Drug_Resistance

Introduction The drug-resistance (R) factors found in Enterobacteriaceae possess two outstanding functions .They render their hosts resistant to antibacterial agents such as antibiotics, and, at the same time, enable them to transmit resistance to other bacteria. The recipient of an R factor thus becomes drug-resistant and a genetic donor in its turn. It may also receive other genetic material from the donor cell, such as segments of chromosome or other extrachromosomal elements which the donor may carry at the time. Chemically, R factors consist of deoxyribonucleic acid (DNA), like the bacterial chromosome, although they behave independently in biophysical and genetic experiments. An R factor is thus an independent linkage group, composed of genes determining drug resistance associated with others conferring the ability to conjugate and to transfer the factor to a new host. The latter have been collectively referred to as the "resistance transfer factor" or RTF , or "transfer factor" , but, as those found in R factors are unlikely to differ essentially from those possessed by other types of transmissible plasmid like colicin factors' a more general term is to be preferred. Of the terms available, "sex factor" seems at present the most suitable and is used here, although, after it was first applied to the F factor, its meaning has been both extended to any transmissible plasmid as a whole and restricted to those plasmids capable of promoting chromosomal transfer . Akiba and Ochiai suggested that the resistance might be transferred to Shigella from strains of Escherichia coli already resistant, and showed that multiple resistance of this kind was indeed transmitted from one bacterium to another by cell contact. Watanabe and Fukasawa confirmed that cell contact was required for the factor to bring about its own transfer, although the resistance genes could also be transduced by phage . Many reports followed, from 1957 onwards, of strains of S. flexneri or E. coli resistant to one or more antibiotics whose resistance was similarly transmissible by growth in mixed culture . Transmission did not depend on the presence of F, the classical sex factor of E. coli K-12 (157), so that the bacteria had evidently acquired an independent mechanism for conjugation. The Meaning of Resistance The susceptibility of organisms to antimicrobial drugs was discussed in Chapter 2. Resistance in an organism can be defined in two ways. First, it can be defined in relation to the population as a whole of which the organism is a part. There is often a "break" between the susceptibility in vitro of susceptible and resistant bacteria within a population (Fig. 3.1). Second, resistance can be defined in relation to the mean serum or tissue concentration of an antimicrobiai drug administered at the usual dosage 1'c the usual route. This relationship can be used to define a breakpoint for interpretation of in vitro susceptibility data, a value that sometimes also incorporates break values obtained by studying susceptible and resistant bacteria within the population of the particular species being examined (see Chapter 2). Types and Mechanisms of Resistance RE•wstance is classified os either Intriinsic (constitutive) or acquired. Intrinsic Resistance Bacteria nriv be mtnnsicallv or naturallv resistant to antibiotics because the organisms 1WE 27 ^actrriaceae to vancom% -cin and of Gram-positive bacteria to polymyxin 6 In addition, bacteria that are susceptible in vitro may be resistant in aiwo. For example, sometimes Gram-positive bacteria mav lose their cell ~sall and subseyuentlv peraist in the bodv as L forms, which makes them resistant to beta-lactam antibiotics. Acquired Resistance Acquired, genetically based resistance can arise because of chromoaomal mutation or, more importantly, through the acquisition of transferable genetic material. There are many mechanisms of acquired resistance, including activation of drug efflux pumps and induction of enzymes that degrade the antibacterial agent. Acquired resistance is not a problem in all bacterial species. Gram-positive bacteria, apart from staphylococci, often lack the ability to acquire R plasmids (resistance plasmids), so that their significanee as causes of disease in animals and people has, for a number of species, declined considerably since the antibiotic era started. Nevertheless, in recent years acquired resistance among Gram-positive bacteria has become increasingly problematic. In contrast to resistance in a few Gram-positive bacteria, resistance to many antibiotics is serious and important in Enterobacteriaceae and occurs increasingly in a broad range of other Gram-negative pathogens, such as Bordetetla, Haernophilvs, Pasfeurella, and Pseudomorras. Acquired resistance has been identified in most but not all pathogenic bacterial genera, as well as in the commensal flora. Two factors contribute to the seriousness of the problem once multiresistant organisms develop: they may persist in the host or the environment in the absence of antibiotic selection, and they may act as reservoirs of resistance genes that may spread to other bacteria. Bacteria that are resistant to one antibiotic are likelv to become resistant to other antibiotics for reasons that are not well understood but may relate to the presence of mutational defects in DNA mismatch repair mechanisms ("mutator