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APPENDIX I INFECTIOUS DISEASE: EFFECT OF ANT IMICROBI~S ON BACTERIAL POPULATIONS Thomas F. O'Brienl EPIDEMIOLOGY OF ANTIBIOTIC RESISTANCE We have known for a century that germs cause infection. For a third of a century we have had antibiotics to control infection and molecular biology to explain, ultimately, how the process works. We can now see each strain of bacteria as a specific collection of genes. Some specify the life cycle. Some code for markers we find convenient for speciation. Others code for virulence factors or for enzymes that inacti- vate antibiotics. The specific linkage of genes in a bacterial strain is not fixed. We know increasingly more about the ways in which bac- teria can lose, gain, or exchange genes in experiments and, pre- sumably, in nature to fill virtually every habitat (Artier, 197 9~. However, there have been few measurements of actual rates of bacterial gene reassociation in natural environments (O'Brien et al., 19773. Such measurements are of critical importance because cost-benefit estimates of the behavior of bacterial populations may depend ultimately on their rates of gene reassociation. We know how most antibiotics work (Kucers and Bennett, 1975~. Each binds to and inactivates a critical specific target site in the bacterial cell. The discovery of each new family of antibio- tics--penicillins, streptomycin, tetracyclines, chloramphenicol, etc.--proved in retrospect, to have also been the discovery of a new target site. Unfortunately, few new classes of antibiotics are being discovered. The last discovery of an antibiotic with a truly new target site may have been rifampicin, which was first marketed 15 years ago (Hartmann _ al., 1967~. Most of the newer antibiotics circumvent resistance mechanisms to reach old target sites (Christensen et al., 1979~. This declining rate of discovery of new target sites for antibiotics suggests that these sites are, like fossil fuels, another unreplenishable resource. Director, Microbiology Laboratory, Peter Bent Brigham Hospital Boston, Mass. 275

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276 Also, it costs increasingly more to reach those target sites. If it were not for the prevalence of intervening resistance mecha- nisms, the less expensive, older agents could continue to be used. These resistance mechanisms force us to use succeeding waves of newer antibiotics that are expensive to develop and promote. These costly products now account for most of the antibiotic dollar volume which is, in turn, a large part of the pharmaceutical dollar volume. Mutational and "Gained" Resistance Our understanding of bacterial resistance to antibiotics has improved over the past two decades. Early concepts were based on studies conducted with an _ -vitro model of acquired resistance to streptomycin. These studies showed that a mutation altered the target site in such a way that it still functioned enzymatically, but that the antibiotic could no longer bind to it. Thus, suscept- ibility was lost. We now realize that it is far more common for resistance to be gained, i.e., the target site remains unchanged, as vulnerable as ever, but that the bacterial cell acquires some- thing like an antibiotic-inactivating enzyme or a permeability barrier that prevents the antibiotic from getting near the target site (Davies, 1979~. We can imagine and have begun to observe the implications of "gained" resistance being more common than lost susceptibility. Mutational loss of susceptibility to an antibiotic might be expected to occur at a measurable rate. So should the subsequent overgrowth replacement of susceptible strains by the mutant-resistant clone in populations exposed to the antibiotic. Consequently, for the muta- tional loss-of-susceptibility model, the relationship between number of bacteria exposed to an antibiotic and the prevalence of antibio- tic-resistant bacteria seems to be relatively simple with few inter- vening variables. It would also seem possible that this model could be used to estimate the prevalence of antibiotic resistance in one population of bacteria that is ascribable to the administration of antibiotics to another population, if the rate of interchange of bacteria between the two populations is known. Gained resistance is more complex. Usually, one of two types of genes is acqu~red--a gene for an enzyme, which inactivates the antibiotic or bypasses a blocked enzyme, or a gene for a protein, which impairs the antibiotic's entry into the cell (Davies, 1979~. These are large, specific molecules that are too big to arise often by chance mutation. Therefore, most strains of bacteria have to acquire such genes from other bacteria (Artier, 1979; Falkow, 1975~.

