Contents

Stuart B. Levy

Abstract. Antibiotic resistance thwarts the treatment of infectious diseases worldwide. Although a number of factors can be identified which contribute to the problem, clearly the antibiotic as a selective agent and the resistance gene as the vehicle of resistance are the two most important, making up a 'drug resistance equation'. Both are needed in order for a clinical problem to arise. Given sufficient time and quantity of antibiotic, drug resistance will eventually appear. But a public health problem is not inevitable if the two components of the drug resistance equation are kept in check. Enhancing the emergence of resistance is the ease by which resistance determinants and resistant bacteria can spread locally and globally, selected by widespread use of the same antibiotics in people, animal husbandry and agriculture. Antibiotics are societal drugs. Each individual use contributes to the sum total of society's antibiotic exposure. In a broader sense, the resistance problem is ecological. In the framework of natural competition between susceptible and resistant bacteria, antibiotic use has encouraged growth of the resistant strains, leading to an imbalance in prior relationships between susceptible and resistant bacteria. To restore efficacy to earlier antibiotics and to maintain the success of new antibiotics that are introduced, we need to use antibiotics in a way which assures an ecological balance that favours the predominance of susceptible bacterial flora.

Julian E. Davies

Abstract. Since the introduction of antibiotics in the late 1940s there has been an inexorable propagation of antibiotic resistance genes in bacterial pathogens (and their relatives). This survival phenomenon was first characterized as the appearance of point mutations that altered drug targets, but in the mid-1950s transmissible antibiotic resistance genes were reported in Japan. Since this time both resistance strategies have been used, often in concert. For some types of antibiotic, only resistance by mutation has been identified, for others only resistance by plasmid acquisition. There is conflicting evidence with respect to the presence of antibiotic resistance in bacterial pathogens in the 'pre-antibiotic' era; however, it is likely that the evolution of antibiotic resistance occurred over short periods. Thus, antibiotic resistance genes must be common in the environment, but their derivation remains to be established conclusively. This paper examines the proposals that antibiotic resistance genes originated in the bacterial population, either as bona fide resistance genes or genes encoding metabolic functions. In addition, the acquisition of heterologous resistance determinants by different genetic elements, their intergeneric exchange mechanisms, and the possible roles of antibiotics in these processes are discussed. Are there prospects for drug intervention that eliminate or retard these natural evolutionary processes?

Pentti Huovinen, Helena Seppälä, Janne Kataja, Timo Klaukka and the Finnish Study Group for Antimicrobial Resistance

Abstract. Because the discovery of new antimicrobial agents cannot be expected in the near future, we will have to manage with the antimicrobials currently available at least for the next decade or two. Therefore, attempts to prevent development of antimicrobial resistance are of major importance. The relationship of local antimicrobial consumption and antimicrobial resistance has been shown in many hospital studies but not in the community, even though this is where most antibiotics are used. At the beginning of 1990s, erythromycin resistance in group A streptococci increased rapidly in Finland. The geographical variations found led to a nationwide study of the possible relation between local erythromycin consumption and variations in erythromycin resistance in the community. Erythromycin resistance was found to be significantly (P=0.006) linked to local consumption of erythromycin. In further experiments, we found that a new erythromycin resistance phenotype belonging to the T4 serotype was spread over the whole country; 83% of the erythromycin-resistant isolates were of this new phenotype in 1994. In 1991, recommendations were given to reduce use of erythromycin in Finland. Following these recommendations, macrolide consumption decreased by 40% from 1991–1994. Studies are now in progress to evaluate the effect of this reduction on erythromycin resistance of group A streptococci.

Abstract. Abundant evidence suggests a relationship between antibiotic resistance and use, including animal models, consistent associations between resistance and antibiotic use in hospitals, concomitant variation in resistance as antibiotic use varies, and a dose–response relationship for many pathogen/antibiotic combinations. Much of the evidence has come from studies performed in single hospitals. Most multicentre studies on resistance have not included data on antibiotic usage. Despite this substantial body of evidence, some studies have failed to demonstrate an association between antibiotic resistance and use, suggesting other contributing factors such as cross-transmission, inter-hospital transfer of resistance, a community contribution to resistance, or a complex relationship between resistance and the use of a variety of antibiotics. A multicentre study, project ICARE (Intensive Care Antimicrobial Resistance Epidemiology), implemented in 1994 by Centers for Disease Control and Prevention and Rollins School of Public Health, Emory University, has found dramatic differences in the patterns of antibiotic usage and resistance in US hospitals. The findings suggest that antibiotic usage is the major risk factor in development of antibiotic resistance in hospitals but the relationship can be complex with additional factors involved. Understanding the problem of antibiotic resistance in a hospital cannot be achieved without knowledge of the hospital's pattern of antibiotic use.