phenotype"), making these strains more prone both to chromosomal mutation and to promiscuous exchange of DNA between species. Factors responsible for bacterial drug resistance ,~r'ta-iactam drugs is an exception to this generalization. The development of chromosomallv based resistance can be a major problem. For example, among the fluoroquinolones a series of small increments in resistance as a result of individual nucleotide mutations in different genes can quickly lead to inefficacy of the drug. Among the beta-lactamases, which cause resistance to beta-lactam drugs, minor differences in molecular structure mw° have dramatic differences in the function of the beta-lactamase en'vme. For example, a mutation producing a single nucleotide change can convert a TEM-7 to a TEM-12 beta-lactamase (see Chapter 6), and a second single nucleotide change can convert this TEM-12 enzyme into a T=M-26 beta-lactamase, with a dramatic increase in resistance to ceftazidime. Mutations are sometimes associated with other cell changes, so that these cells may be at a disadvantage compared with the parent and mav therefore be diluted out from the population in the absence of antibiotic selection. Some mutants, however, are as viable as the parents and are therefore inevitably selected by antibiotic use. Mutations leading to resistance may be dramatic, as in the case of streptomycin, whose minimum inhibitory concentration (MIC) can increase 1,000-fold with a single mutation, or they may be gradual, as in the development of multistep resistance to fluoroquinolones. The variation in mutational events is characteristic for each antibiotic; it occurs at high frequency for streptomycin, nalidixic acid, and riflotic fZ plasmirl mpper lett;, and the compositfon of a transposon upper right Tr.rnsposonc ore a central element in the fermaiion cih R plasmids. r )8i-11 TtiFRAt'Y IN VI FFRINARY MEDIC INF MULTIPLE ANTIBIOTIC RESISTANCE PLASMID retains copies of the plasmid, and the recipient now becomes a potential donor (Fig. 3.2). This transfer mav occur between bacterial strains of the same species, within species of the same genus, or even between species belonging to different families. For example, Stapiuflucoccus arrreus can exchange ~;enetic material with Errterocnccua. fneaatis or Esc/rcricf7ia coll. The genetic elements responsible for transfer of antibiotic resistance are the [Z (resistance) plasrnids, or R factors. Thev are present in bacteria as extrachrornosomal circular DNA, which replicates independentlv of, but svnchronouslv with, chromosomal DNA. Thev are relatively stably inherited but are not required by the bacterium for its surv=ival. They possess repcms with the resistance genes, which mav code for resistance to between 1 and If) different antibiotics (rumttiyle antibiotic resistance) and for the ability of bacteria to transfer genes by conjugation. Use of anv one antibiotic ~to which the plasmid encodes resistance will select for the maintenance of the entire plasmid. In some cases, however, these plasmids mav encode other attributes, such as the ability to colonize or resistance to heavy metals, which mav help ensure their maintenance in the absence of antibiotic selection. Some plasmids mav_ not have the genes required for conjugal transfer but can be mobilized to move by using the conjugal apparatus of other, self-transmissible 1>iasmids in the cell. Transposons. Short sequences of DNA known as transposons ("jumping genes") can transpose from plasmid to plasmid, from plasmid to chromosome, or vice versa. A transposon copy does not remain at the original site. The frequency of transposition is characteristic of the transposon and the bacterial strain. Many transposons (class 1) contain an antimicrobial drug-resistance gene flanked by two insertion sequences, which in turn are made of a central sequence with the genes for transposition flanked by short inverted repeats (Figs. 3.2 and 3.3). Other classes are more complex. The key property of all transposable elements is their ability to move and integrate into foreign DNA sequences by nonhomologous ("illegitimate") recombination. Transposons are readily acquired by plasmids and may readily incorporate into chromosomal DNA. Because of transposons, plasmids of very diverse origin possess identical antibiotic-resistance genes. The rapid transfer of transposons between plasmids in a cell and between chromosomes and plasmids, together with the interbacterial transfer of plasmids, can result in the rapid development of antibiotic resistance within bacterial populations. The phenomenon of nonhomologous recombination means that the same transposon may be found in the genome or plasmids of highly unrelated organisms. In addition, certain conjugative transposons mav excise from DNA to form a circular transfer form, which, like plasrnids, carries the genes needed for conjugation between bacteria. Integrons. lntegrons are a class of mobile genetic element often found on plasmids and are distinct from transposons and insertion sequences. They are associated with antibiotic resistance and other changes in bacteria. An integron is a site-specific recombination system containing an integrase (recombinaae) enwme, a gene-capture (attachment) site, and a captured gene or genes. The csptured genes occur as mobile gene cassettes, a unique familv of small mobile elements that include only a single antibiotic