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277 Thus, when relating antibiotic usage to prevalence of resistance in a bacterial population one would first calculate the probabil- ity of the arrival of the resistance gene from another population instead of determining the mutation rate as with loss of suscepti- bility. Antibiotic Resistance Genes Origin and Evolution. The source of antibiotic resistance genes is not known. Some of them may have developed to protect antibiotic-producing organisms from their own products (Davies and Courvalin, 1977~. The 6-lactamases could have evolved if the active sites of transpeptidases of the bacterial cell wall, which selectively bind but do not hydrolyze penicillins, had begun to acquire a hydrolytic function (Crosa et al., 1977; Tipper and Strominger, 1965~. Techniques for characterizing products of resistance genes are so new, and surveillance so sketchy, that there has been little opportunity to distinguish evolution of a new gene from dissemination of a previously rare one. However, examples that must involve one or the other have been observed, e.g., the emergence of the newer aminoglycoside- inactivating enzymes (Davies and Courvalin, 1977; LeGoffic et al., 1977~. There are also opportunities for evolution towards improved efficacy for existing antibiotic-inactivating enzymes. For exam- ple, a modification of active site configuration might permit binding of an increased range of substrate (antibiotic) analogs as new ones come onto the market (LeGoffic _ al., 1979~. The R1818 6-lactamase, which produces only low-level resistance, could increase that resistance if it could evolve to have a higher sub- strate turnover number (Medeiros et al., 1974~. Evolution of an improved enzyme environment can also help. The TEM S-lactamase produces resistance to ampicillin that is much less dependent on inocula in Escherichia cold than in Haemophilus influenzas because the outer membrane of the latter is more permeable to ampicillin (Medeiros and O'Brien, 1975~. Dissemination. Whatever the origin and rates of evolution of antibiotic resistance genes, their transmission through bacterial populations is increasingly well understood. Clearly, there are multiple levels of genetic mobility and persistence. Many antibio- tic resistance genes, e.g., a majority of the known 6-lactamases of Gram-negative bacilli, have never been shown to be plasmid-borne and are presumably chromosomal (Sykes and Matthew, 1976~. Essentially,

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278 they can go only where their fixed-host bacteria can go, which depends on the specific colonizing and competing capabilities that are built into the other genes on the chromosome. An entirely different level of mobility is open to antibio- tic resistance genes that become located on plasmids, but the extent of the mobility depends greatly on the plasmid on which the gene is carried. Many plasmids are nonconjugative. They can transfer between bacteria only if "rescued" by a conjugative plas- mid (Mitsuhashi, 1979~. Others are conjugative and self-transfer- able but only at a limited rate or to a limited number of host bacteria. If transferred, they become unstable in many bacteria and are readily segregated. Moreover, plasmids cannot transfer to bacteria that already contain plasmids of the same incompatibility group (Falkow, 1975~. Thus, a resistance gene on a plasmid of a group already widely distributed in a bacterial population might achieve only limited dissemination in that population. The ultimate dissemination and persistence of a plasmid- borne resistance gene also depends on the other types of genes that are on the same plasmid. Many of the plasmids bearing re- sistance genes are large and have space for many other genes. Some of these other genes code for resistance to other antibiotic- The prevalence of any one of these resistance genes, and of the plasmid itself, is amplified whenever the plasmid's hosts are ex- posed to one of the antibiotics to which any of the other genes code resistance. A plasmid is essentially a gene cooperative in which an advantage accruing to any member gene is shared by all. In this sense, plasmids can be looked upon as devices that cause an increased prevalence of resistance to several antibiotics to result from the use of one antibiotic. Linkage to Genes for Other Characteristics. Some genes, which code for functions other than resistance to antibiotics, also amplify and sustain the prevalence of an antibiotic resist- ance gene that was associated with them on the same plasmid. Some plasmid-borne genes code for functions that permit host bacteria to survive in specific environments such as those containing specific metallic ions (McHugh et al., 1975; Pinney, 1977~. Others code for colonization (Silver and Mercer, 1978) or for metabolic functions, e.g., lactose catabolism (Kopecko et al., 1979), on which host bac- teria might depend either always or only under certain environmental conditions. The functions of these other genes, which occupy so large a part of so many plasmids bearing resistance genes, are just beginning to be elucidated. The glimpses of them to date, however,

