sou · GLB3132d1 · 1998-01-01 00:00:00 — )N VIVO transfer of pheromone-responsive plasmids carrying genes conferring cytolytic properties and macrolide resistance (ERMB) between strains of % FAECALIS has been studied in a model using Syrian hamsters (Huycke ET AL , 1992). Transfer occurred in high frequencies (10-1 - 10-2 per donor). Even more worrying is the transfer IN of macrolide resistance (ERMBZ) and toxin A, apparently carried on a transposon (Tn ) from # DIFFICILE to non-toxigenic # DIFFICILE strains and to " SUBTILIS reported by Mullaney (1995). Consequently, a selection for antibacterial resistance may, in some cases, also select for virulence. The danger of transfer elements carrying multiple genes is evident; a single transfer event conveys not only resistance to the selector but also other properties to the recipient bacterium. The recipient may acquire multiresistance and/or increase its virulence. advantage given by one of the transferred genes helps to maintain all of the transferred genes in the new host. 4.4.4Resistance genes and animal hosts Highly homologous resistance genes and their associated transfer elements have been found in natural isolates not only of different bacterial species but also in bacteria from a variety of host species. The macrolide resistance gene ERMB has been demonstrated in bacteria of different species originating from pigs, chickens, cattle, dogs and humans (, 1985; , 1987; , 1992; Eady ET AL , 1993; and , 1994; , 1994). As an example, the results of Eady and co-workers (1993) who screened for resistance determinants in macrolide resistant isolates of staphylococci (excluding 3 AUREUS) from humans, pigs and dogs are shown in table 4.VII. Distribution of macrolide resistance genes in staphylococci from various hosts given as percent of total number of genes identified in isolates from respective animal host1 (modified from Eady ET AL , 1993) 4ABELL 6)) &¶RDELNINGEN AV MAKROLIDRESISTENSGENER HOS STAFYLOKOCKER FR¥N OLIKA V¤RDDJUR UTTRYCKT SOM PROCENT AV TOTALANTALET IDENTIFIERADE GENERMODIFIERAT FR¥N %ADY ET AL of genes identified and No. of investigated isolates: swine 35 and 33, dogs 17 and 16, humans group 1 58 and 55, humans group 2 121 and 117, respectively. 2Group 1 includes isolates from patients undergoing ambulatory peritoneal dialysis (n=27) and from blood cultures (n=28); group 2 includes patients with acne (n=117). 1 !NTAL IDENTIFIERADE GENER RESPEKTIVE ANTAL UNDERS¶KTA ISOLAT SVIN OCH HUNDAR OCH M¤NNISKOR GRUPP OCH M¤NNISKOR GRUPP OCH 'RUPP INKLUDERAR ISOLAT FR¥N PATIENTER I AMBULATORISK PERITONEALDIALYS N SAMT BLODODLINGAR N GRUPP INKLUDERAR PATIENTER MED AKNE N The suggested spread of vancomycin resistance between enterococci of various origin is another example of the spread of resistance among gram- positive bacteria. VRE harbouring the VANA gene cluster have been isolated from humans, both in hospitals and community, from pig, rabbit, dog, cat, horse chicken, , pheasant, duck, food of animal origin and sewage (, 1994; Torres ET AL , 1994; , 1995b; Chadwick ET AL , 1996; Devriese ET AL , 1996; DANMAP, 1997). A polyclonal nature of the VRE strains has been demonstrated (, 1995b). The VANA gene cluster consists of 7 gene components and two transposition gene sequences. It is extremely unlikely that such a complicated gene should have developed separately in so many different host populations. Its occurrence therefore strongly suggests a spread between bacteria of different host species. 4.4.5The encumbrance of resistance for bacteria In an environment that contains antibacterials, possession of a corresponding resistance gene is clearly beneficial for the bacterium. The resistance traits have, however, been considered to impose a burden on their bacterial hosts. Synthesis of plasmids require energy and metabolites. The products encoded for by resistance genes may interfere with the bacteriums physiology; alterations of the cell wall may lead to loss of adhesive properties and alternative metabolic pathways may be less efficient. Constitutive expression of resistance genes encoding for such traits would be a handicap in the absence of antibacterials. For such reasons, resistant clones have been thought to suffer a competitive disadvantage in the absence of a selective pressure. Even so, this might not always be the case. Much evidence of the cost for the bacterium of harbouring the resistance trait has been obtained in laboratory experiments with bacteria that have recently acquired the resistance gene. Other experiments show that, given time, bacteria can eliminate the cost of resistance by adapting their