staphylococci from Denmark. They found that the ERMA gene was solely responsible for erythromycin resistance in strains isolated before 1971, but had since then gradually disappeared. Today the ERMC determinant is responsible for 72% of erythromycin resistance in Danish 3 AUREUS, according to the authors. Both constitutive and inducible expression was noticed, with the inducible form being the most common. No ERMA genes were found in
Clostridia
The hitherto described genes encoding for macrolide resistance in clostridia belong to the ERM family. The most common resistance determinant seems to be ERMQ which has been found in isolates from pigs and humans from a wide geographical range (Berryman ET AL , 1994). A second, apparently less
prevalent gene, the
Berryman and Rood, 1995). Genes belonging to this subgroup have earlier been described in numerous bacterial genera, both
negative, indicating that they are readily transferred. Sequencing of the # PERFRINGENS ERMB determinant and its flanking regions by Berryman and
Rood (1995) revealed a close similarity to the corresponding determinant on plasmids from % FAECALIS (pAMb1) and 3TREPTOCOCCUS AGALACTIAE (pIP501)
which are both of the conjugative, broad host range type. It was therefore
| 302 | Annex E | SOU 1997:132 |
postulated that the # PERFRINGENS ERMBP determinant was derived from an enterococcal or streptococcal determinant. The gene ERMB is carried by transposon Tn917, often residing on a self transmissible, broad host range plasmid. This gene is widespread in human and animal isolates of enterococci as well as in other bacteria (Rollins ET AL , 1985; LeBlanc ET AL , 1986).
E.4.2 Enzymatic inactivation
Unlike target modification, which causes resistance to structurally distinct antibiotics, enzymatic inactivation confers resistance only to structurally related drugs.
Macrolide modifying enzymes have been described in lactobacilli of
animal origin (Dutta and Devriese, 1981; Arthur ET AL , 1987) and in 3TREPTOMYCES spp. (Arthur ET AL , 1987), but the genes in question have not
been described (Arthur ET AL , 1987). Inactivation of erythromycin by production of erythromycin esterases or phosphotransferases has been described in human enterobacteria isolated from patients undergoing treatment with erythromycin (Arthur ET AL , 1987). Two types of esterases (I and II) are encoded for by the genes EREA and EREB, respectively (Arthur ET AL , 1987; Leclercq and Courvalin, 1991b).
Resistance to streptogramin antibiotics, caused by the modification of both components, was described in the 70s, in 3 AUREUS (Le Goffic ET AL , 1977a;
Le Goffic ET AL , 1977b). This resistance is coded for by two genes, SBH and SAA located on a large plasmid. Most of the strains are also resistant to low levels of lincosamides although these antibiotics are not inactivated. Since this original description, several different enzymes and genes with similar activity have been described (Rende Fournier ET AL , 1993; Allignet and El Solh, 1995). In staphylococci, resistance to the group A compound will always confer resistance to the mixtures of group A and B compounds. This is not the case with resistance to group B compounds where group A compounds still may remain active (Duval, 1985).
E.4.3 Active efflux
Resistance due to active efflux has been reported in staphylococci. The resistance gene MSRA described in 3TAPHYLOCOCCUS EPIDERMIDIS encodes a
antibiotics. Further, a constitutively expressed resistance of
| SOU 1997:132 | Annex E | 303 |
Courvalin, 1991b). In this case, the streptogramins retain their effect. Active efflux of streptogramin A is mediated by the gene VGA coding for an ATP-
binding protein probably involved in the active transport of the compound. This gene has been found in 3 AUREUS and 3 EPIDERMIDIS (Allignet ET AL ,
1992; Rende Fournier ET AL , 1993).
E.4.4
%NTEROCOCCUS FAECALIS to 3TAPHYLOCOCCUS AUREUS has been shown to occur both IN VITRO and IN VIVO , in a mouse model (Noble ET AL , 1992). Others have
reached similar results. Leclercq (1989) achieved
resistance and ERMAM, located on the plasmid plP819, between different enterococcal species and from enterococci to streptococci and ,ISTERIA MONOCYTOGENES. Further, the transfer of
harbouring virulenceand resistance factors (among others ERMB on Tn ) between strains of % FAECALIS in an animal model has been demonstrated (Huycke ET AL , 1992).
E.4.5 Exposure to AFA and resistance
Evaluating resistance data between different countries or different regions, is difficult without information about the degree of exposure to various antibacterials in various regions. Little information is available concerning statistics on the quantities of MLS antibacterials used for different purposes in different countries. Moreover, differences between countries in climate, animal husbandry and animal health, frustrate comparisons. However, Denmark and Sweden are, in these respects, similar enough for a comparison to be attempted. Recent statistics from Denmark and Sweden are presented in table E.VI. The statistics on human consumption are only available as the sum of macrolides and lincosamides. An estimate of the proportion of macrolides in this group was made based on
| 304 | Annex E | SOU 1997:132 |
Table E.VI. Usage of MLS antibacterials in 1995 in Sweden and Denmark expressed as kg active substance (Apoteksbolaget, 1996; DANMAP, 1997)
| Country | Tylosin | Spiramycin | Macrolides | Virginiamycin |
| (total) | ||||
| Denmark | ||||
| animal therapy | 9500 | |||
| feed additives | 52275 | 507 | 52782 | 2590 |
| human therapy | 65001 | |||
| Sweden | ||||
| animal therapy | 1238 | 565 | 1803 | 575 |
| human therapy | 61001 |
1 Estimated as described above
In the debate concerning the contribution of MLS antibacterials used as growth promoters to development of resistance, it has often been assumed that their therapeutic use dominate the selective pressure. The figures from Denmark show, however, that the amount of macrolides used for growth promotion exceeds the therapeutic use in animals by more than 5 times. Comparing the amounts used for therapy in Sweden and Denmark, the differences can be attributed to different sizes of animal populations. Thus, it is a reasonable assumption that the observed differences in prevalence of MLS resistance between enterococci isolated from Danish and Swedish animal sources (see table E.II) are largely explained by the use of MLS antibacterials, mainly tylosin, for growth promotion.
In a paper by Lacey (Lacey, 1988) data on macrolide consumption in humans (erythromycin) and animals (tylosin) during 1986 in the UK are presented. The quantities used in animals and man are about the same, 47 000 and 51 000 kg active substance, respectively. However, the majority of animals (pigs) would have received a low level of antibiotic for a long time, whereas the humans would have received a high level for a short time. Thus, more individuals were exposed for a longer time period in the animal population, as compared to the human population.
E.5 Effects on specific animal diseases
Virginiamycin may be used for prevention of necrotic enteritis (NE) in poultry In a large study, Jansson and
| SOU 1997:132 | Annex E | 305 |
Stutz and
to
MLS antibacterials are still considered to be alternatives in the therapy and prevention of swine dysentery (Allen ET AL , 1992). Although tiamulin is often the drug of choice for treatment of this disease, tylosin is in many areas important for maintaining the means to control the infection.
There seems to be a good correlation between MIC values of 3 HYODYSENTERIAE for tylosin and therapeutic effect of this substance. Williams
and Shively (1978) found that tylosin at
while it was only partly effective against the disease induced by isolates with higher MIC values for tylosin. Jacks and
Miller and
virginiamycin and tylosin on pigs experimentally infected with 3 HYODYSENTERIAE. They found a good prophylactic effect of virginiamycin at
about 25 ppm but only a moderate effect of tylosin even at about 100 ppm. Antimicrobial resistance pattern of the infecting strain is not presented, but presumably it was resistant to tylosin. Williams and Shively (Williams and Shively, 1978) fed experimentally infected pigs virginiamycin at 50 to 100 ppm. Virginiamycin could not control the disease, although clinical signs were a little less common in medicated pigs than in
McOrist and
| 306 | Annex E | SOU 1997:132 |
E.6 Impact of resistance on animal and human health
E.6.1 Consequences of MLS resistance
As stated earlier, macrolides and streptogramins are important therapeutic or prophylactic substances for animals. Macrolides also play an important role in human medical therapy (Kirst and Sides, 1989). Streptogramins are not, except in some countries, yet widely used in human medicine, but due to the development and spread of antimicrobial resistance among human pathogens these compounds are expected to become more commonly used in the future (Pechere, 1992; Pechère, 1996).
From an animal health point of view resistance to
dysentery can lead to substantial losses and eradication of whole herds may be the only option left (Bouwkamp, 1982). )N VITRO data on resistance of this
bacterium seems to be a good predictor of clinical outcome after treatment (Williams and Shively, 1978; Jacks ET AL , 1986). Unfortunately, such data indicate that the therapeutic potential of the MLS drugs is now limited (Ronne and Szancer, 1990; Gunnarsson ET AL , 1991; Fellström ET AL , 1996; Molnar, 1996). Buller and Hampson (1994) claimed that the present situation presents a potential threat to the pig industry.
Bearing in mind that both macrolides and streptogramins are attractive therapeutic options for several important animal and human diseases, a further increase in resistance prevalence would be most unfortunate
E.6.2 Influence of AFA usage
The possible influence of the use of macrolides as growth promoters on the resistance of human pathogens was intensively discussed in relation to the recommendations of the Swann committee. The debate has, to a large extent, been concentrated on the possible influence on the resistance of staphylococci. Knothe (1977b) concludes, in a review on medical considerations of the use of macrolides in animal feeds, that transfer of staphylococcal strains from animals to humans is possible. However, he considers this to be of no significance since the likelihood of an animal strain actually causing disease in humans is small and the proportion of macrolide resistant strains in human medicine is low. The basis of this assumption is a review of investigations published from
| SOU 1997:132 | Annex E | 307 |
towards a reduction over time. In a more recent report from the same region of Germany, the prevalence of erythromycin resistance in 3 AUREUS from community acquired infections was 8%, from general ward 13% and from intensive care 28% (Shah ET AL , 1993). In some recent reports from other European countries, though, the prevalence of resistance to macrolides in staphylococci and enterococci approaches 50% (Turano ET AL , 1994). At the time when Knothe (1977b) published the review, exchange of genes between
The conclusion is thus based on the assumption that animal strains have to colonise and infect humans in order for resistance to have an impact on human health. This view is no longer predominant (see chapter 4).
