sou · GLB3132d3 · 1998-01-01 00:00:00 — 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 (, 1985; LeBlanc ET AL , 1986). 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; , 1987) and in 3TREPTOMYCES spp. (, 1987), but the genes in question have not been described (, 1987). Inactivation of erythromycin by production of erythromycin esterases or phosphotransferases has been described in human enterobacteria isolated from patients undergoing treatment with erythromycin (, 1987). Two types of esterases (I and II) are encoded for by the genes EREA and EREB, respectively (, 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 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 (, 1985). Resistance due to efflux has been reported in staphylococci. The resistance gene MSRA described in 3TAPHYLOCOCCUS EPIDERMIDIS encodes a transport-related protein that confers inducible resistance to 14- and 15- membered macrolides (Leclercq and Courvalin, 1991b). The strains with this characteristic are, for unknown reasons, cross-resistant to streptogramin B Further, a constitutively expressed resistance of efflux-type mediated by the gene ERPA has been reported in 3 EPIDERMIDIS (Leclercq and Courvalin, 1991b). In this case, the streptogramins retain their effect. efflux of streptogramin A is mediated by the gene VGA coding for an ATP- binding protein probably involved in the 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 Co-transfer of resistance genes Co-transfer of vancomycin resistance and erythromycin resistance, from %NTEROCOCCUS FAECALIS to 3TAPHYLOCOCCUS AUREUS has been shown to occur both IN , in a mouse model (Noble ET AL , 1992). reached similar results. Leclercq (1989) achieved co-transfer of vancomycin resistance and ERMAM, located on the plasmid plP819, between different enterococcal species and from enterococci to streptococci and ,ISTERIA MONOCYTOGENES. Further, the transfer of pheromone-responsive plasmids harbouring virulence- and 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 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. 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, and are, in these respects, similar enough for a comparison to be attempted. Recent statistics from and 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 substance-specified figures from (, 1996). The same figures were also used to estimate an average DDD of 1.7g. Usage of MLS antibacterials in 1995 in and expressed as kg substance (Apoteksbolaget, 1996; DANMAP, 1997) 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 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 and , 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 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, and co-workers (1992) found that virginiamycin given in feed at 20 ppm effectively prevented the experimentally induced NE. Another study by Elwinger and Teglöf (1991) showed similar results; virginiamycin at 20 ppm in feed notably reduced mortality (p<0.001) of chickens in necrotic enteritis, as compared to non- medicated groups. Stutz and co-workers (1983) showed that virginiamycin, at 55 ppm in feed, reduced the amount of # PERFRINGENS in the intestines of chickens compared to non-medicated controls (p<0.05). The numbers of clostridia were inversely correlated with performance data. MLS antibacterials are still considered to be alternatives in the therapy and prevention of swine dysentery (, 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 values of 3 HYODYSENTERIAE for tylosin and therapeutic effect of this substance. and Shively (1978) found that tylosin at 100-110 ppm completely prevented swine dysentery induced by a tylosin-susceptible isolate of 3 HYODYSENTERIAE, while it was only partly effective against the disease induced by isolates with higher values for tylosin. Jacks and co-workers (Jacks ET AL , 1986) also prevented swine dysentery in pigs experimentally infected with a tylosin- susceptible strain, by feeding tylosin at 110 ppm. When a tylosin-resistant strain was used the disease was not prevented, although mortality was lower in the tylosin-medicated group as compared to the non medicated group. A new tylosin compound, 3-acetyl-4"-isovaleryl tylosin was also tested and found to be effective at 50 ppm. and co-workers (1972) studied the effect of different levels of 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. and Shively (and Shively, 1978) fed experimentally infected pigs virginiamycin at 50 to 100 ppm. Virginiamycin could not control the disease, although clinical signs were a less common in medicated pigs than in non-medicated animals. This was supported by the observations of Rønne and co-workers (1992) who investigated the effect of 20 ppm of virginiamycin. McOrist and co-workers (1997) evaluated the efficacy of orally administered tylosin for the prevention and treatment of experimentally induced porcine proliferative enteritis (PPE). They found that tylosin at therapeutic levels could prevent PPE in challenge exposed pigs and could also be used for treatment of previously induced PPE. Even at growth promoting levels, tylosin was effective in preventing development of PPE. and (1996) reported successful prevention of PPE by tylosin at about 100 ppm. and (1994) compared 0, 40 and 100 ppm of tylosin for the treatment and prevention of PPE. The higher level of tylosin was effective as treatment of PPE, while 40 ppm not quite prevented emergence of clinical signs. 