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279 suggest that they may be major codeterminants of the ultimate distribution of resistance genes carried with them on the same plasmids (Novick, in press). The closeness and stability of the linkages between a particular antibiotic resistance gene and other resistance or nonresistance genes on the same plasmid also influence the pre- valence of that specific antibiotic resistance gene. The in- creasingly well understood mechanisms for genetic recombination serve not only to put genes together but also to disrupt and separate them, ultimately segregating genes that no longer pro- vide a survival advantage. Conforming to the analogy of the cooperative venture, genes that never bring any advantage are eventually dismissed, but the time of dismissal varies greatly. A gene coding for resistance to a rarely used antibiotic could persist on a plasmid for long periods during which it offers no survival advantage if it is closely and stably linked to another gene that provides an advantage. The close linkage of genes coding for resistance to streptomycin and sulfonamide on a com- monly encountered plasmid (van Treeck et al., 1979) could be part of the reason why the gene for resistance to streptomycin remains prevalent long after clinical usage of streptomycin in humans has declined. Transposons. The recently discovered transposons provide yet another level of genetic mobility. Transposons are segments of a DNA chain flanked by special insertion sequences. These transposons and genes carried on them are able to transpose from one plasmid to another or from plasmid to chromosome and back (Heffron _ al., 1975~. Location on a transposon could greatly enhance the prevalence of any gene. Genes coding for resistance to a number of commonly used antibiotics have been found on transposons (Campbell et al., 1977~. A resistance gene can be limited in its distribution by its position on the chromosome of, or on a nonconjugative plas- mid in, a poorly colonizing strain; on a plasmid of limited host range; or on a plasmid carrying few other genes with survival advantages. Such a gene could become more prevalent if it could be transposed to a more mobile plasmid carrying a more advantage- ous collection of genes, possibly including other resistance genes, or, better still, to a variety of such plasmids.

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280 Implications of the Resistance Mechanism The entire assemblage of these properties for any anti- biotic resistance gene, including the substrate range and the efficiency of its gene product, and its genetic location and mobility, can be regarded as an antibiotic resistance mechanism. Defined in this way, the mechanism can be viewed as a major var- iable in determining the prevalence of resistance to antibiotics. When a population of bacteria is exposed to an antibiotic, the extent and distribution of the resulting antibiotic resistance will depend largely on the properties of~the mechanisms of re- sistance to that antibiotic in that population. This can be best illustrated by considering some examples. If no mechanism for resistance to the antibiotic is available to the exposed population of bacteria, there will be no resist- ance. This situation prevailed for a number of years in popu- lations of Enterobacteriaceae exposed to gentamicin in the United States (O'Brien et al., 1978~. If the only mechanism available is a gene coding for an enzyme with a narrow specificity includ- ing only that antibiotic and if that gene is the only resistance gene on the chromosome of a bacterial species with poor colonizing and competing abilities, the ensuing resistance would be directed only against the antibiotic used and would be unlikely to persist. However, resistance would become widespread and persistent if the population contains a resistance mechanism consisting of a gene coding for an enzyme with a broad substrate range and if the gene is located on a transposon in a plasmid with a broad host range and is rich with closely linked genes for resistance to other antibiotics and for other survival advantages. Consider a bacterial population in a chemostat or in nature that includes one cell with a resistance mechanism and a similar population that includes one cell with a different mechanism. Each population could be exposed for the same period to an anti- biotic to which its mechanism produces resistance. Each popula- tion could then be scored for the total number of resistant cells times the total number of antibiotics to which each is resistant times the number of generations during which resistance persists. The values could differ greatly and would constitute for each mechanism an index of its efficiency at converting selection pressure into resistance prevalence. A different kind of index could acknowledge the heterogeneity of bacterial populations in nature. Such populations contain many different strains and species of bacteria coexisting, competing,