Genes of the
The rarity of tylosin resistance in human pathogenic bacteria has been used as evidence that the flow of resistant organisms or their genes from animals to man is rare (Lacey, 1980; Lacey, 1981; Lacey, 1984; Lacey, 1988). In the most recent paper on this topic by Lacey (1988), the author presented five statements supporting the view that resistant bacteria in animals and humans are two separate entities;
Firstly, that animal staphylococci survive poorly in human environments. This may well be the case, but as long as resistance genes from these staphylococci (and other bacteria, such as enterococci) can be transferred to human strains, the survival of the bacteria themselves is not necessary. Moreover, other bacterial species have been found to be less
Secondly, he stated that tylosin resistant strains may grow more slowly than sensitive cultures and would therefore disappear as the exposure to the antibiotic stops. Since use of tylosin as a growth promoter means an almost continuous exposure, this arguing seems to support the view that the use should be limited. Moreover, resistant bacteria may persist even in the absence of a selective pressure (see chapter 4). Relieving the selective pressure of antibiotic exposure in humans usually leads to a drop in the prevalence of resistance, but one cannot assume that the pool of resistance genes would disappear as long as a selective pressure is in force in animal husbandry.
Thirdly, the difficulties in transferring resistance between animal and human staphylococci were pointed out. This was supported by an earlier investigation by the same author (Lacey, 1980), showing that transfer between animal and human staphylococci can occur, but at a lower frequency and in fewer strains than between isolates of staphylococci from the same source. The low frequency of transfer in this study is not surprising, since
| 308 | Annex E | SOU 1997:132 |
only phage dependent transfer is studied. Conjugative transfer is generally believed to be more common. Regarding what can be deemed a ”low” transfer frequency, see chapter 4.
Fourthly, it was stated that tylosin resistance requires the presence of erythromycin to occur, a statement that is true regarding phenotypic expression of
Fifthly the author proposed that the low level of tylosin used for growth promotion in pigs may be too low to select for resistance. The concentrations used for growth promotion are above the MICs of naturally susceptible bacteria (see chapter 4). Finally, the author also concluded that since there are no residues in meat, no selection pressure will be exerted in man. Acknowledging that this is true, the real concern about meat would be that it can contain microbes carrying transferable resistance genes. Besides meat, there are numerous direct and indirect contact areas between human and animal bacteria.
Lately, streptogramin resistance in animal and human bacteria has received increased attention as the streptogramin
| SOU 1997:132 | Annex E | 309 |
E.7 Other effects on the microflora
E.7.1 Salmonella
There are several studies available on the possible influence of antibacterial feed additives on colonisation of the intestines and the shedding of salmonella. At least 10 of these studies from the last decade involve virginiamycin and 3 of those also include tylosin.
The main problem with all studies in this area, however, is the variability in study design which makes them difficult to compare and evaluate. Weaknesses that are found in several of the studies undertaken include, among other things, inadequate number of animals in the experimental groups, lack of nonmedicated control and undocumented sensitivity of the bacteriological methods employed to recover the salmonella organisms. Taken together, the design of these studies only allows for extremely large differences in outcome. Some of the studies use commercial feed and do not state whether care has been taken to ascertain that this feed contains no antimicrobials. Since antimicrobial feed additives are very common ingredients in commercial feedstuffs this may otherwise influence the outcome of the experiment. Several authors claim that their experimental models are reproducible, but any well described method is reproducible. The results obtained with the method, however, have to be reproducible too.
Bearing in mind the weaknesses mentioned above there are some studies which are nevertheless worth further comment.
swine (1979; 1983). No significant differences between medicated animals and control groups were found, when comparing prevalence and duration of salmonella shedding. However, the experimental groups were small in both studies, between 5 and 20 animals per group, which would only allow for very large differences between groups to be detected.
In his thesis from 1981, Leuchtenberger (1981) compares the salmonella excretion of experimentally infected broilers treated with avoparcin, virginiamycin or tylosin with nonmedicated controls. In a series of experiments 2 different concentrations of antimicrobial (20 and 30 ppm virginiamycin, 50 and 100 ppm tylosin), 2 types of housing (wired cages and floor housing), 2 methods of experimental infection (direct oral inoculation
and mixed in the feed) and 2 different infectious doses (103 or 104 organisms of 3ALMONELLA Typhimurium) were tried in various combinations. Most
experiments were performed in duplicate, with groups of
| 310 | Annex E | SOU 1997:132 |
occasions, though. The author concludes that feeding avoparcin, virginiamycin or tylosin, at both levels tested, can prolong the persistence of 3 Typhimurium infection in the intestine as well as internal organs, and significantly increases the amount of salmonella found in samples from these sites. It was also found that the duration and frequency of Salmonella excretion depends on the dose and way of infection, as well as the frequency of dosing with infectious organisms and on the housing system.
It is rather surprising that so many of the trials yield significant differences, since the sample sizes are invariably too small for any small differences to be detected. However it is only in the main experiments, where cloacal swabs were taken continuously, that large enough numerical differences were recorded to be interpreted as a strong tendency towards increased salmonella excretion in treated birds. The trials where all birds were killed and cultures made from internal organs must be regarded as inconclusive, due to too small sample sizes and too variable numerical differences in the results to be statistically significant.
In 1981 Gustafson and
avoparcin or virginiamycin or no antimicrobial were compared after receiving 3Typhimurium via the drinking water (GustafsonET AL, 1981).
The authors tried to achieve a level of infection that corresponds to the level of natural infection and the administration of the salmonellae was distributed over several days. The results indicate that the feeding of virginiamycin or avoparcin led to a larger proportion of animals shedding salmonella as compared to controls during the first 3 weeks after infection. This proportion of positive birds then decreased to become lower than that in the control group at about 4 weeks post infection. However, in the samples taken from the caeca after slaughtering the birds at the end of the trial, the proportion of salmonella positive birds was highest in the
The sample size in this study is comparatively large, 100 animals in each group (all animals were sampled on each sampling occasion). The bacteriological method used for isolating the inoculated salmonella from the faecal samples include selective culture on media containing nalidixic acid, as the experimental strain was resistant to nalidixic acid. This isolation procedure would, however, also yield nalidixic
| SOU 1997:132 | Annex E | 311 |
investigated the differences between birds fed monensin and birds fed monensin plus avoparcin or virginiamycin.
Smith and Tucker have published several studies on the influence of various antimicrobial substances on the course of salmonella infection and excretion in broiler chickens (Smith and Tucker, 1975a; Smith and Tucker, 1975b; Smith and Tucker, 1978; Smith and Green, 1980; Smith and Tucker, 1980). One study from 1975 includes both tylosin and virginiamycin (Smith and Tucker, 1975b) and one from 1978 includes tylosin (Smith and Tucker, 1978). Tylosin was given at concentrations of 10 and 100 mg/kg feed in both studies and in the study from 1975 virginiamycin was also given at concentrations of 10 and 100 mg/kg. Duplicate experiments were performed on groups of
animals were experimentally infected with 0.3 ml of a nalidixic
study from 1978, the animals were infected through contact with experimentally infected chickens. Cloacal swabs were taken from all animals throughout the trials and caecal contents were sampled after slaughter at the end of each trial. The bacteriological methods are the same as those employed by Gustafson and
In these two studies tylosin, at both concentrations tested, gave a higher rate and a greater amount of salmonella excretion in the birds given this diet, compared to that of the control groups. Virginiamycin, at the concentrations tested, caused only a slight increase, or no increase, in the rate and amount of salmonella excretion.
No studies addressing the issue of
Taken together, it appears that tylosin and virginiamycin might effect salmonella colonisation, but available studies can not provide the basis for any proper assessment. No information has been found on spiramycin in this matter.
E.7.2 Other enteric pathogens
No data on influence of macrolides on colonisation with other enteric pathogens, such as #AMPYLOBACTER spp. or 9ERSINIA ENTEROCOLITICA has been
found.
| 312 | Annex E | SOU 1997:132 |
E.7.3 Other effects
Continuous administration of MLS antibacterials to animals might interfere with disease surveillance. Kempf and
macrolide (spiramycin or tylosin) treatment might hinder the detection of
samples from subclinically infected chickens. Ronne (1992) reported a decrease in the isolation rate of 3ERPULINA HYODYSENTERIAE after introducing
virginiamycin in the feed of pigs with clinical signs of infection. Such effects need to be considered for disease control based on
surveillance programs, as they may substantially affect the efficiency and the benefit/cost ratio of the program.
E.8 Toxicological aspects
As spiramycin, tylosin and virginiamycin are safely used in clinical therapy, they are not expected to have any obvious toxic effects on the target species at growth promoting dosages.
E.8.1 Residues
Spiramycin and tylosin were evaluated by the Committee of Veterinary Medical Products(CVMP) in 1994 and by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) 1991 (1991). In table E.VII, provisional maximum residue limits (MRL) fixed by the Commission in 1995 for spiramycin and tylosin when used as veterinary medical products are shown. Corresponding values for spiramycin when used in cattle were not given at the time, but temporary MRLs for cattle as fixed by JECFA 1991 to 50, 300 and 200µg/kg for muscle, liver and kidney. respectively.