system in order to counteract the harmful side- effects of the resistance genes. This will result in increased fitness of resistant strains with consequent persistence of the resistance trait in the microbial population even after the selective pressure is removed. Such adaptation has been shown for resistance acquired through mutations as well as through plasmids (for reviews see Lenski, 1996; and McHugh, 1997). As an example, McConnell and co-workers (1991) demonstrated the need of a selective pressure for the of a resistance plasmid in the microflora of mice. However, once established, the strain originally carrying the plasmid adapted and competed well with susceptible strains in the absence of a selective pressure. 4.4.6Some remarks on horizontal transmission of genes Transmission of genes between bacteria occurs readily IN . Experimental transfer of a conjugative element may require manipulation besides the mixing of two cultures. The rates, or frequencies, of such transmissions will depend on the transfer element involved and the donor-recipient combination as well as on the conditions of the transfer experiments. In this respect it should be noted that failure to demonstrate transfer does not mean it cannot take place, simply that it did not occur under the conditions of the experiment. On the other hand, demonstration of transfer indicates that the event can take place. Bacteria are equipped with very efficient gene transfer systems. Similar, or identical, resistance genes and associated transfer elements are present in field isolates from different host species as well as in different bacterial A multitude of transfer elements are found in bacteria isolated from natural settings. Resistance traits arise within species that were formerly considered uniformly susceptible. The experiments demonstrating IN VIVO transfer in laboratory animals have been conducted to test a hypothesis based on firm observations from real life of emerging resistance. An example of this is the studies on macrolide and tetracycline resistance in ,ISTERIA MONOCYTOGENES cited in 4.4.1. settings are, albeit artificial, certainly closer to the real situation than the test tube. The opinion that the transfer rate in nature would be significantly lower than what is obtained IN is based on the assumption that laboratory settings provide optimal conditions, and that such conditions are rare in real life. This is not necessarily true. The laboratory conditions normally used to test DNA transfer employ rapid growth, aerobic conditions and nutrient excess. Such conditions might enhance transfer rates, but it might also repress gene transfer in some bacteria (Salyers, 1995). Further, the event does not have to be frequent in order to have an effect. Once the resistance gene is present in a recipient, given a selective pressure the resistant clone will multiply. Taken together, it is evident that transfer of genes does occur in nature (in/on living hosts or in their environments). The question that remains is how often it occurs. Resistance mechanisms and resistance genes have only been investigated for belonging to containing therapeuticals. Described resistance genes are generally associated with transfer elements. Transfer of resistance has been shown experimentally both IN VIVO Transfer of resistance has been shown between bacteria of different genera. The same resistance genes are found in bacteria from different host species. Co-transfer of multiple genes is common. Resistant bacteria are not necessarily less competitive in the absence of a selective pressure. 4.5Transfer of resistance between various host species There are two theoretically possible ways for transmission of antibiotic resistance from bacteria of one host species to those of another; firstly, direct transmission of resistant bacteria, and secondly, transmission of resistance genes between bacteria colonising different host species. The debate has mostly been concerned with transmission of resistant bacteria from animals (taken as a group) to man. It has been questioned whether such transmission can occur. Logically, if transfer did not occur between bacteria from a specific animal species to man, then transfer between different animal species would not be expected to occur either. After all, humans are the only species in contact with all the other species. Studies concerning transmission of resistant bacteria from animal to man have mainly focused on gram-negative enteric pathogens such as 3ALMONELLA spp. and #AMPYLOBACTER spp. For antibiotic resistant salmonellae, the pathways from animals to man and subsequent infections