resistance gene and a specific recombination site. The recombination site confers mobility because it is recognized by site-specific integrases, which eatalvze integration of the cassettes at i / ANTIMICROBIAL DRUG RESISTANCE 35 Cross-resistance one antibiotic, thereby becomes resistant to another. The classic example is the aminoglycusides, in which chromosomal resistance to a newer drug such as gentamicin is associated xvith resistance to older drugs such as neomycin. Cross-resistance is common among the macrolides and t7uoroquinolcmes. Mechanisms of Resistance Important mechanisms of resistance include (1) enzymatic inactivation or modification of antibiotics; (2') impermeabilitv of the bacterial cell wall or membrane; (3) active expulsion of the drug bv the cell efflux pump; and (4) alteration in target receptors. The mechanisms of resistance to individual antimicrobial drugs, which mav include combinations of the mechanisms listed, are discussed in the chapters describing these drugs. Although most of the mechanisms listed are specific for individual drugs, nonspecific resistance to a wide range of structurally unrelated antibiotics has been described in several common pathogens associated with mutations leading to overexpression of the multi-antibiotic resistance (Mar) locus, which controls multidrug efflux pumps in bacteria. Mar mutations can be selected bv low concentrations of an antimicrobial drug; the clinical importance of Mar mutations leading to broad -spectrum resistance remains, however, to be determined. Extent of Antibiotic Resistance A causal relationship has been shown between antimicrobial use and the development of resistance. This selection is bacterial species specific, so that some species have remained highly susceptible for decades while others have rapidlv become resistant and highly susceptible emerged prominently as pathogens. In veterinary medicine, the tendencv has been to focus cm the well-documented development of resistance in certain Snlmnru•lla strains, which cause zocmotic infection, and in other enteric bacteria, especially Esclmrlchio coli, in part because the intestine is the major site of antibiotic resistance transfer. Relativelv little svstematic studv has been done cm the development of resistance in nonenteric and opportunist pathogens in animals. Antibiotic resistance in opportunist pathogens is a major problem in human hospital practice, but there are few reports of such problems in veterinary hospitals. Resistance in Intestinal E. coli Extensive studv of antimicrobial resistance in intestinal E. cnll has provided information on the mechanisms and ocolop, of such resistance (Hinton et al., 1986). Studies of E. coli isolated from different animal species showed the relationship between the degree of antimicrobial drug use and the extent of resistance (I_inton, 1977). Resistance is extensive in animals kept under intensive conditions where antibiotics are in common use (pigs, broiler chickens). Large numbers of these normal intestinal E. coli show resistance, mostly plasmid mediated, in some cases to as manv as 10 clinicallv useful antibiotics. Although resistant isolates are not more virulent than nonresistant strains, some reports note the acquisition of virulence genes (LT-toxin, ST-toxin) bv fZ plasmids. In England; Smith (1975) recorded the increased resistance of intestinal E. cofi of animal origin that occurred over the vears as a result of the widespread use of antimicrobial drugs in animals. This was first evident in 1957 when Smith showed that feeding rations containing tetracyclines to pigs and poultry resulted in the recovery from their feces of large numbers of tetracvcline-resistant E. coli. The increase in resistance to commonly used antibiotics between 1956 and 1980 was striking. It seems that over the years the tetracycline-resistant E. coh have become increasingly able to compete with sensitive bacteria in the intestine. The linkage of resistance genes on the same plasmid means that the use of any one antibiotic for which resistance was determined bv the plasmid promotes continued resistance to all the antibiotics. Withdrawing the use of all antibiotics in a herd may not result in the loss of resistance by E. coli, because such genes, which are on transposons, mav be incorporated into the bacterial chromosome nnd because possession of R plasmids may not be deleterious to bacterial survival in the intestine. 'In some cases it may even promote colonization. Transfer of R Plasmids in the Intestine In a test tube, drug resistance can be transferred rapidly throughout a susceptible bacterial population, but the frequency of transfer in vivo is lower. Within a short time of treatment of an animal with an antibiotic, the commensal E. coli population becomes resistant to that drug. This mainly results from the selection ot resistant organisms and only to a lesser extent from the transfer of resistance. In general, conditions in the large bowel do not favor the transfer of resistance plasmids. The persistence of resistant bacteria is related to the persistence of the antibiotic. Thus in a cow treated for mastitis with short-term administration of antibiotics, resistant E. coli do not persist long in the intestine. It is the prolonged use of antibiotics that is more likelv to he nssociated with persistence of resistant organisms even after the drug na no longer