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281 and cooperating to occupy all possible niches in the ecosystem. For a resistance mechanism introduced in one strain in such a population, it would be possible to score the number of different strains and specimens into which the mechanism became incorporated after a controlled period of antibiotic exposure. Some mechanisms could remain only in the strain in which they were introduced. Such a strain would presumably overgrow its susceptible neighbors during exposure to the antibiotic but might not by itself be able to occupy all of the niches very fully. After the exposure to the antibiotic, the strain might promptly be displaced and revert to its natural domain in the population, which could be nonexistent. In the same population exposed for the same period to the same antibiotic, a different mechanism of resistance on a plasmid with a broad host range could be incorporated into many strains and species. Or a resistance gene on a transposon in a plasmid of limited host range might be transposed to one or more plasmids with a broader range of hosts and enter many strains and species by this route. In each strain and species the resistance gene would, in effect, be associated with a different assemblage of other genes. Therefore, this score would be an index of gene re- association and, to the extent that the selection pressure pro- motes it, antibiotic-induced gene reassociation. Two consequences of this process are worth examining. If many of the strains and species constituting the original bacter- ial population came to possess the resistance mechanism enabling them to survive exposure to an antibiotic, they would then pre- sumably be better able to fill all niches during the exposure than would a single strain. They would also be less rapidly displaced by susceptible species after the exposure ended. In the hypothe- tically perfect situation, all strains and species in the original population would receive the resistance mechanism so that exposure to antibiotics would not alter their ecological balance. In such a case there would be no tendency after the exposure had ended for reemerging or reentering susceptible species to displace resistant strains in the process of regaining their old niches. In this population, the prevalence of resistance after exposure would be diminished primarily by the segregation rates of the resistance mechanism from each of the strains, which may also be a variable characteristic of the resistance mechanism (Levin, in press) or, possibly, the normal influx of new strains colonizing and dis- placing the older members, although these new strains could also be susceptible to infection by a mobile plasmid widely resident in the population.

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282 Secondly, if a resistance mechanism has evolved to gain a great increment in prevalence per episode of exposure to antibio- tics and to have a high degree of interspecies mobility, it has a greater chance of ultimately getting into a pathogen. The bac- teria that actually infect humans or animals are only a miniscule part of the total bacterial flora of the world and, usually, of the infected individual as well. But they do all the damage. The arrival of a resistance mechanism in a pathogen can be a signifi- cant and tragic event. The recent arrival of the TEM S-lactamase (Medeiros and O'Brien, 1975) into group B Haemophilus influenzas, the commonest cause of bacterial meningitis in children,has led to treatment failure and to the need to treat all children thought to have the disease with a potentially toxic antibiotic, chlorampheni- col (Howard et al., 1978~. All of these considerations lead to one essential question: What is the source of the more efficient and more efficiently distributing resistance mechanisms? Everything suggests that the mechanisms themselves are the ultimate evolutionary products of the cumulative application of antibiotic selection pressures to bacterial populations. Exposure to antibiotics is the only imaginable stimulus that would provide evolutionary survival ad- vantage to antibiotic-inactivating enzymes or to other resistance genes prevalent enough to overcame the odds against their forming favorable linkages with one another or with other advantageous genes or eventually becoming located on plasmids or transposons. Summary The antibiotic resistance observed in a population of bac- teria after exposure to an antibiotic can be attributed to: 1. The net effect of all prior antibiotic exposure on the bacterial flora of this planet in shaping and delivering to this particular population the specific resistance mechanisms it has acquired. 2. The effect of those specific resistance mechanisms in determining the number and types of rests tent bacteria that will be generated by the present episode of exposure to the antibiotic.

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283 INTERCHANGE BETWEEN DIFFERENT BACTERIAL FLORAS Compartmentalization of Floras The preceding considerations have a very practical bearing on how we view the relationship of antibiotic usage in different compartments of the world's bacterial flora. These compartments could be defined as domains within which bacteria can be imagined to circulate more readily than they do between compartments. For example, the flora of each human being, the flora of an intensive care unit as opposed to the rest of the hospital, the flora of an entire hospital (O'Brien et al., 1975) as opposed to the community, or, on a larger scale, the flora of humans in the United States as opposed to that in humans in some other part of the world. Although compartmentalized, the bacterial flora of the world un- doubtedly circulates between all of compartments, but generally less than within them. 1 If the only concern with antibiotic resistance were quan- titative, the resistance in compartment A would change the re- sistance in compartment B only to the extent that bacteria from A moved into B. If A had 100% resistance and B by itself would have had only 10% resistance, yet 1Z of B's strains actually came from A, then they would add 1% resistance for a total of 11% resistance in B. If there are qualitative differences among resistance mecha- nisms, as discussed in the preceding section, then the influence of resistance in one compartment on that of another may be quite out of proportion to the circulation between them. Suppose com- partment A were to contain a mechanism of resistance still absent in B but ideally suited for circulation there because of its plas- mid incompatibility grouping, its special ability to inactivate an antibiotic widely used there, or its linkage to some other genes admirably suited for survival in B. Even if there were very little circulation between the compartments, a few bacteria with the mechanism could transfer from A to B. Once there, the mechanisms could become prevalent. Recent Observations - Recently, an apparent example of this has been observed with the use of gentamicin, which had been widely used since 1970 in

