Patterns and mechanisms of antibiotic resistance.

Symposium on Infectious Diseases

Patterns and Mechanisms of Antibiotic Resistance Barbara E. Murray, MD.* and Robert C. Moellering, Jr., MD.**

The development of effective antimicrobial agents ranks as one of the greatest achievements of modern science. However, these agents are not the panacea they once promised to be. All antimicrobial agents so far developed possess the potential to produce adverse reactions in the host. Likewise, the emergence of organisms which are resistant to its antimicrobial effects has plagued every new antibiotic thus far developed, in many instances limiting the utility of the drug. In our efforts to delineate the major factors involved in antibiotic resistance, we will first review the history of antibiotic use, then follow the subsequent development and spread of resistance, discussing some of the reasons for it, and finally describe some of the known and postulated biochemical events leading to this resistance.

EPIDEMIOLOGY AND EVOLUTION OF RESISTANCE TO ANTIMICROBIAL AGENTS Enteric Pathogens and the First Recognition of R-Factors

Sulfonamides, the first commercially marketed antibacterial agents, were introduced in the 1930's. In Japan, extensive use began in the early 1940's, and because of their initial effectiveness, sulfonamides were widely used against bacillary dysentery (shigellosis). The triumph over the shigellae, however, was short lived, and by 1950, 80 to 90 per cent of Japanese isolates of shigella were resistant to sulfonamides. 89 Fortunately, in the early 1950's chloramphenicol, streptomycin, and tetracycline became available in Japan and were widely and effectively used. Before long, however, shigella strains resistant to these "Research Fellow in Medicine, Harvard Medical School and Massachusetts General Hospital Boston, Massachusetts *'Associate Professor of Medicine, Harvard Medical School; Associate Physician and Consultant in Bacteriology, Massachusetts General Hospital, Boston Massachusetts Supported in part by Grant No. 5 Tol AIO0215-15 from the National Institutes of Allergy and infectious Disease.

Medical Clinics of North America - Vol. 62, No. 5, September 1978

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drugs also began to appear. A strain resistant to tetracycline, streptomycin, and chloramphenicol was isolated in 1952; in 1955, a strain resistant to these agents plus sulfonamides was found. 88 By the late 1950's, there were increasing reports in Japan of multiply-resistant shigellae; isolates of multiply resistant E. coli were also found, often from the same patient who had a multiply resistant strain of shigella. In 1959, Ochiai and Akiba independently reported the transfer of this quadruple resistance between shigella and E. coli. 34 Subsequently, Mitsuhashi and others established that transfer required cell to cell contact, was not mediated by the filterable agents such as phage or DNA, and was independent of chromosomal transmissibility.89 Thus, the concept of transferable, extrachromosomal elements that contain resistance genes was born. Soon, the term R-factor was adopted to describe this type of plasmid. A glossary of plasmid terminology is given in Table 1.60, 101 While the impact of R-factors was not fully realized in 1959, it was not long before it was. The Japanese continued to observe increasing resistance. By 1966, 65 to 75 per cent of shigella strains were quadruply (tetracycline, chloramphenicol, streptomycin, sulfonaInide) resistant; of the multiply resistant strains, from 60 to 90 per cent were capable of transferring this resistance to susceptible recipient organisms. Similar results were obtained in a survey of 12,453 shigella isolated in Japan from 1965 to 1973. 89,139 At this point, it should be emphasized that one cannot assume that plasmid-determined resistance was absent in all of those strains which did not transfer their resistance, since a significant number of R-determinants are nonconjugative. R-factors were not, unfortunately, limited to Japan. Multiply resistant shigella strains appeared and rapidly spread in Israel in 1956. 145 In 1962, Datta reported the transfer of resistance to streptomycin, sulfonamide s, and tetracycline from a strain of Salmonella typhimurium isolated during a 1959 outbreak of salmonellosis in a London hospital. 25 The first transferable resistance to neomycin-kanamycin was found in E. coli; it was accompanied by the transfer of resistance to tetracycline, streptomycin, sulfonamides, and chloramphenicol, presumably all on the same R-factor.88 In 1965, Anderson and Datta reported for the first time the finding of transmissible resistance to ampicillin; it was discovered in a strain of Salmonella typhimurium isolated in 1962.2 The resistance was shown to be mediated by the presence of a beta-Iactamase (penicillinase) which destroys penicillin.25a In 1965 a combination of ampicillin, neomycin, kanamycin, tetracycline, streptomycin, chloramphenicol, and sulfonamide resistance was reported from some of the organisms causing an outbreak of salmonellosis in France. 15 Resistance to kanamycin-neomycin and ampicillin was very rare in Japan, presumably because of the infrequent use of these antibiotics in that country. This observation illustrates the correlation between use of antibiotics and subsequent appearance of resistance, unfortunately a recurrent theme as one studies the epidemiology of resistance to antimicrobial agents.

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In Mexico and Central America, epidemic dysentery continues to pose major health problems. The very large outbreak of dysentery due to S. dysenteriae type 1 which began in 1968 and 1969 was initially caused by antibiotic-sensitive organisms. 44 As the epidemic progressed, the Shiga bacillus acquired an R-factor which mediated resistance to sulfonamides, streptomycin, tetracycline and chloramphenicoP4 Morbidity and mortality from this epidemic were particularly severe, partly because local health authorities were unaware that they were dealing with antibiotic-resistant shigella; as many as 12,500 deaths were recorded in Guatemala during the first year alone. 44 Although Americans traveling within Central America and Mexico have become infected, secondary spread from returning tourists has not been a problem in the United States. A 1972 outbreak of S. dysenteriae type 1 on a children's ward in Mexico City showed that the strain had now become resistant to ampicillin in addition to streptomycin, sulfonamides, tetracycline and chloramphenicol; 106 since ampicillin had been considered the drug of choice for severe dysentery, the finding of high-level resistance to ampicillin in these strains (and from strains in subsequent outbreaks in Central America and Bangladesh) has been of great concern. Studies of strains from all these areas have shown that resistance to ampicillin is on an identical 5.5 million dalton (MdaL) plasmid which codes for a beta-Iactamase designated TEM - the most common plasmid-mediated beta-Iactamase among Enterobacteriaceae. 22 In the United States, epidemic shigellosis is not as great a problem as in certain other countries. Salmonellosis is more frequent than shigellosis in this country, a fact that has been attributed to the prevalence of nontyphoidal salmonellae in animals and animal products. 116 Nonhospital-acquired salmonellosis caused by organisms with Rfactors was reported in 1966 in a survey of cases of pediatric diarrhea at Children's Hospital in Boston.1 31 Since then nontyphoidal strains of salmonellae that are resistant to multiple antibiotics, including ampicillin and chloramphenicol, have been isolated with increasing frequency, both in and out of the hospital. In general, however, salmonella gastroenteritis in the otherwise healthy patient does not require antibiotic therapy, and in such cases, outcome is not affected by resistant to antibiotics. There have been, however, life-threatening outbreaks of hospital-acquired salmonellosis. In 1974 there was a small outbreak of endoscopy-associated infections, including one case with bacteremia, caused by S. oslo resistant to chloramphenicol and ampicillin.16 A serious hospital outbreak of S. heidelberg gastroenteritis, including 25 cases of bacteremia and 8 deaths, spread through two pediatric wards in adjacent hospitals in San Juan, Puerto Rico in 1972; R-factors in these organisms were responsible for resistance to kanamycin-neomycin, streptomycin, and ampicillin. 114 In our own hospital in 1974, an outbreak of S. typhimurium septicemia occurred among burn patients; this pathogen was found to have R-factor mediated resistance to ampicillin, chloramphenicol, tetracycline, sulfonamides, streptomycin, mercuric chloride, and silver nitrate. 84 The pa-