administered. The persistence of IZ hlasmids is generallv o tunction of the bacterial strain, not the plasmlds. I he majority of 1Z plasmid-containing E. coli are not good intestinal mlommers, but the persistent presence of an antibiotic which it is particularlv pathogenic, and from a wide variety of their contacts, including humans. -T he strain appears to have originated in England but has spread to and become common on most continents. Like phage types 29 and 204, it is unusual in that multiple antibiotic resistance is a characteristic of the clone, but unlike the other two phage types, the genes encoding at least some of the multiple resistance have become chromosomally integrated, probably through the activity of an integron. This organism is an example of the association between therapeutic antibiotic use in animals and the development of resistance in bacteria that can cause serious illness in people. Hospital-acquired Infections by Resistant Bacteria Nosocomial infection by multiple-resistant resident bacteria is a major problem in human medicine but has been little studied in veterinary hospitals, although it undoubtedly occurs. Analogy to the ecology of resistance in E. coli and 5alrnormlln in farm animals is useful. There is a causal relationship between the use of antibiotics in hospitals and the selection of resistant pathogens. Colonization of patients by resistant, opportunist bacteria is hard to prevent because of shared air spaces, environment, utensils, and nursing staff; the presence of bacteria that colonize patients easily; and the use of antibiotics that destroy the normally protective bacterial flora of the patient. In addition, the ability of R plasmids SENSITIVE STRAINS p DRUG-RESISTANT STRAINS --1 APPEARANCE OF RESPECTIVE RESISTANCE PATTERNS 3 / ANTIMICROBIAL DRUG RESISTANCE to transfer across bacterial genera is a major cause of the development of resistant bacteria in hospitals. Antibiotic Resistance in Animal Pathogens and Human Health The effect of antimicrobial drug resistance in bacteria of animal origin on human health is the subject of prolonged, acrimonious, and ongoing debate. It is the biggest issue currently affecting antimicrobial drug use in animals. In particular, the focus has been on the unrestricted and often widespread use of antimicrobial drugs important in treating human infections for growth promotional and disease prophylactic ("subtherapeutic") purposes in food animals. The recent emergence of vancomycinresistant enterococci, of multiresistant S. typhimurinam DT104, and of fluoroquinolone-resistant Compylobacter has restimulated discussion of this important issue. Bacteria from animals may reach the human population by many routes (Fig. 3.5). Drug-resistant bacteria of animal origin, such as E. coli, ROUTES OF EXCHANGE OF RESISTANT ENTERIC BACTERIA Fig. 3.5. Koute's cA exchange ()f E. cnli hetween nnimal, anci humans. Note'ho are•os wherr antibiotic seleWicm mr re~IWanc e is mc,st likelv. After I Intwn I')-- , modified !n• K. Iwvin reprrxlu, m1 with Cx•rmlssinn. there is a direct relationship between antimicrobial use and the selection of resistance among bacteria generally, the major approach to control of antimicrobial drug resistance is the reduction of use of these drugs. A number of approaches can be taken to limit the development and spread of antimicrobial drug resistance, but there is need to document the value of such approaches. Control of antibiotic resistance, insofar as it is possible, depends on the careful and appropriate use of antibiotics by knowledgeable veterinarians who cooperate with clinical microbiologists and laboratories of excellent standards in adopting measures to control the problem. Rational Use Antibiotics should be used onlv where a bacterial infection is known or suspected to be present. This determination can be either bv direct demonstration of the infection (Gram- or Wright-stained smear, poly merase chain reaction (PCR), culture) or from clinical data (eg, at least two of fever, leukocytosis, localized inflammation, components of the Wrightstained sample, radiographic evidence, elevated serum fibrinogen) (Hirsh, 1995). In human medicine, up to 70"%, of antibiotic use has been estimated to be either unnecessary or inappropriate. No similar assessments have been attempted in veterinarv medicine. Antibacterial drugs should be administered at therapeutic doses only for short periods, which for acute infections is usually for 48 hours after clinical cure; prolonged use selects for resistant strains that may persist. Selection of antibiotic resistance is more likely with suboptimal antimicrobial exposure, so full therapeutic dosage should be used. Where possible, narrow-spectrum drugs are preferred over broad-spectrum drugs. Drug combinations may overcome the development of chromosomal mutations to resistance or of plasmid-mediated resistance (eg, clavulanic acid or sulbactam resistance to beta-] actamases). Antibiotics should be used in prophvlaxis only where proven effective anJ usually for not more that 48 hours. National policies should ensure that antibiotics be available only bv prescriptions by veterinarians, who should be responsible for educating users about their proper use. There should, however, be no financial gain made bv the provider from prescribing or supplying antibiotics. Antibiotic use should be guided where necessary by use