MRLs are based on ADI (acceptable daily intake). These limits are based on risk assessments and are set to ensure that residues in animal products do not harm the consumers of these products.
| SOU 1997:132 | Annex E | 313 |
Table E.VII. Maximum residue limits of spiramycin and tylosin as veterinary medical products in foodstuffs of animal origin according to Commission regulation (EC) 1442/95
| 3UBSTANCE | !NIMAL | 4ARGET TISSUE | |||
| RESIDUE | SPECIES | uG KG | |||
| 3PIRAMYCIN | spiramycin | swine | 6001 | liver | |
| 3001 | kidney, muscle | ||||
| 2001 | fat | ||||
| 4YLOSIN | tylosin | bovine, swine, | 100 | muscle, liver, | |
| poultry | kidney | ||||
| bovine | 50 | milk | |||
1 Provisional MRL, expired July 1, 1997.
According to a study by Green Lauridsen and
Published studies on residue aspects on spiramycin when given at growth promotion dosages have not been found. Spiramycin fed to pigs at approximately 8 times the maximum concentration permitted for growth promotion resulted in liver residues at zero withdrawal time of 10 times the MRL (FAO/WHO, 1991). Assuming a linear relationship, residues in swine liver, from spiramycin used for growth promotion, could be above MRL at zero withdrawal time (see chapter 5). The pharmacokinetics of spiramycin are not linear in swine (Sutter ET AL , 1992), which could result in higher residue levels than what might be expected. Similar experiments in poultry, with similar assumptions of a linear relationship, indicates that in poultry the MRL would not be reached with spiramycin at growth promotion dosages(FAO/WHO, 1991). For tylosin at growth promoting dosages, MRL is not expected to be reached (see chapter 5).
Further investigations into possible residues from spiramycin as antibacterial feed additives (AFA) in target species are therefore necessary.
E.8.2 Allergy
Allergic reactions triggered by macrolide therapy in humans are rarely reported (Descotes ET AL , 1988; Periti ET AL , 1993). Occupational contact dermatitis and/or asthma is, however, not unusual in farmers (especially pig farmers), feed plant workers, veterinarians and people working in the pharmaceutical industry (Hjorth and Weismann, 1973; Gollins, 1989; Lee ET AL , 1989; Caraffini ET AL , 1994; Danese ET AL , 1994). Hypersensitivity to tylosin and/or spiramycin, indicating a certain but not absolute crossreactivity, has been reported (Hjorth and Weismann, 1973). Airborne antigen is thought to be the main cause of these reactions.
| 314 | Annex E | SOU 1997:132 |
There is no information available on the prevalence of hypersensitivity to virginiamycin, but there can be no doubt that both tylosin and spiramycin are capable of functioning as potent antigens and that people continuously exposed to dust containing these substances may become allergic. Frequent use of macrolides in animal feed may therefore be regarded as a professional hazard for farmers and other people in contact with this feed.
E.8.3 Other immunological effects
An apparent reduction of the response to vaccination following administration of macrolides has been noted in two studies. In a study by Hassan (1990), chickens, vaccinated against Newcastle disease virus (NDV), were challenged with NDV and subsequently treated with spiramycin. This resulted in a twice as high mortality in treated and vaccinated birds as compared to
In one study by Vahl (1985), virginiamycin was given in the feed at a concentration of 20 mg/kg in a floorpen experiment. Immunological response to vaccination with NDV, in the form of antibody production, was measured. The NDV titers in the group receiving virginiamycin were depressed as compared to the
No explanations of the mechanisms behind these findings have been provided by the authors. No other, similar reports have been found. In many countries the control of NDV infections depends on vaccination programs. In view of this, these observations certainly merit further investigation.
E.9 Environmental effects
Like other AFA, if the use of spiramycin, tylosin or virginiamycin reduces the amount of feed consumed per kg weight gain in the target animal, they would also be expected to reduce nitrogen output per kg weight gain.
The effect of tylosin and tylosin fermentation wastes on microbial activity in soil has been investigated (Bewick, 1978). Tylosin from waste was detected in leachates and after 10 weeks
| SOU 1997:132 | Annex E | 315 |
the added spiramycin had been degraded. However, when the manure was mixed with soil in the ratio 1:3, degradation of spiramycin was complete within a week. Gavalchin and Katz (1994) studied the degradation of tylosin and erythromycin in sandy loam from a
E.10 Summary comments
Spiramycin, tylosin and virginiamycin all belong to important antibacterial classes. Increased resistance to spiramycin, tylosin and virginiamycin would hamper the therapeutic use of substances from these classes in both animals and humans. Exposure of bacteria to spiramycin, virginiamycin and tylosin selects for resistant strains, usually carrying one or several transmissible resistance determinants. In order not to further diminish their therapeutic value, these substances should be restricted to therapeutic use. Spiramycin, tylosin and virginiamycin are potent allergens, and may as such represent an occupational hazard for farmers and feedmill workers.
| 316 | Annex E | SOU 1997:132 |
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2EINA * "ORRELL . AND 3ERRA ! , 1992. Emergence of resistance to erythromycin and
fluoroquinolones in thermotolerant Campylobacter strains isolated from faeces
2ENDE &OURNIER 2 ,ECLERCQ 2 'ALIMAND - $UVAL * AND #OURVALIN 0 , 1993.
Identification of the satA gene encoding a streptogramin A acetyltransferase in Enterococcus faecium BM4145. !NTIMICROBIAL !GENTS AND #HEMOTHERAPY 2119-
2125.
2OBERTS - # AND "ROWN - " , 1994.
2OLLINS , $ ,EE , . AND ,E"LANC $ * , 1985. Evidence for a disseminated
erythromycin resistance determinant mediated by Tn
2ONNE ( AND *ENSEN * # % , 1992. Virginiamycin susceptibility of 3ERPULINA HYODYSENTERIAE, IN VITRO and IN VIVO. 6ETERINARY 2ECORD
2ONNE ( AND 3ZANCER * , 1990. In vitro susceptibility of Danish field isolates of 4REPONEMA HYODYSENTERIAE to chemotherapeutics in swine dysentery (SD) therapy.
Interpretation of MIC results based on the pharmacokinetic properties of the antibacterial agents. )N PROCEEDINGS OF TH )063 #ONGRESS July 1 5, Lausanne. p.
126.
| 324 | Annex E | SOU 1997:132 |
3CHALEN # 'EBRESELASSIE $ AND 3TAHL 3 , 1995. Characterization of an erythromycin resistance (erm) plasmid in Streptococcus pyogenes. !CTA 0ATHOLOGICA
3HAH 0 - 3CHAFER 6 AND +NOTHE ( , 1993. Medical and veterinary use of
antimocrobial agents: implications for public health. A clinician’s view on antimicrobial resistance. 6ETERINARY
3MITH ( AND 'REEN 3 ) , 1980. Effect of feed additives on the incidence of naturally acquired salmonella in turkeys. 6ETERINARY 2ECORD 289.
3MITH ( 7 AND 4UCKER * & , 1975a. The effect of antibiotic therapy on the faecal excretion of 3ALMONELLA TYPHIMURIUM by experimentally infected chickens. *OURNAL OF (YGIENE
3MITH ( 7 AND 4UCKER * & , 1978. The effect of antimicrobial feed additives on the colonization of the alimentary tract of chickens by 3ALMONELLA TYPHIMURIUM.
*OURNAL OF (YGIENE
3MITH 3 #
3MITH 7 ( AND 4UCKER * & , 1975b. The effect of feeding diets containing permitted antibiotics on the faecal excretion of 3ALMONELLA TYPHIMURIUM by experimentally infected chickens. *OURNAL OF (YGIENE
3MITH 7 ( AND 4UCKER * & , 1980. Further observations on the effect of feeding diets
containing avoparcin, bacitracin and sodium arsenilate on the colonization of the alimentary tract of poultry by salmonella organisms. *OURNAL OF (YGIENE 137-
150.
3T 'EORGIEV 6 , 1994. Management of toxoplasmosis. $RUGS
3TUART * ' :IMMERER % * AND
3TUTZ - 7 *OHNSON 3 , AND *UDITH & 2 , 1983. Effects of diet and bacitracin on growth, feed efficiency, and populations of #LOSTRIDIUM PERFRINGENS in the intestine of broiler chicks. 0OULTRY 3CIENCE
3UTTER ( - %NGELI *
4AUCH ! +ASSING & +ALINOWSKI * AND 0UHLER ! , 1995. The #ORYNEBACTERIUM XEROSIS composite transposon Tn consists of two identical insertion sequences, designated IS1249, flanking the erythromycin resistance gene ERMCX. 0LASMID
| SOU 1997:132 | Annex E | 325 |
4HAL , ! 7ELTON , ! 0ERRI - " $ONABEDIAN 3
:ERVOS - * , 1996. Antimicrobial resistance in enterococci isolated from turkeys fed virginiamycin. )N PROCEEDINGS OF TH )#!!# New Orleans.
4URANO ! 2AVIZZOLA ' 0ERONI , #ERUTI 4 'RECO , - 0ITZUS % 3ANTINI ' #RESTI 3 AND 3ATTA ' , 1994. A multicentre study: Staphylococcus and Enterococcus susceptibility to antibiotics. %UROPEAN *OURNAL OF %PIDEMIOLOGY
6AHL ( ! , 1985. Vitamin A, iron and virginiamycin in broiler diets. Effects on performance and immune responsiveness. (Thesis).