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tients involved in outbreaks such as these are frequently not "otherwise healthy," and the outcome of salmonella infection, especially when it presents with bacteremia or enteric fever, can indeed be strongly affected by the presence of resistance and the lack of effective antibiotics. Of even greater concern has been the major epidemic of typhoid fever beginning in 1972 in Mexico, which has been described as occurring "on a scale unprecedented in modern times."44 The S. typhi involved in this outbreak contain an R-factor which mediates chloramphenicol resistance, as well as resistance to streptomycin, tetracycline, and sulfonamides. Prior to this, S. typhi had remained sensitive to chloramphenicol and that drug was regarded by many as the treatment of choice for typhoid fever. Chloramphenicol~resistant strains have also been isolated from India and Southeast Asia, as well as from cases imported into the United States. 2a • 14. 111 A few of these strains have been resistant to both chloramphenicol and ampicillin. 107 Although typhoid fever caused by sensitive or resistant S. typhi is generally not considered a significant public health threat in the United States, limited outbreaks can occur, as evidenced by the epidemic of waterborne typhoid fever which occurred in a Florida migrant labor camp in 1973, resulting in 188 clinical cases, 105 of which were bacteremic. 62 Moreover, typhoid fever is always a potential threat even in industrialized nations when breaks occur in the sanitation systems, as was the case in the Zermatt, Switzerland, typhoid outbreak of 1963. 8 Vibrio cholerae is another enteric pathogen which has recently acquired multiple drug resistance. The current cholera pandemic (El Tor strain) was initially caused by a uniformly antibiotic susceptible strain. As it spread, sporadic reports of resistance appeared and Rfactors which transfer resistance to streptomycin, tetracycline, chloramphenicol, sulfonamides, and to ampicillin, streptomycin, tetracycline, chloramphenicol, kanamycin have now been found in strains of V. cholerae. 56 Since the primary treatment of cholera is fluid replacement and not antibiotics, the impact of this resistance has not been great. Its occurrence, however, further demonstrates the widespread emergence of R-factors. It probably also illustrates the selective effect of the antibiotics that have been employed during this epidemic. S. Aureus and the Development of Penicillin Resistance: The Role of Plasmids The occurrence of transmissible antibiotic resistance is by no means confined to the gram-negative bacilli. Penicillin was introduced for clinical use in the early 1940's: This was followed by the rapid emergence of resistance to penicillin by S. aureus. Although the first penicillinase described was produced by a strain of E. COli/ 34 the first cognizance of the clinical significance of penicillinase followed its appearance in coagulase-positive staphylococci. In 1944 McKee described a clinical isolate of S. aureus that produced penicillinasej134 strains made resistant in the laboratory had no such enzyme and some controversy over its importance ensued. By the late 1940's, however,

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Table 1. Glossary60.101 Plasmid-an extrachromosomal genetic element that replicates independent of the host chromosome. Conjugative plasmid-a plasmid that can initiate and cause the transfer of DNA by conjugation. Non-conjugative plasmid-a plasmid that cannot bring about the transfer of DNA by conjugation, by itself. It may be transferrable via transformation, transduction, or mobilization by a conjugative plasmid. Resistance plasmid-a plasmid that carries genetic information for resistance to antibacterial agents. We have used the term R-factor interchangeably with resistance plasmid although to many "R-factor" implies a conjugative plasmid with both a "resistance determinant" sequence and a "resistance transfer" sequence. The term resistance plasmid encompasses conjugative as well as non-conjugative plasmids that can mediate resistance to antimicrobials. Conjugation (mating)-the process of genetic exchange between two organisms after cellular contact has been established. Constitutive enzyme production-enzyme production which occurs whether or not the substrate is present. As used in this paper, substrate refers to an antibiotic which is modified by the enzyme. Inducible enzyme production-enzyme production which is brought about or increased by the presence of the substrate (antibiotic). Transformation-the process of acquiring genetic information by taking up soluble DNA released from another ("donor") cell. Transduction-the process of acquiring genetic information which is transported from another ("donor") cell to a new cell by a phage. Transposon (translocation or transposition sequence) -a well defined genetic element that can translocate intact from one genetic locus to another. TnA - the designation for translocatable ampicillin resistance.

the importance of penicillinase-producing staphylococci was well recognized since such strains were clearly causing clinical disease. In 1950 Finland published data concerning the resistance of S. aureus before and after penicillin was widely used. 38 Of 120 strains isolated at the Boston City Hospital prior to 1946, 82 per cent were susceptible in vitro to :5 0.04 f.Lg per ml and only one strain was resistant to > 5 f.Lg per ml (its MIC was 25 f.Lg per ml). By 1947, only 25 per cent were sensitive to :5 0.04 f.Lg per ml and 32 per cent were resistant to > 25 f.Lg per m!. By 1951, 73 per cent of S. aureus were resistant to > 25 f.Lg per ml of penicillin and most of these were penicillinase producers. 39 For some years, the prevalence of penicillin resistance among hospitalacquired strains of S. aureus was higher than among communityacquired strains; but by 1967, the resistance had equalized at about 82 to 84 per cent for both inpatient and outpatient isolates, and this approximate percentage has been maintained at most centers over the ensuing 10 years.~ In 1975 at the Massachusetts General Hospital, 84 per cent of inpatient and 83 per cent of outpatient isolates of S. aureus were penicillin resistant. 94 The propensity of S. aureus to become resistant to antibiotics was not limited to penicillin. Lepper found that all strains of S. aureus were susceptible to :::; 1 f.Lg per ml of erythromycin in the early 1950's, prior to extensive use of that drug. Erythromycin was then used for all infections thought to be susceptible and 5 months later, 70 per cent of