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284 the United States to treat hospitalized patients infected with Enterobacteriaceae. Through 1975, however, exceedingly few strains of Enterobacteriaceae isolated in the United States had any ability to resist that antibiotic (O'Brien et al., 1978~. For purposes of bacterial ecology, there was a great need for some mechanism of resistance, but apparently none, or none with appreciable genetic mobility, had become available to strains in the United States. In other parts of the world--in France, for example--there were genes for two aminoglycoside-inactivating enzymes (O'Brien _ al., 1978; Witchitz and Chabbert, 1972~. The 2 -aminoglycoside nucleotidyl transferase (AND 2 ~ and the 3-aminoglycoside acetyl transferase (AAC-3) were known to be widely distributed in nosocomial strains. In early 1976 a strain of Klebsiella pneumonias of serotype - 2 with an unusual biotype was isolated from a patient in one of the U.S. hospitals in our international collaborative study (O'Brien _ al., in press). This strain had a distinctive conju- gative plasmid of 56 Mdal belonging to incompatibility group M, which carried genes for resistance to sulfonamides as well as to chloramphenicol acetyl transferase, the TEM 1 6-lactamase, and the AND 2 enzyme, one of the two gentamicin resistance enzymes that are common in other parts of the world. Over the next 4 months this distinctive strain of K. pneu- moniae was isolated from 40 patients in the hospital. Thereafter, this strain was isolated only infrequently, but by then, the plas- mid was being found in isolates of other strains of K. penumoniae and in other species of Enterobacteriaceae as well (O'Brien et al., in press). Ultimately, this one plasmid was found in 49 different biotypes of six different bacterial strains. Restriction endonu- clease digestion analyses conducted on plasmids from Escherichia cold K-12 transconjugants of eight different strains of six different species confirmed the identity of the plasmid through- out the outbreak (O'Brien et al., in press). In this instance, a restrictive mechanism not previously circulating in one compartment (the hospital flora) and probably not circulating in a much larger one (flora of humans in the United States), but which circulated widely in another compartment for several years (European hospital flora) appeared in one hospital compartment and rapidly became prevalent. An almost identical se- quence in one other U.S. hospital has since been reported (Gerding _ al., 1979; Sadowski et al., 1979~. We are beginning to search files in microbiological laboratories in other U.S. hospitals for evidence of further spread.

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285 There is no proof that the mechanism was imported from out- side of the flora compartment of humans in the United States, although it had been prevalent there (O'Brien et al., 1978) and might have been expected to be imported if there had been any appreciable exchange between these compartments. Another item circumstantially suggesting such importation was the plasmid's carriage of the chloramphenicol resistance gene, which tends to be relatively rare in the flora of humans in the United States but common in flora outside that compartment. Proof of its gene- alogy will have to come from studies identifying plasmids on a larger scale, which are beginning to become practical. This experience illustrates that if there are qualitative r differences between resistance mechanisms, the influence of re- sistance in one bacterial flora compartment on that of another may be completely out of proportion to the rate of interchange between them. The compartment in which this particular complex mechanism had evolved could not have had great interchange with the compart- ment of flora in humans in the United States because only traces of it could be found there prior to the outbreak. Apparently, however, this particular mechanism was qualitatively so "fit" for this hospital flora compartment that once introduced it circulated widely and became prevalent. The consequences of the entry and circulation of this one plasmid in a hospital where it had previously been absent were substantial. The resistance of all Gram-negative bacillary iso- lates at the hospital to a set of six antibacterial agents (APAR index) (O'Brien et al., 1978) increased approximately 50% because of this plasmid. The number of isolates with resistance to more than six antibiotics increased nearly 10-fold. A number of cases of Gram-negative sepsis were virtually untreatable. Most of the resistant isolates came, as usual, from patients being treated or having recently been treated with antibiotics. Yet the level of antibiotic usage had not appeared to change appreciably. As dis- cussed previously, the entry and dissemination of a qualitatively different resistance mechanism had reset the equilibrium so that a given amount of antibiotic usage now generated a greater preva- lence of resistance to antibiotics. IMPLICATIONS TO HUMAN HEALTH FROM ANTIBIOTIC USE IN ANIMALS Exchange Between Compartments The considerations discussed in the preceding paragraphs generate concern about the veterinary use of antimicrobial agents.