Abstract. In Greece, antibiotic over-consumption and high resistance rates run in parallel. In the spring of 1989 surveillance of 12500 Gram-negative strains, derived from 55 hospitals from all over Greece, revealed that resistance rates of Pseudomonas aeruginosa, Enterobacter spp., Klebsiella spp. and Acinetobacter spp. to antimicrobial agents introduced after 1985 exceeded 50%. As a consequence, the application of (1) rules of hospital hygiene, (2) educational small group programs, and (3) an antibiotic policy aiming to restrict antibiotic use, was decided in Laiko General Hospital. Since 1989, imipenem, the newer quinolones, vancomycin, aztreonam and third-generation cephalosporins were only ordered by the hospital pharmacy after completion of a specific request form, which since 1991 has been more detailed and which can be signed only by physicians with interest in infectious diseases. In 1991, in cooperation with the pharmacy, an audit program was added requiring a final inspection of the already approved request forms by an infectious diseases specialist. Any disagreement was discussed with the physicians in charge. Consumption data were analysed monthly and discussed with each department. Newer antibiotic consumption in a selected month (November) of three consecutive years, before (1991) and after the application of the audit program (1992–1995) has been analysed. Results reveal a decrease in consumption of restricted antibiotics, especially in surgical departments and in kidney transplantation units, without simultaneous increase in consumption of the non-restricted compounds. Since 1994, resistance has decreased remarkably. However, the resistance of quinolones is increasing steeply. Consequently, for the last 12 months quinolones have been removed from the hospital formulary. An audit program requires close co-operation of physicians, pharmacists and, particularly, of surgeons, in the application of a correct prophylaxis regimen. It seems to be efficacious in reducing both resistance rates and total antibiotic consumption.

Abstract. With the exception of flavomycin and olaquindox, the antibiotics currently used in European countries as feed additives exert a Gram-positive spectrum of activity. Of these, tylosin and virginiamycin are known for cross-resistance to macrolides, lincosamidines and streptogramines, and avoparcin is known for cross-resistance to vancomycin and teicoplanin. The use of avoparcin in animal husbandry creates a potential reservoir of transferable, vanA-mediated glycopeptide resistance in enterococci. A study in a rural area in Germany where vancomycin-resistant enterococci (VRE) were not isolated from infected humans but found in animal husbandry has shown that VRE are disseminated via meat products and are also found in faecal samples of non-hospitalized humans. VRE of different ecological origin from Germany (hospitals, sewage, food, animal husbandry) are polyclonal as evidenced by macrorestriction patterns and multilocus enzyme electrophoresis, suggesting a wide dissemination of the vanA gene cluster. These results confirm earlier observations on the spread of the sat genes, which confer resistance to a streptothricin antibiotic which has only been used in animal feeding. The resistance determinants were later also found in Escherichia coli from human infections and had spread in the absence of selective pressure.

Abstract. The biochemistry and genetics of antibiotic resistance are far better known than the equally important events underlying the selection of resistant populations. The hidden selection of low-level resistant variants may be a key process in the emergence of high-level antibiotic resistance. Different low-level resistant bacterial subpopulations may be specifically selected by different low antibiotic concentrations. The space in the environment (human body) where a given selective concentration exists represents the selective compartment. For pharmacokinetic reasons, low antibiotic concentrations occur in a larger selective compartment and persist longer than high antibiotic concentrations. The specific selection of low-level variants by low concentrations of antibiotic can be reproduced in experimental in vitro models using mixtures of susceptible and low-level resistant populations. We demonstrated this in Escherichia coli strains harbouring TEM-1, TEM-12 and TEM-10 (-lactamases challenged by cefotaxime, and also Streptococcus pneumoniae strains with various levels of penicillin resistance challenged by amoxicillin or cefotaxime. In both cases, four hours of antibiotic challenge produced selective peaks of low-level resistant variant populations at low-level antibiotic concentrations. We conclude that variants with small decreases in antibiotic susceptibility may be fully selectable under in vivo circumstances; on the other hand, low-level antibiotic concentrations may have a considerable selective effect on the emergence of antibiotic resistance.