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S. aureus from patients and personnel were resistant to > 100 ILg per ml. 78 A similar example was seen in a burn unit when chloramphenicol was substituted for chlortetracycline, resulting in an increase in chloramphenicol resistance from 10 to 20 per cent to 55 per cent after only 6 months. The re substitution of chlortetracycline led to a decline in chloramphenicol resistance to 14 per cent over the next 6 months, but at the end of this period, 83 per cent of S. aureus isolates were resistant to chlortetracycline. 47 It has now been demonstrated that resistance of S. aureus to chloramphenicol, tetracycline, erythromycin, and the aminoglycosides is plasmid mediated in many, if not most, clinical isolates. Unlike the multiple resistance mediated by a single plasmid in the gram-negative bacilli, the staphylococci plasmids usually mediate resistance to only one type of antibiotic, although the penicillinase plasmids can include determinants for resistance to erythromycin and to inorganic ions such as mercury, cadmium, lead, and bismuth. Again, unlike the plasmids in gram-negative bacilli which are usually transferred by conjugation, the staphylococcal plasmids appear to be transferred exclusively by phage transduction. 102 The introduction of the penicillinase-resistant, semisynthetic penicillins, such as methicillin, oxacillin and nafcillin, and of the cephalosporins, seemed to overcome the problem of resistance resulting from the production of beta-Iactamase by S. aureus. However, methicillinresistant strains have been found, particularly in Europe. The biochemical and genetic mechanisms of this resistance are poorly understood. 55 In some instances, the organisms appear to be less virulent than their methicillin-sensitive counterparts. In this country, resistance to the semisynthetic penicillinase-resistant penicillins and cephalosporins is unusual, but outbreaks of infection due to such resistant S. aureus have recently occurred, and in some settings, may be increasing. 73, 128 In our own hospital, 99 per cent of 4017 strains of S. aureUs tested in 1977 remained susceptible to both methicillin and the cephalosporins. The Effect of Antibiotics in Certain Special Settings The prevalence of R-factors in normal persons not treated with antibiotics is not clear - prevalence studies vary quite widl:!ly - but in general, there does appear to have been an increase in the antibiotic resistance of normal intestinal flora though less so than for the heretofor mentioned pathogens (reference 34, Tables 4.5 and 4.6). What is more clear, however, is the influence of antibiotic administration on the resistance of a given individual's flora. In an early Japanese study, a large survey of healthy human beings showed a 1.4 per cent carriage of multiply resistant E. coli; but in 61 per cent of patients receiving chloramphenicol, and in 20 per cent of patients with tuberculosis being treated with streptomycin, there were multiply resistant E. coli. 88 A study by Datta in 1969 of organisms from urinary tract infections in outpatients showed that 19 per cent were resistant to more


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than one drug (six of seven were transferable); of perhaps greater interest was the observation that 1 week after therapy, patients treated with tetracycline showed uniform resistance to it in their fecal E. coli, and more importantly, it was coupled with resistance to ampicillin, streptomycin, sulfonamides, and chloramphenicol in many strains. Neither the increase of single nor coupled resistance was significant in the fecal E. coli of patients treated with ampicillin or a sulfonamide. The effect of tetracycline had, however, almost disappeared 1 month after cessation of therapy.24 The long-term use of tetracycline for acne can also lead to the presence of multiply resistant E. coli containing R-factors in the feces of individuals taking the drug. 95 Animal feed lots are sites of particularly high use of and resistance to antibiotics. A large number of studies have documented the increasing prevalence of R-factors and resistance to antibiotics in farm animals, both in their normal fecal flora and in pathogens such as salmonellae. This resistance appears to be directly related to the use of antibiotics in feed and their extensive therapeutic use. It has been suggested that human disease, especially that caused by multiply resistant non typhoidal salmonellae, may be etiologically linked to this increasing pool of antibiotic resistance in food-producing animals, although the extent to which this actually occurs is an area of some controversy.3. 51, 90, 100, 127, 142 A setting of far greater concern is that of the hospital, where "normal" human flora and the flora of the environment (including E. coli, Klebsiella sp., Enterobacter sp., Proteus sp., Serratia sp., and Pseudomonas aeruginosa) have become significant and quite resistant nosocOInial pathogens. Finland's experience at the Boston City Hospital is quite illustrative. In 1935, prior to the introduction of antibiotics, the proportion of bacteremia and death caused by pneumococCi and streptococci far exceeded that due to enteric gram-negative bacilli, which caused only 12 per cent of bacteremia and 8 per cent of bactereInic deaths. The latter percentages increased steadily after 1941 and in 1957, gram-negative bacilli were responsible for more than one third of the cases of bacteremia and about half of the bacteremic deaths. These organisms were frequently hospital acquired. In 1965, gram-negative bacilli caused half of all bacteremias and 57 per cent of bactereInic deaths. 37,40 This shift in etiology of bacteremia is related to at least two factors. One is the control of organisms such as the pneumococcus and streptococcus that previously caused high rates of mortality and were usually the cause of infections present on admission. The second is the ability to prolong life in hospitalized patients with previously fatal diseases. This has involved the use of a number of procedures and invasive techniques such as surgery, respiratory assistance devices, intra-arterial and intravenous cannulas, urinary catheters, and the use of immunosuppressive and anticancer drugs - all of which by themselves may predispose to infection with hospitalacquired organisms. In addition, it is well recognized that seriously ill hospitalized patients can rapidly be colonized by gram-negative bacilli;68,69 couple this with the fact that these hospital-acquired organisms