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290 This problem is a very general obstacle to tracing the spread of resistance mechanisms in naturally occurring bacter- ial populations. Most common phenotypic resistance markers have been so widely distributed in most bacterial populations that their further exchange is extremely difficult to detect, let alone measure overall. Generally, we are able to detect and measure exchange only when a new marker enters a population as it did in the previously mentioned outbreaks, which involved a previously rare aminoglycos ice-inactivating enzyme (O'Brien et al., in press). Similarly, incursions of resistance without gene exchange is usually observed only when a previously rare type of resistance is involved, as with penicillinase-producing gono- cocci, ampicillin-resistant Haemophilus influenzas, methicillin- resistant staphylococci, and penicillin-resistant pneumococci. With regard to the dissemination of antibiotic resistance, it seems likely that only the tip of the iceberg has been observed. A corrolary to this is that improved discrimination of any component in this system--levels of antibiotic resistance, identi- fication of inactivating enzymes, identification of specific transposons or plasmids, or identification of specific biotypes or serotypes of host bacteria--will greatly increase our ability to find distinctive combinations that can be traced through bacterial populations. Every strain of bacteria would be distinctive and traceable if we knew enough about it. Despite these obstacles, some investigators have observed the spread of antibiotic resistance from the flora of animals to the flora of humans. For example, farm workers who work with anti- biotic-fed animals have in their flora a higher prevalence of anti- biotic-resistant, plasmid-containing bacteria than do other humans (Levy, 1978; Wiedemann and Knothe, 1971~. Moreover, multiresistant strains of salmonellae have been traced from infected animals to humans on many occasions (Anderson, 1968; Anonymous, 1979; Center for Disease Control, 1977~. These documented episodes of the flow of antibiotic resistance from the flora of animals to the flora of humans are important because they confirm, in spite of the obstacles, what molecular biologists have implied. In the near future, it should be possible to overcome some of these obstacles and to improve our ability to observe the spread of resistance, including that between compartments of flora, e.g., between animals and humans. Two new techniques should enable us to observe antibiotic resistance as an interrelated system throughout the world.

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291 One technique is computerized analysis of results of tests for antibiotic susceptibility. Every day, thousands of labora- tories in all parts of the world test the susceptibility of tens of thousands of clinical bacterial isolates. In the aggregate, these data, which have already been paid for, represent the work- ings of the antibiotic resistance exchange system. The problems of quality control, accurate sampling, data management, and ana- lytic methods have been examined (O'Brien et al., 1977, 1978), and an international collaborating group now representing 30 centers is pooling these types of data for continuing analysis (International Antibiotic Resistance Survey Group and O'Brien, 1978 and in press). In addition, other sets of susceptibility testing results, such as data observed from isolates of salmo- nellae from humans and animals (Figure 2) and a collection of test files from several hundred U.S. hospitals are being put into computers and similarly analyzed. These analyses provide a detailed view of the distribution of antibiotic resistance and its trends in time. Much of the contro- versy over any aspect of antibiotic use concerns the magnitude of the resistance problem and the direction it is taking. Information on both of these issues should be extractable from the data in the computer files and could be observed continually thereafter if mon- itoring is maintained. Moreover, this type of reconnaissance might serve as a starting point in the development of strategies to mini- mize resistance resulting from the use of antibiotics in humans or animals. Furthermore, these analyses should also provide unprecedented detail about the distribution of resistance mechanisms in the flora of humans in the United States. This is extremely impor- tant to this report because it should enhance the detection of the incursion of individual or a combination of resistance mechanisms into the flora of humans in the United States from another compart- ment. Previously noted incursions of this kind, e.g., penicilli- nase-producing gonococci from the Far East, have been observed, usually because they were extremely conspicuous. The more sensitive and selective are one's techniques for identifying resistance mech- anisms, the greater is one's ability to detect incursion of more subtly distinctive groups from the flora of humans in the United States or from the flora of animals. For example, it was possible to trace the entry and dissemination in the flora of a patient in a U.S. hospital of the AND 2 resistance gene, previously more common in isolates from humans in other countries, because in its early circulation it was linked with a grouping of other resistance genes virtually unique for that hospital (O'Brien et al., in press).