VAN DEN "OGAARD ! * 0
enterococci in humans and pigs in The Netherlands: is the addition of antibiotics to animal feeds to blame? *OURNAL OF !NTIMICROBIAL #HEMOTHERAPY
7ANG 7 , 2ELLER , " AND "LASER - * , 1984. Comparison of antimicrobial susceptibility patterns of #AMPYLOBACTER JEJUNI and #AMPYLOBACTER COLI. !NTIMICROBIAL !GENTS AND #HEMOTHERAPY
7ASTESON 9 (OIE 3 AND 2OBERTS - # , 1994. Characterization of antibiotic resistance in 3TREPTOCOCCUS SUIS. 6ETERINARY
7EISBLUM " , 1985. Inducible resistance to macrolides, lincosamides and streptogramin
type B antibiotics: the resistance phenotype, its biological diversity, and structural elements that regulate the expression - a review. *OURNAL OF !NTIMICROBIAL CHEMOTHERAPY
7EISBLUM " , 1995a. Erythromycin resistance by ribosome modification. !NTIMICROBIAL !GENTS AND #HEMOTHERAPY
7EISBLUM " , 1995b. Insights into erythromycin action from studies of its activity as inducer of resistance. !NTIMICROBIAL !GENTS AND #HEMOTHERAPY
7ESTH ( , 1996. Ertythromycin resistant 3TAPHYLOCOCCUS AUREUS in Denmark. !CTA 0ATHOLOGICA
7ILLIAMS " * AND 3HIVELY * % , 1978. )N 6ITRO antitreponemal activities of carbadox,
virginiamycin, olaquindox, and tylosin as indices of their effectiveness for preventing swine dysentery. 6ETERINARY
7OODFORD . 0ALEPOU - & *OHNSON ! 0 #HADWICK 0 2 AND "ATES * , 1997. Correspondance. 4HE ,ANCET 738.
| 326 | Annex E | SOU 1997:132 |
| SOU 1997:132 | 327 |
Annex F: Carbadox and olaquindox
F.1 Introduction
Carbadox and olaquindox belong to the group of
After oral administration, olaquindox and carbadox are rapidly absorbed and are extensively metabolised (FAO/WHO, 1991; FAO/WHO, 1994). The concentration profiles of the active substances in the gut show a gradual decline from the stomach to the colon where the concentrations are below detectable levels (Baars ET AL , 1988; de Graaf ET AL , 1988; Spierenburg ET AL , 1988c).
The quinoxalines are not used for in human medicine. Carbadox and olaquindox are approved as feed additives for swine for growth promoting purposes at dosages ranging from 20 to 50 ppm and 50 to 100 ppm respectively. Quinoxalines are also used at similar or higher dosages for prevention of intestinal infections (see F.7).
The use of quinoxalines has been questioned for toxicological reasons, mainly due to their genotoxic and/or carcinogenic potential (Anonymous, 1995), they are also known to have toxic effects on the target species (Prescott and Baggot, 1993).
F.2 Mode of action and resistance mechanisms
As mentioned above, the activity of the quinoxalines is markedly improved under anaerobic conditions. The mode of action of the quinoxalines on bacteria was investigated by Suter and
| 328 | Annex F | SOU 1997:132 |
The figures in this report is only available in the printed version
Figure F.I. Tentative sketch of carbadox, olaquindox, nalidixic acid and enrofloxacin
| SOU 1997:132 | Annex F | 329 |
the parent substance or some decomposition products thereof damage DNA without being bound to this target. As a consequence, DNA synthesis ceases, breakdown of chromosome ensues and the bacterial cell dies.
The quinoxalines are somewhat structurally related to the quinolones (see figure F.1) which also exert their antibacterial activity by interfering with the bacterial DNA synthesis. The quinolones inhibit the activity of bacterial topoisomerases, i.e. enzymes controlling the supercoiling of DNA, converting relaxed covalently closed circular DNA to a superhelical form by an energy dependent strand breakage and resealing process (Maxwell, 1997). Although there are still unclarities as to the precise bactericidal mechanism of the quinolones, it is thought that when the gyrase is inhibited, resealing following breakage is prevented and DNA is exposed to and degraded by exonucleases.
Resistance to quinolones develop through alterations of the target of the drug and to some extent through altered permeability or increased efflux.
As the information on mechanisms of action of the quinoxalines predates the discovery of bacterial gyrases, possible similarities with the precise mechanism of action of the quinolones cannot be determined. No information on the molecular background of observed quinoxaline resistance, nor on
F.3 Development of resistance
An experimental study on
A prospective study to monitor the development of olaquindox resistance in coliforms following the introduction of olaquindox as a feed additive was conducted in commercial farms in Suffolk by Linton and
| 330 | Annex F | SOU 1997:132 |
neighbouring farms not using olaquindox as well, but to a lesser extent. The latter finding is not surprising as the herds were not isolated from the environment.
Table F.I. The average percentage of coliforms resistant to 50 µg/ml olaquindox in each year of the survey (from Linton ET AL , 1988)
| 9EAR | 2ESISTANCE | |
| #ONTROL FARMS | 4EST FARMS | |
| 19811 | 0.00 | - |
| 1982 | 0.03 | 0.04 |
| 1983 | 0.12 | 5.63 |
| 1984 | 0.68 | 6.14 |
1 Sampled before olaquindox was used in the UK
The ecological aspects of olaquindox resistance were investigated by Hedges and Linton (1988). In a prospective study, chosen pens in four pig farms, two of which used olaquindox were sampled weekly. Coliform bacteria were isolated from the samples and biovariant along with sensitivity to olaquindox and other antibacterials was determined. The overall proportions of coliforms resistant to olaquindox (counts on selective medium divided by counts on
An increase in carbadox resistance in salmonellae over time was noted in a study from Kansas by Mills and Kelly (1986). The study included clinical isolates from necropsied swine in Kansas during
Ohmae and
| SOU 1997:132 | Annex F | 331 |
Pohl (1987) reported a 5 times higher prevalence of
antibacterials, in a material collected in various European countries. Several investigations report on a uniform sensitivity of 3ERPULINA spp to
carbadox (Molnar and Magyar, 1987; Walter and Kinyon, 1990; Molnar, 1996) with reported MICs generally considerably below 0.4µg/ml. Contrary
to this, in a study from Canada, a MIC90 (minimal inhibitory concentration for 90% of the isolates) of carbadox for 3ERPULINA HYODYSENTERIAE of
>6µg/ml was reported (Messier ET AL , 1990). This figure is considerably higher than those reported by other authors, indicating an emerging resistance. In an investigation from Sweden, carbadox had a MIC of >0.1µg/ml for 3 out of 67 (4%) of the Serpulina isolates investigated. Although MICs in this range might not cause the isolates to be classified as resistant, their sensitivity seems reduced as the modal MIC in this study was 0.012µg/ml. This might indicate a gradual increase in MICs, a phenomenon which is seen when stepwise mutations are needed for full resistance. Interestingly, carbadox was not used in Sweden at the time when the isolates were collected.
Two reports show the MICs of olaquindox and carbadox for individual strains of 3ERPULINA spp (Williams and Shively, 1978; Fellström ET AL , 1996).
No clear relation between MICs of carbadox and olaquindox can be noted in the data presented. In the study by Williams (1978), a good correlation between MICs obtained IN VITRO and therapeutic efficacy of respective drug in experimental infections was noted. Infection with the three strains with MICs for olaquindox of 2.5µg/ml or more resulted in considerable morbidity in spite of olaquindox treatment, while the treated pigs infected with a strain
with a MIC of olaquindox of 0.3µg/ml remained healthy. The same 4 strains were uniformely sensitive to carbadox both IN VITRO and IN VIVO Taken
together, these studies indicate that strains resistant to olaquindox are not
Concerning the activity of carbadox against
publications are available. Devriese (1980) reported on the carbadoxsensitivity of 950 isolates of 3TAPHYLOCOCCUS AUREUS from cattle, poultry and
pigs. All the isolates had MIC values <1.6µg/ml and no indications of acquired resistance were found. Similarly, in an investigation of MIC of carbadox for clostridia from the same animal species, all 68 isolates had MIC values below 0.25µg/ml (Dutta ET AL , 1983).
Indications of a simultaneous increase in minimum inhibitory concentrations for olaquindox and the quinolone enrofloxacin was recently detected in a Swedish survey (Greko, 1997). The investigated strains originated from three different cathergories of piglet producing herds; 10 herds were using olaquindox as medicated feed (160 ppm), 10 herds were using zinc oxide (2500 ppm) and 10 herds used no medication. The observed
| 332 | Annex F | SOU 1997:132 |
phenotypes with respect to olaquindox and enrofloxacin sensitivity are shown in table F.II. 16 out of 61 isolates with resistance to olaquindox according to the
Table F.II. Phenotypes with respect to olaquindox and enrofloxacin sensitivity in % COLI isolated from piglets in herds using different regimes (Greko, 1997)
| 0HENOTYPE | 0ROPORTION OF ISOLATES WITH PHENOTYPE | |||
| FROM SAMPLING GROUP | ||||
| /LAQUIN | :INC | .O MEDI | !LL GROUPS | |
| DOX | OXIDE | CATION | ||
| n=60)2 | n=73) | n=85) | n=218) | |
| 0 | 0 | 1 | <1 | |
| 15 | 9 | 0 | 7 | |
| 20 | 9 | 30 | 20 | |
| 0 | 1 | 0 | <1 | |
| 0 | 0 | 0 | 0 | |
| 65 | 80 | 68 | 71 | |
1Determined according to MICs (µg/ml); Olaquindox R >32, OlaquindoxS<64, EnrofloxacinR >0.25, EnrofloxacinI=0.25, EnrofloxacinS<0.25
2n= number of isolates
F.4 Acquisition of resistance
Omahae and
and ampicillin. The resistance determinants were shown to reside on a conjugative plasmid. No transfer to 3ERPULINA spp was observed. Spanoghe
and Pohl (1987) tested 61 % COLI strains with anaerobic MIC values for carbadox of >8µg/ml for their ability to transfer resistance (including carbadox). The experiments were successful in 55 and 69% of the attempts when two % COLI strains were used as recipients. Transfer of carbadox resistance to salmonellae was achieved only in 10% of the experiments. Transfer of carbadox resistance was constantly associated with
| SOU 1997:132 | Annex F | 333 |
resistance to at least one other antibacterial. The most frequently linked transfer was
Linton and
Transfer of resistance did not correlate to the appearence of a plasmid nor was it linked to transfer or other resistance traits. The evidence therefore suggests that the gene(s) conferring olaquindox resistance were located on the chromosome.