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are becoming exceedingly resistant to antibiotics and one can begin to understand why noscomial infections are such a serious problem. The newer aminoglycosides have been the backbone of therapy for hospital-acquired infections caused by gram-negative bacilli. However, the emergence of organisms resistant to these agents has been a troublesome problem. Streptomycin was the first of the aminoglycosides (amino glycosidic aminocyclitols, to be exact) to be released for clinical use. Its introduction in the late 1940's was followed by the rapid emergence of resistance among gram-negative bacilli. Initially, it was felt that the resistance among clinical isolates was related solely to the fact that exposure of gram-negative bacilli to streptomycin in vitro resulted in the appearance of mutants which were highly resi~tant because of ribosomal alterations. 6 Subsequent studies, however, have established that R-factors mediating resistance to streptomycin may also be found and produce. resistance by coding for a streptomycininactivating enzyme. Unlike the case with streptomycin, single step mutations to high-level resistance to the aminoglycosides which contain 2 deoxystreptamine (neomycin, kanamycin, gentamicin, tobramycin, amikacin) do not occur with high frequency. Nonetheless, resistance to kanamycin became prevalent in the 1960's after its release for clinical use. Indeed, R-factors mediating resistance to kanamycin were described as early as 1962,88 even though the drug had first been isolated only 5 years before. The prior use of neomycin may have resulted in the selection of organisms resistant to kanamycin since many organisms contain R-factors which mediate resistance to both kanamycin and neomycin by similar mechanisms. Gardner and Smith in 1967 reported a nosocomial outbreak of serious renal and pulmonary infections caused by klebsiella that were resistant to multiple drugs including kanamycin. 46 Patterns of resistance in klebsiella isolated from all sources during this time period showed that about 56 per cent were resistant to kanamycin and over 90 per cent of these were also resistant to streptomycin, chloramphenicol, and tetracycline. Fifty per cent of these transferred the multiple resistance. Nursery epidemics caused by R-factor mediated kanamycin-resistant bacteria, especially E. coli and Klebsiella pneumoniae, were also frequent.31, 43, 54, 135 The development of resistance to (or the selection of resistant strains by the use of) gentamicin has occurred at a slower pace than was true for streptomycin and kanamycin. Nonetheless, the extensive use of gentamicin since its release for clinical use in the late 1960's and early 1970's has resulted in the emergence of significant numbers of gentamicin-resistant organisms in many locations. Reports of increasing resistance and infection began as early as 1971. 52 At the University of Virginia Hospital, the use of gentamicin began in 1969. In 1971, only 0.8 per cent of more than 900 isolates of bacteria were resistant to gentamicin, but by 1975, 7.7 per cent were resistant. In 1974, 20 per cent of nosocomial bacteremias at this hospital were due to gentamicin-resistant gram-negative bacilli (primarily Klebsiella sp., but also P. aeruginosa, Enterobacter sp. and Serratia Sp.).53 A similar pattern of increasing resistance to gentamicin has been noted at

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the Massachusetts General Hospital, where gentamicin was released for clinical use in March 1971. As can be noted in Figure 1, there has been a progressive increase in the prevalence of gentamicin-resistant organisms since that time. The Manhattan Veterans Administration Hospital reported an abrupt increase in gentamicin-resistant isolates in 1973 and 1974; bacteremia caused by such organisms occurred. in 17 patients. Resistance was transferable in 34 of 36 clinical isolates. Numerous other outbreaks of infection caused by gentamicin-resistant organisms have occurred, involving E. coli, Klebsiella sp., Serratia sp., Proteus sp. (especially P. rettgeri), P. aeruginosa, and a variety of less virulent pathogens such as Acinetobacter sp. (herellea and mima).21, 30. 41. 48, 63, 115. 120. 121. 140 In many, but not all instances, resistance to gentamicin has proven to be plasmid mediated. Tobramycin and amikacin are the newest aminoglycosides to be released for clinical use in the United States. Even though neither has been used as extensively as gentamicin or kanamycin, there are already disturbing signs that resistance to these agents will also be a problem. In the case of tobramycin, we have seen increasing numbers of tobramycin-resistant bacteria since 1972 (see Fig. 1) even though the clinical use of tobramycin at the Massachusetts General Hospital has been very limited. This suggests that the use of gentamicin and other aminoglycosides (which has been extensive) may result in the selection of organisms resistant to both gentamicin and tobramycin. The possible reasons for this observation will be better understood when we examine mechanisms of resistance in a subsequent seetion of this paper. Amikacin, the newest of the aminoglycosides, is a semisynthetic compound which is effective in vitro and in vivo against many organisms which have plasmid-mediated resistance to the other aminoglycosidesY3 However, even this drug is not a panacea. We have already found a large number of bacteria from clinical specimens which are resistant to amikacin, and the prevalence of these appears to have in1400 (/)

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Figure 1. Emergence of aminoglycoside-resistant gram-negative bacilli, Massachusetts General Hospital. Abbreviations: GMR = gentamicin resistant: TMs = tobramycin susceptible; AM' = amikacin susceptible; AM" = amikacin resistant. Data collected in collaboration with Dr. L. J. Kunz ..

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creased over the past several years. Since the use of amikacin has been limited, it seems likely that pressures of selection exerted by the use of gentamicin and kanamycin may also, in part, explain the increase in amikacin-resistant organisms noted (figure 1, ref. 91). Many of the amikacin-resistant organisms isolated so far have been "non pathogens" or organisms of low pathogenicity, but amikacin-resistant strains of P. aeruginosa, Serratia sp., Klebsiella pneumoniae, and other potential pathogens have also been found. 9l Minshew et al. 87 have recently reported an increase in amikacin-resistant organisms isolated from burn patients. Thus, it appears that the emergence of resistant organisms is a problem which will plague all presently known amino glycoside antibiotics. Fortunately, the widespread emergence of organisms resistant to gentamicin, tobramycin, and amikacin appears so far to be limited to iarge hospital centers where the selective pressure of the use of antibiotics is greatest. Transposons and Their Importance in Some Common Pathogens: H. Influenzae and Gonococci Plasmids have been found in almost all bacteria. Resistance plasmids (R-factors) have also been found, as noted in the preceding paragraphs, in a wide range and variety of bacteria. Certain specific types of plasmid-mediated resistance are peculiarly common. The best example is the penicillinase called TEM beta-Iactamase which appears to be the most prevalent type of plasmid-mediated beta-Iactamase in gram-negative bacilli,34 It has been identified not only in widely varying bacteria but also in association with many different types of plasInids. In the early 1970's, the probable mechanism for the disseInination of a particular genetic element among plasmids, and indeed for the very evolution of plasmids themselves, became more clear. It was found that a segment of DNA could translocate, or transpose, itself from one DNA site to another. The genes for the TEM beta-Iactamase (now called TnA for Transposon-ampicillin) were found to be able to jump (transpose) from one plasmid to another, from plasmid to chromosome, and then again from chromosome to plasmid. 18, 19, 119 The phenomenon is detected by phenotypic acquisition of ampicillin resistance, its transfer by plasmids that formerly did not have the resistance marker, and most definitively by DNA homology studies showing identical (homologous) areas between the DNA of the known ampicillin resistance genes and the plasmids that have newly acquired the penicillinase property, but not with the parents of these new plasmids which lacked the ampicillin resistance marker (penicillinase).58,59 Certain genes for kanamycin resistance have also been found capable of translocation between plasmid, phage, and chromosome DNA.7 A similar type of genetic sequence carrying tetracycline resistance has been acquired from R-factors by phage, and then observed to insert into a large number of different sites on the salmonella chromosome. 5 Linked streptomycin-trimethoprim resistance genes have also been shown to translocate. 72 The mechanism of transposition seems to involve the insertion of these "jumping genes" at a variety of