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 therapy (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 MLS-antibacterials in 3 HYODYSENTERIAE is perhaps most alarming. Uncontrollable outbreaks of swine dysentery can lead to substantial losses and eradication of whole herds may be the only option left (Bouwkamp, 1982). )N data on resistance of this bacterium seems to be a good predictor of clinical outcome after treatment (and Shively, 1978; Jacks ET AL , 1986). Unfortunately, such data indicate that the therapeutic potential of the MLS drugs is now limited (and Szancer, 1990; , 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 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 debate has, to a large extent, been concentrated on the possible influence on the resistance of staphylococci. Knothe (1977b) concludes, in a review on 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 1953-1971 and an accompanying paper describing the prevalence of resistance in from 1968-1976 (Knothe, 1977b; Knothe, 1977a). In the latter study, prevalences of erythromycin resistance between 13 and were reported with a tendency towards a reduction over time. In a more recent report from the same region of , the prevalence of erythromycin resistance in 3 AUREUS from community acquired infections was , from general ward and from intensive care (, 1993). In some recent reports from other European countries, though, the prevalence of resistance to macrolides in staphylococci and enterococci approaches (Turano ET AL , 1994). At the time when Knothe (1977b) published the review, exchange of genes between gram-positive cocci was thought to be a rare event in natural environments. 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). of the ERM-family conferring resistance of the MLSB type have been demonstrated in both human and animal bacteria (, 1987; and , 1994; , 1994). This may be due either to common ancestry of the bacteria and/or to genetic exchange in more recent times. 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 host-specific. 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 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 erythromycin-inducible -encoded resistance, but overlooks the lack of correlation between genotype and phenotype in MLSB resistant staphylococci (see table E.I). 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 quinpristin-dalfopristin has recently been introduced for human therapy. Woodford and co-workers (1997) reported streptogramin resistance in vancomycin-resistant enterococci (VRE) isolated from raw chicken (3 isolates) and from a hospital patient (1 isolate) in the UK. The resistance trait included cross-resistance to macrolides and lincosamides and was transferable to other enterococci. The authors commented on the fact that no streptogramin is yet licensed for use in human therapy in UK, whereas virginiamycin is widely used for growth promotion in animals. A reservoir of streptogramin resistance may be present in animal bacteria. Since infections with VRE is one of the main indications for quinpristin-dalfopristin therapy, acquisition of streptogramin resistance by those organisms is most alarming. Similar observations have been reported from The (van den Bogaard ET AL , 1997). In this study, enerococci were isolated from healthy humans and pigs. The prevalence of streptogramin, macrolide and vancomycin resistance in human samples was , and , respectively. The corresponding figures for the samples from pigs were , and . The higher prevalences observed in the material from pigs indicate a higher selective pressure in this population. No information on consumption of macrolides and streptogramins in either humans or pigs in The were given. E.7 Other effects on the microflora 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. and co-workers studied the effect of in-feed virginiamycin on experimental infection with 3ALMONELLA Typhimurium in chickens and 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. experiments were performed in duplicate, with groups of 20-30 animals. Sampling was performed on several occasions throughout the trials, by cloacal swabs on live birds and from the heart, liver, duodenum and caecum of killed birds at the end of the experiment. Not all birds were sampled on all 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 and co-workers published a study where chicks fed 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 virginiamycin-fed group and lowest in the control group. 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 acid-resistant mutants of other lactose negative enteric bacteria, and one cannot be certain that the proportion of coliforms mistakenly identified as salmonellae would be the same in all experimental groups. No further identification of the presumed salmonellae isolated in this way is reported in the article. The major objection against the design of this study is, however, that all birds received monensin at 100 ppm in the diet. Since monensin has antibacterial effects, this must be regarded as a possible confounder. Thus, the study only investigated the differences between birds fed monensin and birds fed monensin plus avoparcin or virginiamycin. and have published several studies on the influence of various antimicrobial substances on the course of salmonella infection and excretion in broiler chickens (and , 1975a; and , 1975b; and , 1978; and , 1980; and , 1980). One study from 1975 includes both tylosin and virginiamycin (and , 1975b) and one from 1978 includes tylosin (and , 1978). Tylosin was given at concentrations of 10 and /kg feed in both studies and in the study from 1975 virginiamycin was also given at concentrations of 10 and /kg. Duplicate experiments were performed on groups of 33-45 chickens receiving either antibiotic-containing feed or control feed without antimicrobial additives. In the study from 1975, the animals were experimentally infected with of a nalidixic acid-resistant strain of 3ALMONELLA Typhimurium at a concentration of 109 CFU/ml. 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.