An apparent suppression of coliforms carrying
No information on the precise genetic nature of either carbadox or olaquindox resistance has been found. Plasmid mediated resistance to nalidixic acid, a quinolone, was reported from Bangladesh in 1987 (Munshi ET AL , 1987). This was later found to be due to a mutation of the recipient strain. Possibly, the transferred plasmid induced mutations leading to nalidixic acid resistance (Ahmed, cit. by Courvalin, 1990). Although speculative, a similar phenomenon could explain why the transconjugant strains in the experiments cited above had markedly lower MICs than those of the donor strains (Ohmae ET AL , 1983; Baumgartner ET AL , 1985). If stepwise mutations are necessary to reach higher MICs, single or
F.5 Effects on specific animal diseases
The preventive effect of the quinoxalines, especially carbadox, against certain intestinal diseases in animals, especially swine dysentery, is well documented.
Studies on the pharmacokinetics of the quinoxalines have shown that the intestinal concentrations gradually decline from the stomach to the colon (Baars ET AL , 1988; de Graaf ET AL , 1988; Spierenburg ET AL , 1988c). As swine dysentery is primarily a disease of the lower intestine, a preventive rather than a therapeutic effect is to be expected.
Williams and Babcock (1978) investigated presence and development of
experimentally induced infection by carbadox. No
| 334 | Annex F | SOU 1997:132 |
were found and carbadox at 5 ppm above maximum growth promoting level was found to be effective in preventing swine dysentery. Similar results were
obtained by Williams and Shively (1978), who found no
carbadox at, or slightly above, growth promoting levels. Jenkins and Froe (1985) prevented experimentally induced swine dysentery by feeding carbadox at growth promoting levels to pigs. In a study by Jacks and coworkers (1986), carbadox was fed at 5 ppm above growth promoting level and was found to successfully prevent swine dysentery.
Raynaud and
Taylor and Davey (1980) found that carbadox at maximum growth
promoting levels prevented the onset of swine dysentery and eliminated 3 HYODYSENTERIAE infection in experimentally infected pigs. Rainier and co-
workers (1980b) investigated therapeutic effects and prevention of the carrier state in pigs experimentally infected with 3 HYODYSENTERIAE. Carbadox at 5
ppm above permitted growth promoting levels effectively eliminated the infectious agent from the host, thereby curing the disease without leaving any asymptomatic carriers. Similar results were shown in a similar study by the same authors (Rainier ET AL , 1980a). Biehl and
same dosage of carbadox and were able to successfully treat pigs experimentally infected with 3 HYODYSENTERIAE.
In a large scale trial in commercial pig herds in France (Raynaud and Bretheau, 1973), 50 ppm carbadox was found to be sufficient for both prophylaxis and treatment of swine dysentery.
As mentioned under F.3, olaquindox at growth promoting levels completely prevented swine dysentery induced by an
These experimental results have been confirmed by various field studies. In a study from Pfizer Technical Information Service (Anonymous, 1980), carbadox at the maximum growth promoting dosage totally prevented occurrence of swine dysentery. Hunneman (1980) reported a notable decrease in outbreaks of swine dysentery in an area when carbadox was introduced as a feed additive in local swine herds. Molnar (1987) reported an attempt to eradicate swine dysentery from swine herds by the aid of carbadox at growth promoting levels. The eradication program failed, but on an
| SOU 1997:132 | Annex F | 335 |
individual basis, medication was successful both as a preventive and therapeutic measure. No
Colibacillosis and salmonellosis were also successfully prevented in the medicated herds. Olson (1986) reported successful eradication of swine dysentery with
Some information is also available concerning the preventive effect of quinoxalines for other diarrhoeal conditions of pigs. Bertschinger (1976) found that olaquindox at low dosages (50 ppm) in feed was effective for prevention of experimentally induced %
carbadox at the maximum growth promoting concentration reduced clinical signs and intestinal lesions in pigs experimentally infected with 3ALMONELLA
Cholerasuis Winkelman and Hawkins (1996) evaluated carbadox for the control of proliferative enteropathy in swine. Carbadox at growth promoting
levels was found to reduce clinical signs and pathological lesions in pigs experimentally infected with ,AWSONIA INTRACELLULARIS (the causative agent of
porcine proliferative enteritis).
Stutz and
enteritis) in the intestines of chickens. Carbadox is not used in poultry, so this
may be of limited interest, but it is clear that carbadox is effective against clostridia, both IN VIVO and in VITRO.
F.6 Impact of resistance on animal and human health
Carbadox, and to some extent olaquindox, are used for prevention or therapy in veterinary medicine. Neither of those, nor related substances, are used in human medicine. As no information on possible
Quinoxalines are valuable for prevention of swine dysentery and weaning diarrhoea. Development of resistance in 3ERPULINA spp. to carbadox or
olaquindox would entail reduced effectiveness of strategies employing
| 336 | Annex F | SOU 1997:132 |
quinoxalines for prevention of this disease. Few drugs are available for this purpose. However, available data indicate that the development of resistance to quinoxalines is slow.
F.7 Other effects on the microflora
The influence of carbadox treatment on salmonellosis was investigated by
Troutt (1974). When fed at 55 ppm to pigs experimentally infected with 3ALMONELLA Choleraesuis, carbadox diminished the clinical signs and reduced
the extent and severity of lesions. Development of resistance in salmonella towards carbadox has been documented (Mills and Kelly, 1986). No
investigations concerning possible effects of quinoxalines on infections with resistant strains of 3ALMONELLA spp. have been found.
No information on the influence of quinoxalines on other
found.
F.8 Toxicological aspects
F.8.1 Target species
A series of investigations concerning adrenal toxicity of quinoxalines, particularly carbadox, has been published. Field observations of intoxications in pigs fed high doses of carbadox (up to 150 ppm) were confirmed by an experimental study where 150 ppm carbadox was given to weaned pigs for up to 10 weeks (van der Molen ET AL , 1985). Clinical signs of dehydration and impaired growth were observed from 3 weeks and onwards. Histopathological examination revealed atrophy of the glomerular zone of the adrenal glands.
In a subsequent experiment, pigs fed carbadox at doses between 25 and 200 ppm were investigated (van der Molen ET AL , 1986). The results confirmed the observation of adrenal damage as lowered
Further investigations into the pathomorphological changes showed that after 10 weeks of carbadox at 25 ppm or more, damage to the cells of the zona glomerulosa of the adrenal gland could be observed histologically (van der Molen, 1988). Again, the effects were dose and time dependent. Only two animals per dosage group were examined histologically.
| SOU 1997:132 | Annex F | 337 |
The hormonal changes induced by carbadox were further investigated (van der Molen ET AL , 1989). After 9 weeks, the levels of plasma renin were higher in all groups fed carbadox than in the control group.
Taken together, these experiments show that carbadox supresses the
experiments (Spierenburg ET AL , 1988a; Spierenburg ET AL , 1988b; Jager ET AL , 1994).
In a study comparing the effects of carbadox, cyadox and olaquindox similar toxic effects were noted. For olaquindox, adrenal toxcicity was observed, although less pronounced, at dosages of 100 ppm or more (Nabuurs ET AL , 1990). Again, the effects were doseand time dependent. The clinical signs observed were also
In the IN VIVO studies cited above, the
Accidental overdosing of olaquindox (Köfer ET AL , 1990; Stockhofe- Zurwieden ET AL , 1991) and carbadox (Power ET AL , 1989) has been reported to cause death and severe adrenal damage in piglets.
As the main early sign of intoxication with quinoxalines, i.e. dry faeces, may be mistaken for recovery from enteric disease, mild intoxications are expected to be overlooked by farmers and farm workers. Further, as the symptoms are diffuse, a definite diagnosis might be difficult to reach.
| 338 | Annex F | SOU 1997:132 |
F.8.2 Adverse effects in humans
Carbadox
In a study where radiolabelled carbadox was fed to swine at 55 ppm for 5 consecutive days, total residues 30 days after withdrawal were around 5, 74 and 15 µg/kg in muscle, liver and kidney, respectively. After 70 days, the measured concentrations in liver were 13µg/kg (FAO/WHO, 1990).
Carbadox is rapidly metabolised to, among other metabolites, quinoxaline-
Detection of QCA is dependent on the extraction method used, and it has been suggested that this is due to other unknown intermediate metabolites in the pathway of carbadox to QCA (Baars ET AL , 1991). QCA, being the major residual metabolite, has been suggested as a marker substance for residue studies (FAO/WHO, 1990).
Long term toxcicity studies on carbadox in rats have shown
Positive results were reported in 14 out of 15 mammalian and nonmammalian genotoxicity studies (FAO/WHO, 1990), clearly indicating a genotoxic potential of carbadox.
Chronic administration of the meatabolite desoxycarbadox to rats has resulted in an increase of liver tumour incidence in all dosage groups (FAO/WHO, 1990; FAO/WHO, 1991). Most tests for genotoxicity have produced negative results, but positive findings were recorded in the cell transformation test and in Ames test using liver cells
With respect to QCA, no studies indicate reasons to suspect carcinogenic or genotoxic properties (FAO/WHO, 1990; FAO/WHO, 1991)
In the 36th report of JECFA (FAO/WHO, 1990) it was concluded that an ADI could not be established, due to the carcinogenic and genotoxic nature of carbadox and some of its metabolites. MRLs set in 1990 (FAO/WHO, 1990) for QCA as marker substance were apparently based on the detection limit of the analytical method.
| SOU 1997:132 | Annex F | 339 |
Olaquindox
Olaquindox is rapidly absorbed from the gut and mainly excreted via urine. No bound residues appear to be present in tissue (FAO/WHO, 1995). In a study where radiolabelled olaquindox was given to pigs as a single oral dose of 2 mg/kg body weight, <1, 2 and 1 µg/kg of total residues were detected in muscle, liver and kidney, respectively after 28 days. (FAO/WHO, 1990). At doses corresponding to recommended feed inclusion doses (2.5 mg/kg body weight) and a 28 days withdrawal period, olaquindox residues were below 5µg/kg in muscle and below 10µg/kg in kidney (FAO/WHO, 1995). The substance is extensively metabolised the animal. The metabolites found vary between tissues and between animal species (FAO/WHO, 1995). In pigs given 60 ppm olaquindox in the feed up to 16 weeks of age, and with a withdrawal period of 28 days, olaquindox residues were below 0.005 ppm in muscle and below 0.01 ppm in kidney (FAO/WHO, 1995). One of the metabolites,
In
The genotoxicity of olaquindox has been investigated in a range of IN VITRO and IN VIVO studies. Both positive and negative findings were reported in assays using various mammal and
The marker substance, MQCA, is known to be responsible for the bacterial mutagenecity of other quinoxaline derivatives (FAO/WHO, 1995). No studies specific for olaquindox are available.