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possible sites and is not dependent upon general recombination functions. The existence of transposons implies that R-factors may increase their resistance by picking up genes from various sources such as phage, chromosomes, other plasmids, and then disseminate them. Indeed, the recognition that "new" R-factors frequently turn out to be known plasmids that have acquired new resistance genes could easily be explained by the insertion of a transposon. The importance and recognized prevalence of transposons will undoubtedly grow as knowledge increases. The impact of the TnA has already shaken some of our longest established therapeutic principles in pediatricsand venereology, with H. injluenza and N. gonorrheae respectively. For over 10 years, ampicillin has been considered the drug of choice for meningitis due to H. injluenza type b. In early 1974, reports of ampicillin-resistant Hemophilus injluenzae began to appear from the United States; by 1976, these organisms had been reported in over 20 states, Canada, Germany, and England. 71, 99, 141 Although marked variation in the level of resistance was noted among different strains, the resistance was found to be due to the TEM betalactamase - the same enzyme that is so widespread in enteric gramnegative bacilli. 86, 130 Elwell and Falkow examined the plasmids of four ampicillin-resistant strains of H. injluenzae; 3 had a 30 Mdal. plasmid and one had a 3 Mdal. plasmid. The ampicillin resistance, mediated by the production of beta-Iactamase, could be transferred to sensitive strains by conjugation or transformation. DNA hybridization with E. coli containing the TnA sequence showed considerable homology between the TnA sequence and regions of both the large and small plasmid from H. injluenzae. 28 ,32 Thus, there exist at least two different plasmids in H. injluenzae which contain the same genetic sequence (or at least a significant portion of it) that codes for the beta-Iactamase (TEM) which is found so commonly in other gram-negative organisms. Whether H. injluenzae acquired the plasmid de novo, or had preexisting plasmids into which was inserted the ampicillin transposon is unknown. What does seem clear is that H. injluenzae acquired resistance to ampicillin from the existing pool of resistance in enteric gramnegative bacilli. Currently, ampicillin-resistant H. injluenzae (type b as well as non-typable strains)126 are widely distributed and have necessitated a change to chloramphenicol, or ampicillin plus chloramphenicol, as initial therapy for serious H. injluenzae infections. At least one chloramphenicol resistant type b, and several nontypables, have also been reported. 97,144 So far, ampicillin and chloramphenicol resistance have not occurred in the same hemophilus type b strain. The gonococcus is another organism that has recently acquired penicillin resistance via beta-Iactamase production. Penicillin was first used for gonorrhea in the early 1940's and during the next 30 years, a gradual stepwise increase in resistance to penicillin occurred. This resistance was probably chromosomally mediated and it was only relative; that is, the minimal inhibitory concentrations of penicillin for

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these strains was greater than that of their more sensitive counterparts, but infections still responded to a single, but larger, intramuscular injection of penicillin. In 1975, however, true resistance to penicillin began to emerge, first in the Far East, then in the United States, especially among Far East contacts, in England, the Netherlands, Africa, and other locations. The resistance was shown to be due to the production of TEM beta-Iactamase. Although still rare in the United States, up to 30 to 50 per cent of gonococci in certain parts of the Philippine Islands are beta-Iactamase producers. 83 • 96. 112. 133 Studies of gonococci from the Far East have shown that they contain a 4.4. Mdal plasmid, and those from London a 3.2 Mdal plasmid, both of which mediate resistance to penicillin. DNA hybridization studies of these plasmids have demonstrated significant homology with the TnA sequence found in the enteric bacilli and H. injluenzae. 33 Thus once again it appears that the resistance transposon for TEM beta-Iactamase has found its way into a pathogen which had long been' sensitive to penicillin. Its presence threatens to change drastically the traditional therapy of venereal disease. Resistance Plasmids -

Where Did They Come From?

The origin of R-factors is not known. The beginning of the commercial era of antibiotics was obviously not the beginning of antibiotics themselves since all true antibiotics are produced by microorganisms,especially the Actinomycetales, and have likely existed in nature for eons. The massive outpouring of antibiotics that began after 1940 has surely done much to select and disseminate R-factors, and may have hastened their evolution, but almost certainly did not create them. The finding of a tetracycline-streptomycin R-factor in an E. coli strain lyophilized in 1946 (prior to the clinical introduction of these drugs) is evidence for this reasoning. 132 Two out of three studies of "antibiotic virgin" populations have also encountered R-factors.26. 45. 80 In one of these, a tetracycline-streptomycin R-factor was found in a fecal E. coli strain from a Solomon Islands bushman, and another was found in an organism from the soil, which is the natural habitat for many antibiotic producing organisms. 45 An interesting finding by Davies is that some streptomyces that produce amino glycoside antibiotics also produce enzymes, similar to the ones found in gram-negative bacilli, that inactivate these aminoglycosides. 6 One could postulate from these observations that either (a) other bacteria acquired the genes that code for these enzymes from these natural enzyme producers, or Cb) exposure to antibiotics, for example, in the soil, has through the ages led to the evolutionary acquisition of the survival capacity to inactivate these compounds. Davies also suggests that since the aminoglycosides are "amino sugars" it is possible that inactivating enzymes for these antibiotics may have evolved from enzymes that at one time were involved in the metabolism of various other amino sugars. 6 Thus, although the exact origin of R-factors is unknown, it continues to be a subject of much speculation and investigation.


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MECHANISMS OF RESISTANCE TO ANTIMICROBIAL AGENTS In the preceding sections we have emphasized the importance of plasmids in the spread of resistance to antibacterial agents. Not all R-plasmids, however, mediate resistance by the same mechanism and not all resistance is plasmid mediated. In the following three sections, we will outline the current knowledge of the biochemical events leading to resistance. In some instances, chromosomally mediated resistance by mechanisms identical to plasmid-mediated ones appears to be important; and in some instances, it has not yet been clearly shown which genetic component is responsible. Enzymatic Inactivation PENICILLINS AND CEPHALOSPORINS. As was mentioned earlier, over 80 per cent of S. aureus isolates in most parts of the United States are resistant to penicillin because of the inducible production of plasmid mediated penicillinase, which hydrolyzes the beta-lac tarn ring present in the antibiotic and thus inactivates it. There are at least three distinct staphylococal penicillinases, and all have virtually no effect on the semisynthetic penicillins, such as oxacillin, or on the cephalosporins.1 7 Penicillinases also occur in several Bacillus species. 6 Penicillin G is not generally an effective agent against the enteric gram-negative bacilli. Therefore the production of beta-Iactamase by these organisms was not of clinical concern until the more effective semisynthetic penicillins (e.g., ampicillin) and the cephalosporins were discovered. Transferable ampicillin resistance in gram-negative bacilli was first found in an isolate from 1962 and the role of betalactamase in producing this resistance was recognized by 1965. Since then, other penicillin derivatives such as carbenicillin, tic arcillin , amoxycillin, and various cephalosporin derivatives have become available; these are more resistant to the effect of most beta-Iactamases. It appears, however, that for each derivative so far created, there is somewhere a beta-Iactamase to hydrolyze it. Our knowledge of the diversity and extensive distribution of the beta-Iactamases of gram-negative bacilli is rapidly growing. Attempts have been made to classify these enzymes based on various properties including substrate profile, molecular weight, immunologic reactivity with certain antisera, sensitivity to various inhibitors, isoelectric focusing, and so forth.35, 66, 82, 85 All of the classification schemes are plagued with ambiguities and there are many enzymes whose physical and biochemical properties do not permit their precise assignment to anyone category. One convenient scheme divides beta-Iactamases into two broad types. (1) The "cephalosporinases" which are much more active against cephalosporins than ampicillin, are often inducible and are mostly, but not always,IO chromosomally mediated. (2) The "penicillinases" which have broad activity against penicillin, ampicillin, and cephalosporins,. are produced constitutively, and are mostly