have been clearly demonstrated (Bezanson ET AL , 1983; , 1984; Spika ET AL , 1987; Mishu ET AL , 1991). Several reports suggest the transfer of other antibiotic-resistant enteropathogens such as quinolone-resistant campylobacter (Endtz ET AL , 1991) and chloramphenicol-resistant 9ERSINIA ENTEROCOLITICA (AL , 1988). Transfer of % COLI between various animals and humans has been demonstrated in various studies. The spread of a labelled multiresistant % COLI from cow to pig, mouse, fowl, fly, water and human, and from pig to fly, mouse, fowl, water and human was reported by and co-workers (1990). (, 1976) reported the transfer of a labelled multiresistant % COLI from chicken to a farm worker. and co-workers (1977) demonstrated the transfer of sulphonamide-resistant % COLI from a chicken carcass to the faecal flora of five human volunteers handling the chicken. Although the above cited studies concern resistance to antibacterials not used as in the EU, they demonstrate that bacteria can be transmitted from animals to man directly, via food, or by other routes and in the case the bacterium resistance this trait will not disappear along the line. Transfer of gram-positive bacteria has been less studied. Hummel and (1981) found macrolide-resistant 3 AUREUS of human biovariant in farm workers and in occasional pigs on farms using in-feed tylosin and tetracycline. This seems to indicate a human to animal transmission. As staphylococci are known to be relatively host specific, it is likely that the finding was merely the result of a transient colonisation. Interestingly, no macrolide-resistant strains were found among the family members of the farm workers, indicating the need for a selective pressure for transferred bacteria to persist in the new host. In the case of the farm-workers, they were likely to be exposed to tylosin through dust. Direct transmission from chicken to man has been implicated in a recent report on a case of wound infection with a vancomycin resistant % FAECIUM in a worker at a chicken packaging plant (Das ET AL , 1997). Available information strongly suggests that the infection was occupationally derived (contracted from chicken carcasses). Human % FAECIUM strains have been widely used as probiotics and do, at least transiently, colonise animal intestines. Further, 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. Another report on occupational exposure provides further evidence (van den Bogaard ET AL , 1997). The prevalence of VRE in , farmers, slaughterers and urban residents was found to be , , and , respectively. Further investigations showed that VRE isolated from one of the farmers and his could not be differentiated by phenotypic or genotypic (pulsed-field gel electrophoresis) methods. Investigations of the VANA-gene cluster by polymerase chain reaction (PCR) and hybridisation showed the two strains to be identical in the tested areas, having an insertion in a previously nondescribed position, between the VANX and VANY gene, and a deletion in the right end of the cluster. The majority of the bacteria in the animal intestinal microflora are relatively host specific and would not be expected to colonise other animal species or man. Similar restrictions do not apply to the bacterial host range of resistance genes. Therefore, transfer of genes between animal and human bacteria is more likely to have an impact on interspecies exchange of resistance. Transfer of resistance genes between animal and human microflora has been shown in several studies. Again, many of those deal with resistance genes related to therapeutical antibacterials. However, in the light of the close relatedness between resistance genes in human and animal bacteria and the current knowledge of gene transfer mechanisms and genetic drift, it would be naïve to presume that there is no exchange of resistance genes between animal and human microflora. On the contrary, this is the most, if not the only, plausible explanation for the spread of antibacterial resistance against both therapeuticals and in various populations. The majority of the studies addressing this topic concern resistance in gram-negative bacteria against therapeutic antibacterials. Nonetheless, they illustrate the fact that transmission of genes occurs between animal and human bacteria. Resistance genes identified in both animal and human bacteria and co-workers (, 1976) reported spread of an % COLI multi- resistance plasmid between chickens and from chicken to man. The spread of streptothricin resistance from animal to human bacteria has been documented in former Eastern . A streptothricin antibiotic, nourseothricin, was