sou · GLB3132d3 · 1998-01-01 00:00:00 — Depending on the exact mechanism, a variety of resistance phenotypes are found, many of which are cross-resistant to most or all members of the group (table E.I). Most studies on MLS resistance have been conducted on gram- positive cocci and clostridia. Regarding macrolide and streptogramin resistance in #AMPYLOBACTER spp. and 3ERPULINA spp., is known about the mechanisms and the genes conferring resistance. Further information on resistance mechanisms and corresponding genes are found in E.4. Examples of phenotypic and genotypic expression of some resistance genes (R=resistant, ) -inducible in: 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 promotion are well above the 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. Several studies by the research group of (, 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 (, 1985; Kaukas ET AL , 1988). Considering the potential of virginiamycin to select for MLSB-type resistance, it cannot be excluded that the gradual increase in macrolide resistance for the control groups in the former studies was related to the antibiotics in the diet. In one of the studies (, 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. 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 () 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. 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 respectively) and % FAECIUM (88 and 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 (, 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. The influence of virginiamycin on the prevalence of resistant enterococci within flocks of has been reported by Thal and co-workers (1996). Different from the same flock were found to harbour the same type of streptogramin-resistant % FAECIUM and the prevalence of resistance to both streptogramins and ampicillin increased with the age of the flock. In a German study (Hummel and , 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 non-exposed herds were sensitive to macrolides. This indicates that tylosin may exert a selective pressure, not only on the microflora of animals consuming the feed with the added tylosin but also 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 non-exposed humans could indicate that a selective pressure has to be in force in order to promote further transmission. The dose of tylosin given and the precise administration principles were not detailed in the publication but from what is noted a therapeutic concentration seems most likely. In addition, all animals included in the study were fed tetracyclines, apparently on routine basis. The development of tylosin resistance in 3ERPULINA HYODYSENTERIAE was reported by Molnar (1996) in an investigation from . The proportion of strains classified as resistant to tylosin in this investigation increased from during 1978-82 to during 1983-87. The author assumed that the high level of resistance to tylosin was to a large extent due to the fact that since the 1980s most pig rations in have contained tylosin as an additive. However, this assumption is not substantiated in the article and no 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 (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 to depending on animal source. In the second study from 1991-1992, the corresponding figures were to . The number of animals in these studies is to small for any clear trends to be deducible, but there is no evidence of increase in the resistance rates over the years. 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 ERM-genes. In table E.II, data from several studies on resistance to macrolides in enterococci from poultry and swine have been compiled. The prevalence of resistance appears to be high in the bacterial populations studied, although somewhat lower in the material presented from . Resistance in enterococci to MLSB antibacterials as reported in different studies et al., et al., al., 1996 al., 1996 et al., al., 1996 al., 1996 1When several macrolides have been investigated, the figures for erythromycin have been used. 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 macrolide- resistance between staphylococci from different animal species. The majority of the staphylococcal strains reported as resistant in table E.III expressed the resistance constitutively. Resistance in 3TAPHYLOCOCCUS AUREUS to erythromycin as reported in different studies # 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. 1984; Burridge ET AL , 1986; Lacey, 1988; Jacobs-Reitma ET AL , 1994; DANMAP, 1997). It has been implied that the widespread resistance to macrolides in # COLI may be linked to the use of macrolides (, 1996; , 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 , and in pigs receiving therapeutic doses of in-feed tylosin it was . In contrast, # JEJUNI 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. Prevalence of erythromycin resistance in campylobacter in various studies al., 1994 et al., et al., 1992 et al., al., 1994 et al., et al., 1992 et al., Cabrita et al., Cabrita et al., 111 isolates from humans, 69 isolates from swine 293 isolates from humans, 5 isolates from swine 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 (, 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 (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, the chromosome to a plasmid or from one plasmid to another plasmid. Further, they may be carried on self-transmissible plasmids. Examples of resistance to MLS antibacterials by different mechanisms and genes. (Data compiled from , 1987; , 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) S. aureus, coagulase Tn551, pAM77, S. aureus, S. intermedius, S. Tn917, Tn1545, hyicus, S. pneumoniae, Streptococcus spp., E. faecalis, Lactobacillus spp. pE194, plM13, S. aureus, coagulase negative staphylococci, S. hyicus, Bacillus subtilis S. aureus, S. epidermidis Cross-resistance to all MLSB antibiotics due to target modification is widespread although the prevalence differs regionally (, 1985; , 1987). The bacteria equipped with this property produce rRNA methylases which are enzymes capable of altering the ribosomal binding site of MLSB-antibiotics. The alteration inhibits binding of the antibiotic and hence allows the bacterial protein synthesis to continue. The genes coding for this property in bacteria are designated (erythromycin resistance rRNA methylase). Various different genes have been identified. substantial sequence diversity but studies on their evolutionary relationship suggest that they are of ancient presence in at least 3TREPTOMYCES spp, gram- positive cocci and "ACILLUS LICHENIFORMIS (, 1987). transfer of genes occurs under natural conditions to bacteria which are phylogenetically remote from the above mentioned bacteria, such as %SCHERICHIA COLI (Brisson , 1988). This transfer is believed to be a recent event although the genes are already disseminated in