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Table 2. Major Mechanisms of Resistance to Antibiotics I. Enzymatic Inactivation A. Beta-lactamases 1. Gram-negative bacilli: these enzymes can variably hydrolyze penicillins, oxacillin, cephalosporins 2. Gram-positive bacteria: oxacillin, cephalosporins are resistant to these beta-lac tamases B. Chloramphenicol acetyl transferase 1. Staphylococci and gram-negative bacilli C. Aminoglycoside-modifying enzymes 1. Staphylococci, enterococci, gram-negative bacteria

Il. Decreased Permeability A. Naturally occurring 1. Penicillin G resistance of enteric gram-negative bacilli 2. Aminoglycoside resistance of enterococci 3. Polymyxin resistance of gram-positive bacteria B. Acquired 1. Plasmid mediated resistance to tetracycline 2. Amikacin and other amino glycoside resistance in some gram-negative bacilli 3. ?plasmid-mediated chloramphenicol and sulfonamide resistance in some bacteria Ill. Alterations in the Target Site Components A. Increased concentration of competing substances 1. Increased production of P ABA by some sulfonamide-resistant bacteria B. Synthesis of a more resistant target site 1. Ribosomes resistant to streptomycin: gram negative bacilli, certain strains of enterococci 2. Ribosomes resistant to erythromycin: plasmid-mediated alteration in rRNA in staphylococci 3. Resistant enzyme: sulfonamide resistance caused by an enzyme which-has a decreased affinity to sulfonamide C. Synthesis of an alternative target site 1. Sulfonamide resistance and trimethoprim resistance due to plasmid-mediated production of a second enzyme in the folate synthesis pathway, which is resistant to the effects of these drugs: E. coli.

plasmid-mediated. Within this latter group two major subtypes can be identified: (a) The so-called TEM penicillinase, already mentioned, is the most common R-factor mediated penicillinase among gramnegative bacilli, and has broad activity against penicillin and cephalosporins, but very little activity against oxacillin; (b) The "0" type penicillinase has much higher activity against oxacillin than penicillin or cephalosporins and is less common. This latter group in turn has various subtypes. 10. 23. 34. 57.81 CHLORAMPHENICOL. In 1961, it was reported that some chloramphenicol-resistant shigella could inactivate chloramphenicol, but the mechanism was not defined until 1967, when it was found that cell-free extracts of E. coli containing R-factors for chloramphenicol resistance could inactivate chloramphenicol by acetylation of hydroxl groupS.105 It now appears that the majority of these R-factors mediate the constitutive production of chloramphenicol acetyl transferase (CATase).6 Despite the fact that determinants for chloramphenicol re-


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sistance occur in a large number of different plasmids, geographic locations, and species of gram-negative bacteria, the enzymes isolated from such strains have been remarkably similar, although not identical.42, 89, 123, 125 Certain gram-negative bacilli without R-factors have been found that are sensitive to chloramphenicol but produce low levels of CATase, which is most" likely chromosomally mediated; some of these strains, especially P. mirabilis, easily produce mutants with much higher levels of the enzyme and concomitantly greater resistance. 105 The relationship between plasmid-mediated and chromosomally mediated CATase may be interlinked in some instances in which the chloramphenicol gene can move from plasmid to chromosome and back again by either recombination or transposition. 50, 65 Another suggested mechanism for chloramphenicol R-factor resistance is decreased permeability (see below).98 S. aureus resistant to chloramphenicol also can produce a CATase, distinct from that of the gram-negative organisms. In most strains, this is an inducible enzyme coded for by a transducible plasmid. 124 AMINOGLYCOSIDES. The enzymatic inactivation of an aminoglycoside was first reported in 1965 by Okamoto and Suzuki, who observed that a cell-free extract of E. coli, containing an R-factor for resistance to kanamycin, was able to inactivate kanamycin in the presence of acetyl COA.104 Subsequently, the following three plasmid-mediated enzymatic modifications of aminoglycosides have been described: 6, 26, 34 1. Phosphorylation of hydroxyl (- OH) groups with ATP as the phosphate donor. 2. Acetylation of amino (- NH2) groups with acetyl coenzyme A as the acetyl donor. 3. Adenylylation of hydroxyl groups with ATP as the adenyl donor. With the exception of spectinomycin, these antibiotics are all aminoglycosidic aminocyclitols, that is, they consist of aminocontaining sugars linked by a glycosidic bond to an aminocyclitol ring. These compounds contain many hydroxyl and amino groups which are susceptible to enzymatic modification by the above mechanisms; such modification usually results in resistance of the enzyme-producing bacterium to that drug. The amino and hydroxyl groups are numbered (e.g., 6', 2", 3), depending upon the ring they are in and their location within that ring. A variety of enzymes exist and are often named for the site and the type of reaction involved, although other nomenclature is also used. Frequently the enzyme is also assigned the name of an amino glycoside upon which the enzyme acts, for example, gentamicin 2' acetyltransferase, although in most instances, more than one aminoglycoside is a good substrate. It appears that many of the enzymes have different forms and different substrate profiles, despite the same name. Knowledge of the mechanism by which aminoglycosides are enzymatically inactivated has permitted a more rational approach to the search for and synthesis of more effective compounds. Amikacin is a semisynthetic derivative of kanamycin A, which has an alpha-