Abstract. For tuberculosis and a number of other bacterial infections, treatment with a single antimicrobial drug frequently fails due to the ascent of mutants resistant to that drug. To minimize the likelihood of this occurrence, multiple drugs with independent resistance mechanisms are used simultaneously. None the less, multiply resistant bacteria sometimes emerge even when patients are simultaneously treated with two or more drugs, and the ascent of these multiply resistant mutants may result in treatment failure in the patient and spread of these resistant bacteria to other hosts. We consider two mathematical models of antibacterial chemotherapy which can account for the ascent of multiple antibiotic resistance within hosts treated with multiple antibiotics. In both, multiple resistance evolves because of selection favouring mutants resistant to fewer than all of the chemotherapeutic agents employed, intermediates. In one model, this occurs because of temporal fluctuations in the concentrations of the antibiotics in the course of normal treatment and/or because of non-adherence to the treatment regime. In the other, intermediates are favoured and multiple resistance evolves because of tissue and somatic cell heterogeneity in the effective concentrations of the antibiotics and physiological variation in the sensitivity of subpopulations of bacteria to different antibiotics. We discuss the limitations (and assets) of this model and approach and the implications for the design of antibiotic treatment regimes. Finally, we consider how the assumptions behind this model and the predictions made from its analysis could be tested experimentally.

Abstract. The possession of an antibiotic resistance gene clearly benefits a bacterium when the corresponding antibiotic is present. But does the resistant bacterium suffer a cost of resistance (i.e. a reduction in fitness) when the antibiotic is absent? If so, then one strategy to control the spread of resistance would be to suspend the use of a particular antibiotic until resistant genotypes declined to low frequency. Numerous studies have indeed shown that resistant genotypes are less fit than their sensitive counterparts in the absence of antibiotic, indicating a cost of resistance. But there is an important caveat: these studies have put antibiotic resistance genes into naïve bacteria, which have no evolutionary history of association with the resistance genes. An important question, therefore, is whether bacteria can overcome the cost of resistance by evolving adaptations that counteract the harmful side-effects of resistance genes. In fact, several experiments have shown that the cost of antibiotic resistance may be substantially diminished, even eliminated, by evolutionary changes in bacteria over rather short periods of time. As a consequence of this adaptation of bacteria to their resistance genes, it becomes increasingly difficult to eliminate resistant genotypes simply by suspending the use of antibiotics.

Abstract. Beta-lactamases, the enzymes often associated with resistance to beta-lactam antibiotics, are found in most bacterial species. Although these enzymes protected bacteria from naturally occurring beta-lactams long before the introduction of synthetic antimicrobial agents, the numbers and varieties of beta-lactamases have increased dramatically with the introduction of modern penicillins and cephalosporins. Over the past twenty years it has become apparent that families of beta-lactamases have been selected as the result of antimicrobial usage. Outbreaks of beta-lactam-resistant bacteria can be traced to the introduction of specific classes of beta-lactams or to the introduction of a specific agent. Many of the most serious epidemics can be related to transferable beta-lactamase genes that are harboured on multi-drug-resistant plasmids. The separation of beta-lactamases into three major functional groups or four structural classes has been proposed. Stepwise selection of variants within several of these classes has been documented both in the clinical setting and in the laboratory, e.g. the extended-spectrum (TEM and SHV) beta-lactamases and the inhibitor-resistant (TEM) beta-lactamases. Close relationships among the recently described plasmid-mediated 'cephamycinases' and the common chromosomal cephalosporinases have been identified. Carbapenem-hydrolysing metallo-beta-lactamases with broad spectrum hydrolyzing activity have become serious concerns as they begin to be described on plasmids. Factors contributing to selection of beta-lactam-resistant strains include decreased outer membrane permeability and increased beta-lactamase production.

Abstract. Methicillin-resistant Staphylococcus aureus (MRSA) is an intractable nosocomial pathogen. The chemotherapeutic intransigence of this organism stems from its predilection to antimicrobial resistance as a consequential response to selective pressures prevailing in the clinical environment. MRSA isolates are frequently resistant to all practicable antimicrobials except the glycopeptide, vancomycin. Although antimicrobial resistance sometimes arises via chromosomal mutation, the emergence of multiply-antibiotic-resistant staphylococci is primarily due to the acquisition of pre-existent resistance genes; such determinants can be encoded chromosomally or by plasmids and are often associated with transposons or insertion sequences. Clinical staphylococci commonly carry one or more plasmids, ranging from small replicons that are phenotypically cryptic or contain only a single resistance gene, to larger episomes that possess several such determinants and sometimes additionally encode systems that mediate their own conjugative transmission and the mobilization of other plasmids. The detection of closely related plasmids, elements and/or genes in other hosts, including coagulase-negative staphylococci and enterococci, attests to interspecific and intergeneric genetic exchange facilitated by mobile genetic elements and DNA transfer mechanisms. The extended genetic reservoir accessible to staphylococci afforded by such horizontal gene flux is fundamental to the acquisition, maintenance and dissemination of staphylococcal antimicrobial resistance in general, and multiresistance in particular.