An ADI could not be allocated by JECFA 1995, because of the genotoxic potential of the parent compound and the absence of specific toxicity studies on the metabolites. No MRL has been set.
Photoallergy
Carbadox, olaquindox and other quinoxalines induce both photoxicic and photoallergic mechanisms through formation of a reactive oxaziridine reacting with proteins upon exposure to light (de Vries ET AL , 1990b).
The photoallergic properties of olaquindox has been confirmed in studies on rats (de Vries ET AL , 1990a).
| 340 | Annex F | SOU 1997:132 |
Hochsattel ET AL , 1991; Schauder ET AL , 1996). In a report by Schauder (1996), twelve out of 15 patients habitually mixed mineral feed containing 1000 ppm olaquindox on the farm, two of the patients only handled pellets. The average interval of exposure at onset of clinical signs for the patients who mixed feed was 2.5 years.
The clinical symptoms are characterised by eczema worsened by sunny weather. At worst, the affected persons have to stay indoors during daytime. The reaction may be persistent and can be severely disabling (Schauder ET AL , 1996). In spite of the experimental findings indicating similar properties of carbadox, no clinical reports have been found. Nonetheless, carbadox should be regarded as a potential photoallergen (de Vries ET AL , 1990b).
Some comments on toxicological aspects
The quinoxalines are suspected of having carcinogenic and genotoxic properties (Cihák and Srb, 1983; Nunoshiba and Nishioka, 1989). Carcinogenic and genotoxic effects are not acceptable since the effect could occur at very low intake levels, especially if the substance in question is ingested regularly over a number of years. Farm and feedmill workers are a special risk group, frequently exposed to AFA when handling animal feed. If appropriate protection cannot be ensured, the handling of animal feed containing quinoxalines and other AFA with potentially genotoxic effects must be regarded as an occupational hazard both due to potential genotoxicity and to photoallergenicity. Althogh the precise exposure cannot be determined, a conservative approach is often recommended for genotoxic substances in order to prevent underestimation of the risks.
F.9 Environmental effects
No information on the environmental fate of quinoxalines has been found. The quinoxalines are mainly excreted via urine (FAO/WHO, 1990; FAO/WHO, 1995). They are sensitive to photodegradation. Considering the potential genotoxicity of some of these substances, not only the fate of the parent substance but also of relevant metabolites should be investigated.
F.10 Summary comments
Although transferable resistance to carbadox and olaquindox has been reported, the mechanisms and molecular biology of this or other resistance phenomena are still unclear. The precise target in the bacterium of the
quinoxalines or their metabolites is still unknown. The development of resistance in important animal pathogens such as 3ERPULINA spp. seems slow.
| SOU 1997:132 | Annex F | 341 |
No information is available concerning
The preventive effects of carbadox and olaquindox against enteric diseases of pigs is well documented.
Olaquindox and carbadox have several unwanted properties related to toxicology. Both substances have toxic effects on the adrenal glands of exposed animals at the dosages used for growth promotion. Profound disturbances in the stereoid balance of the animals, resulting in clinical signs of dehydration has been reported.
The quinoxalines and some of their metabolites are, or are suspected to be, genotoxic. Olaquindox is a well known photoallergen and according to IN VITRO data, carbadox shares this property. Therefore, exposure to quinoxalines must be regarded as an occupational hazard.
| 342 | Annex F | SOU 1997:132 |
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DE 6RIES ( "EYERSBERGEN VAN (ENEGOUWEN ' - +ALLOE & AND "ERKHUYSEN - ( , 1990a. Phototoxicity of olaquindox in the rat. 2ESEARCH IN 6ETERINARY 3CIENCE
| SOU 1997:132 | Annex F | 343 |
DE 6RIES ( "OJARSKI * $ONKER ! ! "AKRI ! AND "EYERSBERGEN VAN (ENEGOUWEN ' - * , 1990b. Photochemical reactions of quindoxin, olaquindox, carbadox and cyadox with protein, indicating photoallergic properties. 4OXICOLOGY
$EVRIESE , ! , 1980. Sensitivity of staphylococci from farm animals to antibacterial agents used for growth promotion and therapy - a ten year study. !NNALES DE 2ECHERCHES 6ETERINAIRES
$UTTA ' . $EVRIESE , ! AND 6AN !SSCHE 0 & , 1983. Susceptibility of clostridia
from farm animals to 21 antimicrobial agents including some used for growth promotion. *OURNAL OF !NTIMICROBIAL #HEMOTHERAPY
&!/ 7(/, 1990. 36th report of the Joint Expert Committee on Food Additives: Evaluation of Certain Veterinary Drugs. 4ECHNICAL REPORT SERIES .O
WHO, Geneva.
&!/ 7(/, 1991. 36th Meeting of the Joint FAO/WHO Expert Committee on Food
Additives. Toxicological evaluation of certain veterinary drug residues in food.
7(/ &OOD !DDITIVES 3ERIES Vol. 27,
&!/ 7(/, 1994.
Food Additives. Toxicological evaluation of certain veterinary drug residues in food. 7(/ &OOD !DDITIVES 3ERIES pp.
&!/ 7(/, 1995.
Food Additives. Evaluation of certain veterinary drug residues in food. 7(/ 4ECHNICAL 2EPORT 3ERIES .O pp.
&ELLSTR¶M # 'UNNARSSON ! (OLMSTR¶M ' AND &RANKLIN ! , 1996. In vitro
activity of 7 antibacterials against 4 phenotypical variants of Serpulina species. )N PROCEEDINGS OF TH )063 #ONGRESS
'EDEK " , 1979. Bewertung der Leistungsfähigkeit von Carbadox als Wachstumsforderer nach mikrobiologischen Kriterien. :ENTRALBLATT FUR 6ETERIN¤RMEDIZIN B
'REKO # , 1997. Sensitivity to antibacterials in % COLI and enterococci isolated from piglets in Sweden (in manuscript).
(EDGES ! * AND ,INTON ! ( , 1988. Olaquindox resistance in the coliform flora of pigs and their environment: an ecological study. *OURNAL OF !PPLIED "ACTERIOLOGY
(OCHSATTEL 2 'ALL ( 7EBER , AND +AUFMANN 2 , 1991. [Photoallergic reaction to olaquindox]. (AUTARZT
(OLMGREN . , 1994. Profylaktiska effekter av zinkoxid eller olaquindox mot avvänjningsdiarré hos svin. 3VENSK 6ETERIN¤RTIDNING
(UNNEMAN 7 ! , 1980. Swine dysentery. From acute outbreaks to an almost subclinical situation. "ULLETIN DE L /FFICE )NTERNATIONAL DES %PIZOOTIES
| 344 | Annex F | SOU 1997:132 |
*ACKS 4 - *UDITH & 2 &EIGHNER 3 $ AND ,IKOFF 2 / , 1986.
*AGER , 0 DE 'RAAF ' * 7IDJAJA 'REEFKES ( # ! !CCORD "URLESON # # & VAN DEN $UNGEN ( - AND "AARS ! * , 1994. Effects of feed additives and veterinary
drugs on aldosterone production and release by porcine adrenal cells IN VITRO.
*OURNAL OF 6ETERINARY 0HARMACOLOGY AND 4HERAPEUTICS
*ENKINS % - AND &ROE $ , ))), 1985. Comparing carbadox and bacitracin in the prevention of clinical signs of swine dysentery. 6ETERINARY
+¶FER * (¶CHER ( AND (INTERDORFER & , 1990. Olaquindoxvergiftung bei Ferkeln.
7IENER 4IER¤RZTLICHE
,INTON ! ( (EDGES ! * AND "ENNETT 0
antimicrobial resistance during the use of olaquindox as a feed additive on commercial pig farms. *OURNAL OF !PPLIED "ACTERIOLOGY
2350.
.ABUURS - * ! VAN DER
signs and performance of pigs treated with different doses of carbadox, cyadox and olaquindox. *OURNAL OF 6ETERINARY
.UNOSHIBA 4 AND .ISHIOKA ( , 1989. Genotoxicity of quinoxaline
/HMAE + 9ONEZAWA 3 AND 4ERAKADO . , 1983. Epizootiological studies on R plasmid with carbadox resistance. *APANESE *OURNAL OF 6ETERINARY 3CIENCE 165-
170.
| SOU 1997:132 | Annex F | 345 |
/LSON , $ , 1986. Probable elimination of swine dysentery after feeding ronidazole, carbadox or lincomycin and verification by feeding sodium arsanilate. #ANADIAN *OURNAL OF 6ETERINARY 2ESEARCH
0OWER 3 " $ONNELLY 7 * #
1989. Accidental carbadox overdosage in pigs in an Irish
6ETERINARY 2ECORD
0RESCOTT * & AND "AGGOT * $ EDS , 1993. !NTIMICROBIAL THERAPY IN VETERINARY MEDICINE 2nd ed. Iowa State University Press, Ames. pp. 612.