enterobacteria. Transfer of ERM-genes has been demonstrated experimentally in numerous Poyart-Salmeron and co-workers (1990) showed by way of IN mating experiments the conjugative transfer of ERMB, carried on plasmid pIP811 together with other resistance determinants, between among others ,ISTERIA MONOCYTOGENES and % FAECALIS. Doucet-Populaire and co-workers (1991) demonstrated transfer IN and in an IN VIVO model of transposon 4N1545, harbouring ERMB, APHA3 (kanamycin resistance) and TETM (tetracycline resistance) between % FAECALIS and , MONOCYTOGENES McConnell and co-workers (1991) demonstrated transfer of plasmid pAMb1, carrying ERMB, from ,ACTOBACILLUS REUTERI to % FAECALIS IN VIVO. Inducible or constitutive expression Expression of genes can be inducible or constitutive. When constitutively expressed, the methylase encoded for by the ERM-gene will always be produced. On the other hand, when inducibly expressed, the methylase is only produced when the gene is activated by an inducer. Various macrolides may function as inducers, depending on the class of gene and bacterial host. The type of expression is related to the class of 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). The figures in this report is only available in the printed version 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 genes and between different bacterial species, but 14-membered macrolides are generally the best inducers. Inducibly resistant strains can convert to constitutively resistant ones by deletions or single nucleotide changes in the regulatory gene, either in the leader peptide or in the inverted repeat sequences. 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 and have shown a trend from predominantly inducible resistance to predominantly constitutive resistance in staphylococci whereas in British and German reports inducible type still dominate (, 1987). A non- inducing 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 genes could survive exposure to a non-inducer. Investigations of Swedish isolates from humans of 3TREPTOCOCCUS PYOGENES revealed that the resistance determinant was present on an ERMB-carrying conjugative plasmid, possibly originating from plasmid pAMb1 of % FAECALIS (Schalen ET AL , 1995). In an Italian study on the same species, in addition to the MLSB type mediated by ERM-genes, a second phenotype, possibly due to an efflux-mechanism, was reported (Coranaglia ET AL , 1996). 3TREPTOCOCCUS SUIS originally described as a cause of outbreaks of arthritis and meningitis in piglets is today more commonly associated with bronchopneumonia in weaners and fatteners. In a study from (, 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. and co-workers (1994) investigated a collection of Norwegian isolates and demonstrated MLS-resistance in 3 SUIS as 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). In staphylococci harbouring ERMC or genes, inducible resistance is triggered by 14- and 15-membered macrolides only. )N , those strains are resistant to 14- and 15- membered macrolides. As long as the induction persists (presence of, for instance, erythromycin) the methylase will be produced and the bacterial cell will be resistant to all MLSB antibiotics. The 16-membered macrolides, lincosamides and streptogramin B cannot induce expression of the gene and will, when tested separately, remain IN . When expressed constitutively in staphylococci, the ERM-genes confer cross-resistance to all MLSB. No information has been found on whether staphylococci constitutively expressing MLSB resistance due to mutations are likely to mutate back to the inducibly expressed form of resistance in the absence of a selective pressure for the constitutive variety, i.e. the presence of a 16-membered macrolide. Experimental data indicate that the inactive structure of the inducibly expressed resistance gene is energetically favoured (Horinouchi and Weisblum, 1981), so a mutation back to this state would not be totally unexpected. Moreover, it is not known whether constitutively expressed genes would still be constitutively expressed after transfer into a new host bacterium. As a comparison, transfer experiments with enterococcal strains expressing VANB constitutively have shown that the resulting transconjugants can be either of constitutive or of inducible type (, 1997). Eady and co-workers (1993) investigated the distribution of , ERMB and ERMC in staphylococci from human and animal sources. In addition, the presence of MSRA was studied. One hundred and seventy-two human isolates of coagulase-negative staphylococci from dialysis patients, blood cultures and untreated acne patients were analysed, along with 33 isolates of coagulase-negative staphylococci from pigs and 16 isolates of 3TAPHYLOCOCCUS INTERMEDIUS from dogs. Number of staphylococcal strains from various sources harbouring macrolide-streptogramin-resistance genes. Data from Eady and co-workers (1993) Fourteen of the human isolates, but none of the isolates from pigs, and only one isolate from a dog harboured . 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. 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 genes circulate in both human and animal populations. Since pigs, as opposed to dogs, are not exposed to 14-membered macrolides, neither for therapy nor growth promotion, only 16-membered macrolides, the constitutive expression of genes in isolates from these animals is not surprising. Dogs, like people, may be given 14-membered macrolides for therapeutic purposes and would therefore be expected to harbour a much higher proportion of isolates with inducibly expressed genes. and co-workers (1996) investigated the presence of and ERMC in human isolates of 3TAPHYLOCOCCUS AUREUS and coagulase negative They found that the 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 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 genes were found in coagulase-negative staphylococci and in paired isolates, i.e. isolates of 3 AUREUS and coagulase-negative staphylococci originating from the same sample, the same gene was only found in both species in 4 out of 15 pairs. This study illustrates the variability over time in the presence of different genes, a fact that emphasises the importance of identifying the genes involved in the resistance patterns studied. The hitherto described genes encoding for macrolide resistance in clostridia belong to the 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 ERMB-ERMAM (also called ERMBP) gene, has also been described in # PERFRINGENS (Daube ET AL , 1992; Devriese ET AL , 1993; Berryman and , 1995). belonging to this subgroup have earlier been described in numerous bacterial genera, both gram-positive and gram- negative, indicating that they are readily transferred. Sequencing of the # PERFRINGENS ERMB determinant and its flanking regions by Berryman and (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.