Abstract. In Gram-negative pathogens, multiple antibiotic resistance is common and many of the known resistance genes are contained in mobile gene cassettes. Cassettes can be integrated into or deleted from their receptor elements, the integrons, or infrequently may be integrated at other locations via site-specific recombination catalysed by an integron-encoded recombinase. As a consequence, arrays of several different amtibiotic resistance genes can be created. Over 40 gene cassettes and three distinct classes of integrons have been identified to date. Cassette-associated genes conferring resistance to beta-lactams, aminoglycosides, trimethoprim, chloramphenicol, streptothricin and quaternary ammonium compounds used as antiseptics and disenfectants have been found. In addition, most members of the commonest family of integrons (class 1) include a sulfonamide resistance determinant in the backbone structure. Integrons are themselves translocatable, though most are defective transposon derivatives. Integron movement allows transfer of the cassette-associated resistance genes from one replicon or another into another active transposon which facilitates spread of integrons that are transposition defective. Horizontal transfer of the resistance genes can be achieved when an integron containing one or more such genes is incorporated into a broad-host-range plasmid. Likewise, single cassettes integrated at secondary sites in a broad-host-range plasmid can also move across species boundaries.

Abstract. Since 1953, tetracycline-resistant bacteria have been found increasingly in humans, animals, food, and the environment. Tetracycline resistance is normally due to the acquisition of new genes and is primarily due to either energy-dependent efflux of tetracycline or protection of the ribosomes from its action. Gram-negative efflux genes are frequently associated with conjugative plasmids, whereas Gram-positive efflux genes are often found on small mobilizable plasmids or in the chromosome. The ribosomal protection genes are generally associated with conjugative transposons which have a preference for the chromosome. Recently, tetracycline resistance genes have been found in the genera Mycobacterium, Nocardia, Streptomyces and Treponema. The Tet M determinant codes for a ribosomal protection protein which can be found in Gram-positive, Gram-negative, cell-wall-free, aerobic, anaerobic, pathogenic, opportunistic and normal flora species. This promiscuous nature may be correlated with its location on a conjugative transposon and its ability to cross most biochemical and physical barriers found in bacteria. The Tet B efflux determinant is unlike other efflux genes because it confers resistance to tetracycline, doxcycline and minocycline and has the widest host range of all Gram-negative efflux determinants. We have hypothesized that mobility and the environment of the bacteria may help influence the ultimate host range of specific tet genes. If we are to reverse the trend towards increasingly antibiotic-resistant pathogenic bacteria, we will need to change how antibiotics are used in both human and animal health as well as food production.

Mitchell L. Cohen

Abstract. Antimicrobial resistance is becoming an important public health problem for both hospital- and community-acquired infections. In the hospital, infections caused by drug-resistant Staphylococcus aureus, Mycobacterium tuberculosis, enterococci, and a variety of gram-negative rods are resulting in increased morbidity, mortality and costs, in part because of prolonged hospitalization and the use of more expensive antimicrobial agents. Drug-resistant, community-acquired infections are also causing important problems in both the developed and the developing world. Although the relative importance of specific pathogens varies with the geographical area, community-acquired pathogens including Salmonella, Shigella, Neisseria gonorrhoeae, Haemophilus influenzae and Streptococcus pneumoniae are causing both sporadic cases and outbreaks of drug-resistant illness. The emergence of antimicrobial resistance is being attributed to a series of societal, technological, environmental and microbial changes. These include increasing populations of susceptible hosts, international travel and commerce, changes in technology and industry, microbial adaptation and change, and the breakdown of public health measures. Addressing emerging problems and antimicrobial resistance will require enhanced surveillance, prudent use of existing antimicrobial drugs, development of new antimicrobial agents, increased emphasis on infection control and hygienic practices, effective disease control programs, better use of existing vaccines, and development of more and better vaccines.