2AINIER 2 ( (ARRIS $ , #LOCK 2 $ +INYON * - AND "RAUER - ! , 1980a.
Carbadox and lincomycin in the treatment and carrier state control of swine dysentery 4REPONEMA HYODYSENTERIAE. !MERICAN *OURNAL OF 6ETERINARY 2ESEARCH
2AINIER 2 ( (ARRIS $ , 'LOCK 2 $ +INYON * - AND "RAUER - ! , 1980b.
Prevalence of asymptomatic carriers of swine dysentery following withdrawal of carbadox or lincomycin treatments. )N PROCEEDINGS OF TH )063 #ONGRESS June 30-
July 3, Copenhagen.
2AYNAUD * 0 AND "RETHEAU ( , 1973. Essais a large echelle du carbadox dans les elevages porcins en France. Bilan en 1972. 2EVUE DE
2AYNAUD * 0 "RUNAULT ' AND 0ATTERSON % " , 1980a. A new swine dysentery
model for evaluation of drug prophylaxis: I. Model development - oral infection plus pen contamination. )N PROCEEDINGS OF TH )063 #ONGRESS 30
Copenhagen. p. 248.
2AYNAUD * 0 "RUNAULT ' AND 0ATTERSON % " , 1980b. A new swine dysentery
model for evaluation of drug prophylaxis: II. Efficacy of various drugs in the control of swine dysentery. )N PROCEEDINGS OF TH )063 #ONGRESS 30
Copenhagen. p. 249.
2UTALJ - "AZULIC $ 3APUNAR 0OSTRUZNIK * :IVKOVIC * AND ,JUBICIC ) , 1996.
3CHAUDER 3 , 1989. [The dangers of olaquindox. Photoallergy, chronic photosensitive
dermatitis and extreme increased photosensitivity in the human, hypoaldosteronism in swine]. $ERM "ERUF 5MWELT
3CHAUDER 3 3CHR¶DER 7 AND 'EIER * , 1996.
photoallergic contact dermatitis followed by transient or persistent light reactions in 15 pig breeders. #ONTACT $ERMATITIS
3PANOGHE 0 AND 0OHL 0 , 1987. Comparison between the influence of carbadox and that
of traditional antibiotics on the resistance of enterobacteria in pigs. !NNALES DE 2ECHERCHES 6ETERINAIRES
| 346 | Annex F | SOU 1997:132 |
3PIERENBURG 4 * "AARS ! * DE 'RAAF ' * AND *AGER , 0 , 1988a. Carbadox
induced inhibition of aldosterone production in porcine adrenals IN VITRO. )N PROCEEDINGS OF TH #ONGRESS OF %UROPEAN !SSOCIATION FOR 6ETERINARY 0HARMACOLOGY AND 4OXICOLOGY August
3PIERENBURG 4 * "AARS ! * DE 'RAAF ' * AND *AGER , 0 , 1988b. Carbadox-
induced inhibition of aldosterone production in porcine adrenals IN VITRO.
4OXICOLOGY IN 6ITRO
3PIERENBURG 4 * VAN ,ENTHE ( DE 'RAAF ' AND *AGER , 0 , 1988c. Liquid
chromatographic determination of olaquindox in medicated feeds and in contents of porcine gastrointestinal tract. *OURNAL OF THE !SSOCIATION OF /FFICIAL !NALYTICAL #HEMISTS
3TOCKHOFE :URWIEDEN . "RUNCKHORST $ 7ALDMANN + ( AND 0OHLENZ * , 1991.
Pathomorphologische Verlaufsuntersuchungen nach Olaquindoxintoxikation bei Mastschweinen. 4IER¤RZTLICHE 0RAXIS
3TUTZ - 7 AND ,AWTON ' # , 1984. Effects of diet and antimicrobials on growth, feed
efficiency, intestinal Clostridium perfringens, and ileal weight of broiler chicks.
0OULTRY 3CIENCE
3UTER 7 2OSSELET ! AND +NUSEL & , 1978. Mode of action of quindoxin and substituted
4AYLOR $ * AND $AVEY , ! , 1980. Elimination of 4REPONEMA HYODYSENTERIAE infection from pigs by medication with carbadox. )N PROCEEDINGS OF TH )063 #ONGRESS June
30- July 3, Copenhagen. p. 251.
4ROUTT ( & (OOPER " % AND (ARRINGTON 2 , 1974. Effect of carbadox in experimentally induced salmonellosis of swine. *OURNAL OF THE !MERICAN 6ETERINARY
5HLIN " % AND .ORDSTR¶M + , 1985. Preferential inhibition of plasmid replication IN VIVO by altered DNA gyrase activity in %SCHERICHA COLI by
7ALTER $ ( AND +INYON *
VAN DER
VAN DER
induced changes in aldosterone, sodium and potassium levels in the blood of weaned pigs. *OURNAL OF 6ETERINARY
| SOU 1997:132 | Annex F | 347 |
VAN DER
VAN DER
and renal immunohistochemically demonstrated renin in carbadox treated pigs.
2ESEARCH IN 6ETERINARY 3CIENCE
7EISSER * AND 7IEDEMANN " , 1987. Inhibition of
7ILLIAMS " * AND "ABCOCK 7 % , 1978. )N 6IVO and )N 6ITRO susceptibility of
4REPONEMA HYODYSENTERIAE to carbadox before and after repeated in vitro passage in sublethal concentrations of drug. 6ETERINARY
7ILLIAMS " * AND 3HIVELY * % , 1978. )N 6ITRO antitreponemal activities of carbadox,
virginiamycin, olaquindox, and tylosin as indices of their effectiveness for preventing swine dysentery. 6ETERINARY
7INKELMAN . AND (AWKINS 0 ! , 1996. Evaluation of carbadox and neomycin-
oxytetracycline for control of proliferative enteropathy (ileitis) in swine. )N PROCEEDINGS OF TH )063 #ONGRESS
7OOD % . , 1987. Financial aspects of swine dysentery eradication without depopulation. )N PROCEEDINGS OF TH )063 #ONGRESSCopenhagen. pp.
| 348 | Annex F | SOU 1997:132 |
| SOU 1997:132 | 349 |
Annex G: Buget sheets to 3.5
G.1 Budget sheets for piglet production
(SLU INFO - OMRÅDESKALKYLER)
Basic assumptions
Herd size 50 sows, a sow produces 20 piglets per year, pigs are 100 % Scan- H piglets, 5 weeks to weaning, piglets sold at 25 kg (at 85 days of age), purchased feed, the feed for gilts until farrowing included, new buildings and interest rate 7%, results per sow
Assumptions on the effect from the use of antibiotics as additives in animal feed on growth 6.8%, feed conversion 4.6%, and reduction of mortality 0.6%.
Gross margins (from table G.I)
| 7ITHOUT | 7ITH | 'AIN | |
| !&! | !&! | ||
| Gross margin (TB 1) = Income | 3092 | 3281 | 189 |
| minus variable costs | |||
| Gross margin (TB 2) = income | 1376 | 1581 | 205 |
| minus variable and capital costs | |||
| Gross margin (TB 3) = income | 205 | ||
| minus variable, capital and fixed | |||
| costs |
| 350 | Annex G | SOU 1997:132 |
Table G.I Budget sheet for piglet production
| )NCOME | |||||||||||
| 5NIT | 1UAN | 0RICE | !DDED 0RICE !DDED | 3UM | |||||||
| TITY | UNIT | QUAN | 3%+ | BENEFIT | ADD | ||||||
| TITY | BE | ||||||||||
| NEFIT | |||||||||||
| Piglets | n. | 20 | 454 | 9080 | 0.12 | 454 | 54 | ||||
| Premium | SEK | 20 | 19.00 | 380 | 0.12 | 19.00 | 2 | ||||
| Meat from culled | n | 0.50 | 1076 | 538 | |||||||
| sows | |||||||||||
| Total income | 9998 | 10054 | 56 | ||||||||
| 6ARIABLE COSTS | |||||||||||
| Replacement | n | 0.5 | 2000 | 1000 | |||||||
| Sow feed | kg | 1289 | 1.70 | 2191 | |||||||
| Piglet feed | kg | 938 | 2.16 | 2026 | 55 | 2.16 | 93 | ||||
| Straw, bedding | kg | 400 | 0.35 | 140 | |||||||
| Electricity | kWh | 710 | 0.45 | 320 | |||||||
| Health control fee | SEK | 58 | |||||||||
| Sow production | SEK | 150 | |||||||||
| control fee | |||||||||||
| Cost of boar | SEK | 351 | |||||||||
| Insurance | n | 70 | |||||||||
| Miscellaneous | SEK | 600 | |||||||||
| Additional vet costs | 20 | 2.00 | 40 | ||||||||
| if AB prohibited | |||||||||||
| Sum variable costs | 6906 | 6773 | 189 | ||||||||
| Buildings, | SEK | 73200 | 2.1% | 1537 | |||||||
| maintenance | |||||||||||
| Costs of animal | SEK | 1538 | 7% | 108 | |||||||
| capital | |||||||||||
| Costs of operational | SEK | 1009 | 7% | 71 | 14 | 7% | 1 | ||||
| capital | |||||||||||
| Savings due to shorter | 14 | 1.06 | 15 | ||||||||
| period to 25 kg | |||||||||||
| 3UM OF VARIABLE | 8622 | 8473 | 205 | ||||||||
| AND CAPITAL COSTS | |||||||||||
| Buildings, | SEK | 73200 | 8.3% | 6076 | |||||||
| maintenance and | |||||||||||
| depreciation | |||||||||||
| Labour | hours | 23 | 115.00 | 2645 | |||||||
| 3UM OF VARIABLE | 17343 | 17194 | 205 | ||||||||
| CAPITAL AND FIXED | |||||||||||
| COSTS | |||||||||||
| SOU 1997:132 | Annex G | 351 |
G.2 Budget sheets for pig meat production
(SLU INFOS OMRÅDES KALKYLER)
Assumptions
Herd size 288 pigs per batch, purchased feed, dry feeding, live weight at slaughter 107 kg, slaughter weight 73% of live weight, new buildings, interest rate 7%, 2.7 batches per year, and feed conversion 2.8 kg feed per kg weight gain, results per slaughter hog
Assumptions on the effect of antibiotics used as additives in animal feed on growth 1.86%, and feed conversion 1.65%.