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hydroxy-gamma-aminobutyric acid side chain on the C-l-amino group.113 This side chain prevents enzymatic inactivation, perhaps by steric changes, at most of the sites that can be attacked in kanamycin. However, even this compound has some vulnerability; for example, a 4' adenylyltransferase which uses amikacin as a substrate has been reported in amikacin-resistant staphylococci; 6' acetyltransferases can also inactivate amikacin. A 3' phosphotransferase which phosphorylates amikacin has been described in staphylococci but it does not lead to in vitro resistance of the bacteria to this drug. 26, 113 The amino glycoside-inactivating enzymes are most often associated with R-factors although this has not always been documented; Kayser has reported a 3' phosphotransferase in staphylococcal. isolates, whose production is under the control of a chromosomally located gene. 70 Plasmid-mediated enzymes have been found in E. coli, Ps. aeruginosa, staphylococci, K. pneumoniae, providencia, Proteus sp., Enterobacter sp., Citrobacter sp., serratia, enterococci and other bacterial species. 6, 26, 67, 113 Decreased Permeability NATURALLY OCCURRING. The beta-Iactam antibiotics have been thought to exert their antibacterial effect primarily by inhibiting transpeptidase, thereby inhibiting the synthesis of the cell wall peptidoglycan, or murein, layer. In gram-negative bacilli, the murein layer lies between an outer covering, which is a phospholipid membrane that also contains lipopolysaccharide and proteins, and an inner (cytoplasmic) phospholipid membrane. 20,110 It is now clear that other enzymes found in the cell envelope besides transpeptidase, are inhibited by penicillin; moreover, recent studies have delineated other specific penicillin-binding proteins whose function is not completely known. 9 , 122 In order to reach any of these proteins and exert its effect, penicillin must penetrate the cell envelope's outermost phospholipid membrane, which is known to be able to retard entry to various substances, including penicillins;77 gram-positive bacteria do not possess the outer membrane layer. The ability of the membrane to retard the entry of different pencillins and cephalosporins varies from species to species. In general, the outer membrane is less permeable to penicillin G than to its semisynthetic derivatives such as ampicillin and carbenicillin. l03, 137, 138, 1,,0 The permeability barrier is important not only because it may result in relative or absolute resistance to penicillin G among gram-negative bacilli but also because it enhances the resistance of beta-Iactamase producers, apparently by controlling the rate at which the beta-Iactam antibiotic is delivered to the beta-Iactamase enzyme. 11 Lack of an effective permeability barrier for certain agents presumably explains why some gram-negative bacilli which produce beta-Iactamase capable of hydrolyzing a particular drug in vitro nonetheless remain susceptible to that drug. 36, 86, 108 Another well recognized and clinically important example of a naturally occurring permeability barrier is that of the enterococci to the aminoglycosides. Enterococci frequently are inhibited in vitro only by


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concentrations of aminoglycoside that are much higher than those clinically achievable; yet they are synergistically inhibited and killed in vitro by low concentrations of an amino glycoside when combined with an agent that interferes with cell wall synthesis, such as penicillins, cephalosporins, cycloserine, bacitracin, or vancomycin.49.93.146 The mechanism of this synergism in enterococci has been shown to involve the enhanced uptake and accumulation of streptomycin, and presumably the other aminoglycosides as well, when enterococci are exposed to streptomycin in the presence of penicillin, vancomycin, cycloserine, or bacitracin, but not when grown without these inhibitors of cell wall synthesis. 92 Presumably, the inhibition of the cell wall formation allows the aminoglycosides to cross an otherwise quite effective permeability barrier. The polymyxins, including polymyxin E (Colistin) and polymyxin B, are cationic detergents whose bactericidal activity is limited to gram-negative bacilli, although Proteus sp., Serratia marcescens, and Providencia stuartii are resistant. The lethal effect of these detergents is due to their ability to disrupt the phospholipids and lipoproteins of the bacterial cell envelope, especially the inner (cytoplasmic) membrane. Studies of three P. mirabilis mutants (one resistant and two sensitive to polymyxin) suggest that the resistance to polymyxin is due to a permeability barrier which prevents access of polymyxin to the critical phospholipids, and not to any intrinsic difference in these phospholipids themselves. 136 Gram-positive bacteria, which are intrinsically resistant to polymyxins, may be so by virtue of decreased permeability to polymyxin through their thicker cell wall. ACQUIRED PERMEABILITY CHANGES. R-factor mediated tetracycline resistance in both gram-negative bacilli and S. aureus involves an inducible decrease in uptake of tetracycline, although the biochemical mechanism for this decrease is not yet understood. 43a This resistance does not include minocycline, at least in E. coli, and there appear to be different mechanisms by which tetracycline and minocycline are transported intracellularly.29 The report of a new protein in membranes of tetracycline R-factor containing E. coli minicells incubated in the presence of tetracycline, suggests that this inducible resistance may involve the synthesis of a new protein which acts at the cell membrane to decrease influx or increase efflux. 78 In 1972 N agai and Mitsuhashi reported finding four clinical strains of E. coli with transferable resistance to chloramphenicol that demonstrated no enzymatic inactivation of chloramphenicol and concluded that the resistance must involve decreased permeability.98 Subsequently four strains of P. aeruginosa were found with transferable resistance to chloramphenicol which also were unable to inactivate the drug. Uptake of 14C-chloramphenicol was 25 per cent less for strains with these R-factors than for a parent strain without them, though this degree of decreased uptake may not fully explain the resistance. 139 There have also been studies suggesting that the sulfonamide resistance mediated by certain R-factors is due to decreased permeabili-

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ty. This reasoning has been based primarily on the inability to demonstrate other known resistance mechanisms for sulfonamides (see below); 35S-sulfathiazole uptake studies showed a one-third decrease in uptake for an R-factor containing E. coli, but this accompanied a 200 to 500 increase in the level of resistance and may be only part of the resistance mechanism. 89 Decreased permeability accounts for some of the resistance of P. aeruginosa to aminoglycosides; this permeability change may be acquired through exposure in vivo and is not associated with enzymes or tranferability. Bryanhas worked extensively with the uptake of aminoglycosides by P. aeruginosa and found that most of the low level resistance of these bacteria is due to decreased permeability; high level resistance is generally associated with resistant ribosomes (for streptomycin) or presence of inactivating enzymes. 12. 13. 143 Price and coworkers have described a number of gram-negative bacilli which are resistant to amikacin (and all other aminoglycosides as well) and which exhibit decreased permeability as the only identifiable mechanism of resistance. 113