commonly used in pig feed from the beginning of the 80s. A novel transposon-encoded resistance mechanism was identified in % COLI from pigs in 1983. Subsequently, this transposon, Tn , has been found in the normal microflora of pig farmers and their families and of healthy unrelated adults in the community, and in urinary tract infections in humans. Finally, the resistance determinant has been detected in salmonella and shigella from human cases of diarrhoea (Hummel 1986, Tschäpe 1994). Streptothricin antibiotics are not used in humans. In areas where the antibiotic was not used in animals, the resistance determinant was not found. Another example is the spread of aminoglycoside-acetyl-transferase IV (apramycin resistance) and hygromycin B phosphotransferase (hygromycin B resistance). The corresponding genes, AACC4 and HPHB, form part of one resistance gene operon associated with an insertion sequence IS probably part of a transposon, that is found on plasmids. The organisation of AACC4 and HPHB is such that they are always co-transferred. hygromycin B are used exclusively in animals. The gene AACC4, conferring cross-resistance to gentamicin, was first identified in % COLI and 3ALMONELLA TYPHIMURIUM from animals in and the (Chaslus- Dancla ET AL , 1986; , 1986). It has since been demonstrated in various enterobacteria, including salmonella, from human as well as environmental sources in different countries (Threlfall ET AL , 1986; Chaslus- Dancla ET AL , 1989; , 1993; , 1994). of genetic homology between plasmids harbouring AACC4 and HPHB of human and animal origin has been demonstrated (Salauze ET AL , 1990; Chaslus-Dancla ET AL , 1991). In a prospective study on a pig farm, (1994) demonstrated a widespread dissemination of plasmids harbouring AACC4 in % COLI from pigs, calves, the stockman and a variety of environmental sources including rainwater puddles and water from a stream nearby. +LEBSIELLA PNEUMONIAE with a slightly smaller conjugative plasmid and similar resistance pattern was isolated from the stockman's wife. As mentioned in 4.3.4, investigations concerning antimicrobial resistance tend to focus on a limited range of microbes. Few investigations concerning transfer of genes between the predominant, anaerobic, part of the microflora of man and animals are available. Nikolich and co-workers (1994) examined the possible gene transfer between microflora of ruminants and man, by using the TETQ gene, conveying tetracycline resistance, as indicator. By DNA sequencing of the gene from "ACTEROIDES spp. and 0REVOTELLA INTERMEDIA of human origin and 0REVOTELLA RUMINICOLA of bovine origin, they showed identical, or nearly identical, gene sequences in bacteria of the two hosts from different geographic origin. The findings indicate that extensive transmission of the TETQ gene has occurred in nature between bacteria normally colonising different hosts and that the transfer is recent. No similar investigations with direct relevance for used in Europe have been found, nor studies concerning dominant microflora of other animal )N co-transfer of macrolide and tetracycline resistance between "ACTEROIDES spp. and 0REVOTELLA RUMINICOLA has been shown (Shoemaker ET AL , 1992), indicating a strong possibility of similar IN VIVO transfer events related to macrolides and streptogramins. Further, successful transfer of ERMF genes from 4REPONEMA DENTICOLA to %NTEROCOCCUS FAECALIS has been demonstrated (, 1996a) The question of identity of genes has been a matter of debate in relation to the discussion on the use of avoparcin in animal husbandry. It is important to point out that the bacterial genome is subject to changes over time. Mobile gene-sequences may insert into or close to non-essential parts of a gene, and deletions or point-mutations may take place. Consequently, the sequence of a specific gene may differ from one point in time to another. The VANA gene cluster contains 9 genes (7 and two transposition genes). Between those genes are intergenic, non-coding regions. The coding regions would be expected to be highly conserved once their sequences are optimal for function. As the intergenic regions are not essential for the function of the gene cluster, they are more likely to vary. Several recent studies have addressed the matter by amplification by polymerase chain reaction (PCR) and sequencing of the genes and/or their intergenic regions (, 1996; Haaheim ET AL , 1997; , 1997). In a study from (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 VANS-VANH region were used to analyse the vanA gene cluster from VRE of Norwegian poultry and humans of various