Gross margin per pig (from table G.II)
| 7ITHOUT | 7ITH | 'AIN | |
| !&! | !&! | ||
| Gross margin (TB 1) = | 155 | 162 | 7 |
| income minus variable costs | |||
| Gross margin (TB 2) = | 62 | 71 | 9 |
| Income minus capital and variable | |||
| costs | |||
| Gross margin (TB 3) = | 14 | ||
| Income minus variable, capital and | |||
| fixed costs |
Gains in a farm with 500 pigs per batch
| 7ITHOUT | 7ITH !&! | 'AIN | |
| !&! | |||
| Pigs produced per | 1350 | 1375 | |
| year | |||
| Gross margin TB1 | 209250 | 227750 | 13500 |
| (SEK) | |||
| Gross margin TB2 | 83700 | 97625 | 13925 |
| (SEK) | |||
| Gross margin TB3 | 12575 | ||
| (SEK) |
| 352 | Annex G |
Table G.II. Budget sheet for pig meat production
| 0ER SLAUGHTER PIG | 5NIT 1UANTITY 0RICE | 3%+ | !DDED | |||
| QUANTITY | ||||||
| )NCOME | ||||||
| Meat kg | kg | 78 | 13.80 | 1076 | ||
| Delivery premium | SEK | 26 | ||||
| 1102 | ||||||
| 6ARIABLE COSTS | ||||||
| Piglets costs 25 kg | n | 1.00 | 454.00 | 454 | ||
| Delivery fee | SEK | 39 | ||||
| Feeding stuff | kg | 229.60 | 1.76 | 404 | 3.8 | |
| Service fee | SEK | 3 | ||||
| Energy | SEK | 24 | ||||
| Mortality (2 %) | SEK | 627 | 2.0% | 13 | ||
| Miscellaneous | SEK | 10 | ||||
| 3UM VARIABLE COSTS | 947 | |||||
| #APITAL COSTS | ||||||
| Buildings | SEK | 3407 | 2.1% | 72 | 64 | |
| maintenance | ||||||
| Cost of animal | SEK | 183 | 7% | 13 | 4 | |
| capital | ||||||
| Cost of operational | SEK | 111 | 7% | 8 | 2 | |
| capital | ||||||
| 3UM VARIABLE | 1040 | |||||
| CAPITAL COSTS | ||||||
| #APITAL COSTS | ||||||
| Buildings | SEK | 3407 | 8.3% | 283 | 63 | |
| depreciation and | ||||||
| interest payment | ||||||
| Labour | hours | 0.4 | 115.00 | 46 | ||
| 3UM VARIABLE | 1369 | |||||
| CAPITAL AND FIXED | ||||||
| COSTS | ||||||
SOU 1997:132
0RICE !DDED 3UM OF BENE ADDED FIT BENEFIT
1102 0
1.76 7
940 7
2.1% 1.3
7% 0.3
7% 0.1
1031 9
8.3% 5
1355 14
| SOU 1997:132 | Annex G | 353 |
G.3 Budget sheets for egg production
(SLU INFOS OMRÅDES KALKYLER)
Assumptions
Batch size 10000 layers, production period 66 weeks, age at start in flock 16 weeks, and at slaughter 80 weeks, purchased feed, feed consumption 2.25 per kg eggs.
New buildings, caged birds, three story cages, annual egg production 20 kg per introduced young bird and interest rate is 7%, results per 100 hens
Assumptions on the effect of antibiotics used as additives in animal feed on egg performance 1.63%, and feed conversion 1.32%.
Gross margins (from table G.III)
| 7ITHOUT !&! | 7ITH !&! 'AIN | ||
| TB1 = Income minus variable costs | 4372 | 4535 | 163 |
| TB 2 = Income minus variable and | 3570 | 3913 | 163 |
| capital costs | |||
| TB 3 = Income minus variable, capital | 132 | 163 | |
| and fixed costs | |||
| 354 | Annex G | SOU 1997:132 |
Table G.III. Budget sheet for egg production
| 5NIT 1UANTITY | 0RICE | 3%+ | !DDED | 0RICE !DDED 3UM | ||||||||
| QUANTITY | BENEFIT ADDED | |||||||||||
| BENEFITS | ||||||||||||
| )NCOME | ||||||||||||
| Eggs | kg | 1564 | 9.30 | 14545 | 21 | 9.30 | 195 | |||||
| Meat for slaughter | n | 69 | 1.38 | 95 | ||||||||
| Quality deductions | kg | 1564 | 21 | |||||||||
| Washing costs | kg | 126 | 2 | |||||||||
| Sum income | 13779 | 13962 | 183 | |||||||||
| 6ARIABLE COSTS | ||||||||||||
| Replacement birds | n | 79 | 33.00 | 2607 | ||||||||
| Growing feed | kg | 79 | 1.75 | 138 | ||||||||
| Laying feed | kg | 3519 | 1.76 | 6193 | 1.76 | |||||||
| Misc. costs | SEK | 1564 | 0.30 | 469 | 21 | 0.30 | ||||||
| 3UM VARIABLE COSTS | 9407 | 9427 | 163 | |||||||||
| #APITAL COSTS | ||||||||||||
| Buildings | SEK | 22000 | 2.1% | 462 | ||||||||
| maintenance | ||||||||||||
| Cost of animal | SEK | 1351 | 7% | 95 | ||||||||
| capital | ||||||||||||
| Operational capital | SEK | 922 | 7% | 65 | 1 | 7% | 0 | |||||
| 3UM VARIABLE AND | 10029 | 10049 | 163 | |||||||||
| CAPITAL COSTS | ||||||||||||
| #APITAL COSTS | ||||||||||||
| Buildings, | SEK | 22000 | 8.3% | 1826 | ||||||||
| depreciation and | ||||||||||||
| interest payments | ||||||||||||
| Labour | hours | 17 | 115 | 1955 | ||||||||
| 3UM VARIABLE | 13810 | 13830 | 163 | |||||||||
| CAPITAL AND FIXED | ||||||||||||
| COSTS | ||||||||||||
| SOU 1997:132 | Annex G | 355 |
G.4 Budget sheets for poultry meat production
(SLU INFOS OMRÅDES KALKYLER)
Assumptions
Flock (batch) size 80000, weight at slaughter 1.65 kg, new buildings, purchased feed, batches per year 6.75, annual production 540000 broilers, number of day old chicks per 1000 broilers slaughtered 1050, feed conversion 2.85 kg feed per kg broiler, interest rate 7% and age at slaughter 36 days, the results in a farm producing 80000 birds per batch (expected values).
Assumptions on the effect of antibiotics used as additives in animal feed on growth 2.09%, and on feed conversion 1.47%.
Gross margins (from table G.IV)
| 7ITHOUT !&! | 7ITH !&! | 'AIN | ||
| TB 1 | = Income minus | 988616 | 1065977 | 77361 |
| variable costs | ||||
| TB 2 | = Income minus | 909608 | 988677 | 79069 |
| variable and capital costs | ||||
| TB 3 | = Income minus | 26890 | 88022 | |
| variable, capital and fixed | ||||
| costs | ||||
| 356 | Annex G | SOU 1997:132 |
Table G.IV. Budget sheet for poultry meat production
| 5NIT | 1UANTITY | 0RICE | 3%+ | !DDED | 0RICE | 3%+ | .ET | ||
| QUANTITY | BENEFITA | ||||||||
| )NCOME | |||||||||
| Broilers | kg | 891000 | 7.42 | 6611220 | 16500 | 7.42 | 122430 | ||
| Quality penalty | SEK | 6611220 | 122430 | ||||||
| Sum income | 6545108 | 6666314 | 121206 | ||||||
| 6ARIABLE COSTS | |||||||||
| Day old chicks | n | 567000 | 2.88 | 1632960 | 2.88 | ||||
| Chicken feed | kg | 1563300 | 2.13 | 3329829 | 2.13 | ||||
| Electricity | kWh | 194940 | 0.45 | 87723 | 39910 | 0.45 | |||
| Oil | SEK | 631 | 34070 | ||||||
| Straw, bedding | kg | 50 | 1.00 | 27000 | |||||
| Insurance | SEK | 157 | 84780 | ||||||
| Misc. costs | SEK | 99 | 53460 | ||||||
| 3UM VARIABLE | 5556492 | 5600337 | 77361 | ||||||
| COSTS | |||||||||
| #APITAL COSTS | |||||||||
| Buildings, | SEK | 5720000 | 1.3% | 74360 | 124348 | 1.3% | 1617 | ||
| maintenance | |||||||||
| Cost of | SEK | 66406 | 7% | 4638 | 1298 | 7% | 91 | ||
| operational capital | |||||||||
| 3UM VARIABLE AND | 5635500 | 5677637 | 79069 | ||||||
| CAPITAL COSTS | |||||||||
| #APITAL COSTS | |||||||||
| Buildings | SEK | 5720000 | 7.2% | 411840 | 124348 | 7.2% | 8953 | ||
| depreciation and | |||||||||
| interest rate | |||||||||
| Labour | hours | 4860 | 115.00 | 558900 | |||||
| 3UM VARIABLE | 6606240 | 6639424 | 88022 | ||||||
| CAPITAL AND FIXED | |||||||||
| COSTS | |||||||||