of susceptibilitv tests, and users should be aware of the errors possible in the performance and interpretation of these tests. Although antibiotic policies are often a source of conflict, a number of hospitals have developed policies in which antibiotics are divided into those,freely available for prescription, those used for special purposes, and those whose use is allowed only after agreement by infectious dis-ease specialists. Such policies have been developed in hurnan medicine in collaboration with hospitals' antimicrobial drug susceptibilitv testing. I'o prevent antibiotic resistance from spreading in some human hospitals, strategies of rotating their use have been advocated. For example, one or more antibiotics would not be used for a vear or two, and unrelated drugs would be administered instead. The effectiveness of such strategies in their application into veterinary use requires confirmation. Decreased use or withdrawal of certain drugs from human hospitals has been followed sometimes b_v dramatic reductions in resistance to these and other antibiotics. Reduction of Spread of Resistance Hygiene and management approaches should aim to limit the spread of antibiotic resistance, where this is a problem. For example, isolating sick animals prevents transmission of bacteria that may be resistant. Because many diseases are self-limiting, hygienic and management measures to prevent spread of bacteria during infection are important. Approaches other than antibacterial use to control infections, such as vaccines or management methods, should be favored over antibiotics. Surveillance Data on the prevalence of antimicrobial drug resistance in veterinary pathogens are generally fragmentary and unbalanced. There have been only varied and limited attempts to monitor drug resistance on national levels but no such attempts internationally. Agreement on standard methods for resistance testing and reporting on a global level are critical (see Chapter 2), although the data resulting from such studies may be difficult to interpret and to use. Resistance data from accredited clinical microbiological laboratories using common, internationallv agreed upon and qualitv-controlled methods and interpretive criteria could be collated and published annuallv bv a designated national organization. National and international surveillance schemes for monitoring resistance might include "sentinel" bacteria isolated from the intestine of healthy animals (eg, for food animals, enterococci, Snlrnon(°/la, C. je°jurri) in a systematic sampling process and tested in an internationally agreed upon manner. The total quantity of different types of antibiotics used in a country should be determined and made public cm an annual basis bv a designated national organization. The availability of data of this tvpe will provide the evidence on which national and international policies for control of antibiotic resistance might be based. Conclusion The use of antibacterial drugs selects for resistant bacteria, and that selection tends to occur in stepwise increments. For reasons that are not fullv clear, resistant bacteria have a tendency to acquire multiple resistant mechanisms. Once a bacterium has acquired a resistance mechanism, it tends to retain it. The use of these drugs does not create resistance but rather eliminates the susceptible bacteria in the host and spares the resistant ones. Use of antibacterial drugs thus provides the selection force for the Darwinian process of natural selection. Only the "fittest," resistant bacteria survive antibacterial drugs. Indeed, antibacterial use over the last 50 years has significantly changed the frequency of different types of bacterial infections observed in animals and humans. The potential for mutation by bacteria and for genetic exchange between bacteria, combined with the short generation time of bacteria, can rapidly produce resistant populations, which will be selected by the use of antibacterial drugs, although this selection effect is often bacterial species-specific and is not inevitable. The development of resistance has commonly followed the introduction of new antibacterial drugs (see Fig. 1.1). Increasing resistance to commonl_v used antibiotics in common Cram-positive aerobic pathogens of medical interest has led to the apparent crisis of antibiotic resistance in human medicine, with important implications for use of certain antibacterial drugs in animals, as described later. Bibliography Aarestrup FM, et al. IA9H. Sum°eillance of antimicrobial resistance in bacteria isolated from food animals to antimicrobial growth promoters and related therapeutic agents in Denmark. A['MIS 106:(,tlh. Anderson ES. 1968. 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Ditterenres in the occurrence of two base pan- variants of Tn1S46 From wnrumvcin-re,rsWnt e,ntercxocci from humans, pigs, and poultry. Antimicrob Agents Chumothc•r k2:2-kbs lensen I-B, et al IQ"') \%ancumvuin-resistant EntrrncnirnI timciurrr strains with highlv wmilar pulsesl-tu•IH gel clectrciphoresis patterns wontaining similar Tn1S}(;-like elements iaulated tnmv o hospitalrzed patlOnt md ln,r;~ in Denmark. Antimicrob Agents Chemmthc•n 1 • "'4 Joint Committee on the ( 'e ~)t Antibiotics m Animal Iiu>handrv and Veterinarv Medicine (Sw'ann RepurU Ivr,v 1 amdnn: }ler Maje,tc s statiunerv Office -\NTLMI( K()RLAt TIiFRAPY 11 VFTFKINARY MIDI( IN[