nationality (Swedish, Norwegian and American). In 9/12 human isolates and 7/10 poultry the results were identical, indicating horizontal transfer of the gene cluster. and co-workers (1997), investigated 37 VRE isolates from one UK hospital and 36 VRE isolates from poultry meat bought in national By PCR, three intergenic regions and three genes of the VANA- cluster were investigated (VANS-VANH, VANX-VANY, VANY-VANZ, VANX, VANY and VANZ) 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 in this study were clearly atypical. The focus of a Danish study, reported by (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 and co-workers (1997), found evidence of an insertion sequence in the VANX-VANY region in 7 of 12 British human isolates. Based on sequencing of coding and non-coding regions, the remaining isolates could be divided into 3 groups, each containing isolates both from man and animals from different countries. designation of one of the groups was based on the presence of a point mutation in VANX and an insertion sequence (IS 6) in a specific position in the transposon (Tn ). The group contained isolates from humans (and ) and pigs (and UK). Mutations within the coding regions appear to be rare. Insertion sequences are highly mobile, and frequently vary in their location. The occurrence of an insertion sequence in the same location in strains of different origin could either be interpreted as evidence of an epidemiological relationship, or as the site being a hot spot for insertion of the specific sequence. However, the likelihood of both a point mutation and an insertion in a specific location occurring independently in different strains is extremely low. Therefore, the genes present in those isolates must be very closely related and their presence the result of horizontal transfer. Lacey (1980) examined the possibility of transfer of macrolide resistance between animal and human isolates of 3TAPHYLOCOCCUS AUREUS. phage conjugation from human to animal isolates was shown at low frequencies (10-8-10-9) for some of the isolates (see comments on transfer rates under 4.4.6 and 4.5.4). No transfer from animal to human isolates could be demonstrated. The genetic nature of the resistance traits for which transfer was attempted is not known. The same author also investigated transfer of resistance between animal and human streptococci, including 3TREPTOCOCCUS group D (now %NTEROCOCCUS) (Lacey, 1984). In about of the animal strains investigated, transfer to human recipients was demonstrated. Transfer of resistance occurred more readily from the enterococci. No transfer to group A or group C streptococci was demonstrated. The most common transfer frequencies were between 10-6 and 10-8. However, the experiments were conducted in broth, filter matings being used only for isolates that had failed to transfer initially. Under optimal conditions, the transfer rates would be expected to be considerably higher (by up to 3 logarithms). Further, the experiments also failed to demonstrate conjugative transfer from human enterococci to human group A or group C streptococci. Such transfer has, contrary to the statement of Lacey, been demonstrated by other authors (, 1979; Horodniceanu ET AL , 1981; Ravdonikas, 1983; Horaud ET AL , 1985). This indicates that the conditions of the experiment may not have allowed optimal transfer rates. 4.5.3Routes of transmission - food as a vehicle Direct contact with animals carrying resistance genes provides excellent opportunity for uptake of these genes. Examples of farmers carrying the same resistance genes in their microflora as do their animals have been given in 4.5.1 and 4.5.2. Logically, this transfer works both ways, resistance genes may also spread from humans to animals. However, as animals are in many ways part of the human food chain, transfer from animals to humans would be expected to occur more easily. Enteric pathogens are readily transmitted through foods as are antibiotic resistant pathogens and commensals. It has been suggested that, in the normal human population, most resistant enterobacteria in faeces come from contaminated food (Corpet, 1988). In an experiment (Corpet, 1988), 7 healthy volunteers were given a control diet for 3 weeks followed by a sterile diet for 2.5 weeks. During both periods, total and antibiotic resistant %NTEROBACTERIACEAE in stools were counted. A drastic drop in faecal concentrations of antibiotic resistant enterobacteria was observed during the sterile diet period (p<0.05). Antibiotic resistant gram-negative bacteria are present in animal products as well as on vegetables and fruits (, 1983).