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294 Surveillance of the prevalence of antibiotic resistance mechanisms in the compartment of flora of humans outside of the United States Will continue to have an important bearing upon the issue surrounding the use of antibiotics in animal feed for two reasons. First, this is the major source other than animal flora of the importation of distinctive resistance mechanism- host combinations into flora of humans in the United States, and it has thus far provided the most conspicuous examples of such importation, including penicillin-resistant gonococci, penicillin-resistant pneumococci, chloramphenicol-resistant typhoid, methicillin-resistant staphylococci, etc. Therefore, knowledge of what is prevalent in human flora abroad could help greatly in deciding the likelihood that a particular mechanism observed in the flora of humans came from animals, rather than from humans from another country. Figure 2 shows that resistance to sulfonamide in salmonella isolates from humans in the United States could have come from animal sources or from foreign human sources, in both of which it appears to be highly prevalent. Examination of the serotype distribution of sulfonamide resistance in the three compartments might provide further evidence for exchange. In contrast, the low-level resistance to tetracycline (zone diameters of approxi- mately 10 mm) observed in several of the isolates of salmonella from humans in the United States appears more likely to be related to strains from animals, which have a high proportion of this type of resistance, than to isolates of salmonella from humans outside the United States, in which only a very small fraction of resist- ant isolates appear to be of this type. The second reason for the pertinence of foreign data is that different countries, in effect, have been testing grounds for a variety of antibiotic uses and food procurement and distri- bution practices, which have developed in them. The prevalence of various resistance mechanisms in the flora of humans from these different countries are presumably largely the consequences of these practices. Observation of these results could, therefore, be an indispensable guide to the development of optimal antibiotic usage policy. For example, the prevalence of resistance to chlor- amphenicol, presumably due to the chloramphenicol acetyl transfer- ase gene, was found to be more than 10 times more prevalent among isolates from humans in France than in the United States (O'Brien _ al., 1978~. The French investigator collaborating in this study believes that the use of chloramphenicol in humans is not excessive in France but that chloramphenicol is the antibiotic most widely used in food-source animals in that country. If this could be

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295 shown to be true, it could constitute a powerful argument for the influence of antibiotic use in food animals on the prevalence of antibiotic resistance in human flora. Similarly, resistance to streptomycin remains relatively com- mon in isolates of Enterobacteriaceae from humans in-the United States long after its use in treating humans has declined. This could represent fortuitous linkages of the streptomycin resistance gene, as discussed previously, but it might also reflect continuing use of streptomycin in livestock in the United States. The second resource for improved future observation of the antibiotic resistance system is the newer techniques for character- izing resistance mechanisms, ranging from the detailed characteri- zation of inactivating enzymes to analyses conducted by incubating the plasmid~s) with restriction endonucl~ases to establish the identity, diversity of, or to map, plasmids. The increasing avail- ability of these methods should improve discrimination of resistance mechanisms, which, as mentioned previously, should greatly enhance our ability to trace their spread in bacterial populations. These techniques may be particularly useful when coupled with computer survey of the large data bases. Discriminant analysis of these data bases may result in the identification of specific antibiotic- inactivating enzymes (Guzman, and O'Brien, 1978; Kent et al., in press). The computer analyses could indicate isolates of special interest for these laboratory studies and then project the results of the studies back to a large population.

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