Alteration of the Target Site Complex INCREASED CONCENTRATION OF COMPETING SUBSTANCES. Sulfonamides exert their bacteriostatic effect by competing with paraaminobenzoic acid (P ABA) for the enzyme dihydropteroate synthetase which is involved in the first step of folic acid synthesis. There exist clinical isolates of S. aureus that are resistant to sulfonamide because they produce up to 20 times as much P ABA as their sensitive counterparts and are therefore able to overcome the competitive inhibition of sulfonamides; some laboratory mutants of S. aureus also develop resistance by the same mechanism. 147 A similar phenomenon has been reported in strains of sulfonamide-resistant gonococci and in one strain of meningococci. 64. 76 SYNTHESIS OF A RESISTANT TARGET SITE. Rapid emergence of resistance to streptomycin can be seen in many bacteria Within several days exposure (in vivo) and is easily demonstrated in vitro. This resistance has been shown to result from a single amino acid change in a protein of the 30S ribosome to which streptomycin normally binds; this amino acid change prevents the 30S ribosomal binding of streptomycin and the subsequent inhibition of protein synthesis and misreading of the genetic code that it would produce with sensitive ribosomes. Resistance of ribosomes to the effects of macrolides (such as erythromycin and lincomycin) has been demonstrated in S. aureus and is due to alteration of the ribosomal RNA. Specifically, methylation of a nucleotide sequence of 23S rRNA decreases the binding of erythromycin to the 50S ribosome. Plasmid-mediated resistance to erythromycin in S. aureus is of two types: (1) inducible resistance to erythromycin and lincomycin, but inducible only by erythromycin, and (2) constitutive resistance to both. These two types appear to correspond to inducible or constitutive production of methylation of the 23S rRN A. 6. 74. 75. 118

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A third example of a resistant target site appears to occur in the first step of folic acid synthesis, in some sulfonamide-resistant bacteria. The alteration is manifest as a decreased affinity of the enzyme dihydropteroate synthetase for sulfa but an unchanged affinity for P ABA, which means that the ability of sulfonamides to serve as a competitor is impaired. This affinity change of the synthetase has been strongly suggested by studies in pneumococci as well as some clinical isolates of gonococci and meningococci. 61, 109, 148 These observations do not appear to be due to synthesis of a second enzyme as will be described below. SYNTHESIS OF ALTERNATE TARGET SITES. Recent studies have documented that R-factors can lead to synthesis of an alternate enzyme which circumvents the inhibitory action of a drug on the chromosomal enzyme. Clinical isolates of several species of gram-negative bacilli with R-factors for sulfonamides have been found to have two dihydropteroate synthetases, which are easily separable because of their physical properties. One enzyme is plasmid mediated and is resistant to the in vitro inhibitory effects of sulfonamides on folic acid synthesis; the other is the wild type, chromosomal enzyme and remains susceptible to these inhibitory effects. 129, 149 Trimethoprim inhibits folate synthesis by inhibiting the enzyme dihydrofolate reductase. An R-factor which increases trimethoprim resistance of a host E. coli 10,000 times has been found to mediate synthesis of a new dihydrofolate reductase which is 10,000 times less susceptible to the in vitro effects of trimethoprim than is the host bacterium's own chromosomal enzyme. 1

CONCLUSION From the foregoing, it may be clearly seen that there are many and diverse mechanisms by which bacteria can resist the activity of antimicrobial agents. In most cases these represent processes already present in nature. The "emergence" of resistant organisms in many, if not most instances, represents the end result of the selective pressure applied by the extensive use of antibiotics. It is quite clear that for the present, the only practical solution to the problem of the emergence of bacterial resistance is to control the indiscriminate use of antimicrobial agents. ACKNOWLEDGMENT

The secretarial skills of Florence Larson are gratefully acknowledged.

REFERENCES 1. Amyes, S. G. B., and Smith, J. T.: R-factor trimethoprim resistance mechanism: an insusceptible target site. Bichem. Biophys. Res. Comm., 58 :412, 1974.

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2. Anderson, E. S., and Datta, N.: Resistance to penicillins and its transfer in Enterobacteriaceae. Lancet, 1 :407, 1965. 2a. Anderson, E. S.: The problem and implications of chloramphenicol resistance in the typhoid bacillus. J. Hyg.(Camb.), 74 :289, 1975. 3. Babcock, G. F., Berryhill, D. L., and Marsh, D. H.: R-factor of Escherichia coli from dressed beef and humans. Appl. Microbiol., 25 :21, 1973. 4. Barrett, F. F., Casey, J. 1., Wilcox, C. et al.: Bacteriophage types and antibiotic susceptibility of Staphylococcus aureus. Arch. Intern. Med., 125 :867, 1970. 5. Barth, P. T., Datta, N., and Hedges, R. W.: Transposition of a DNA sequence encoding trimethoprim and streptomycin resistances from R483 to other replicons. J. Bact., 125:800, 1976. 6. Benveniste, R., and Davies, J.: Mechanisms of antibiotic resistance in bacteria. Ann. Rev. Biochem., 42:471, 1973. 7. Berg, D. E., Davies, J., Allet, B., et al.: Transposition of R-factor genes to bacteriophage. Proc. Nat!. Acad. Sci., 72:3628, 1975. 8. Bernard, R. P.: The Zermatt typhoid outbreak in 1963. J. Hyg. (Camb.), 63:537, 1965. 9. Blumberg, P. M.: Penicillin binding components of bacterial cells and their relationship to the mechanism of penicillin action. Ann. N.Y. Acad. Sci., 235:310,1974. 10. Bobrowski, M. M., Matthew, M., Barth, P. T., et al.: Plasmid-mediated betalactamase indistinguishable from the chromosomal f3-lactamase of E. coli. J. Bact. 125:149, 1976. 11. Boman, H. G., Nordstrom, K., and Normark, S.: Penicillin resistance in E. coli K12: Synergism between penicillinases and a barrier in the outer part of the envelope. Ann. N.Y. Acad. Sci., 235:569, 1974. 12. Bryan, L. E., and van den Elzen, H. M.: Streptomycin accumulation in susceptible and resistant strains of E. coli and Ps. aeruginosa. Antimicrob. Agents Chemother., 9:928, 1976. 13. Bryan, L. E., van den Elzen, H. H., and Shahrabadi, M. S.: The relationship of aminoglycoside permability to streptomycin and gentamicin susceptibility of Ps. aeruginosa. In Mitsuhashi, S. and Hashimoto, H., eds: Microbial Drug Resistance. University Park Press/Univ. of Tokyo Press, 1975, pp. 475-490. 14. Butler, T.: Chloramphenicol resistant typhoid fever in Vietnam associated with Rfactors. Lancet, 2:983, 1975. 15. Chabbert, Y. A., and Baudens, J. G.: Transmissible resistance to 6 groups of antibiotics in salmonella infections. Antimicrob. Agents Chemother., 1965:380, 1966. 16. Chmel, H., and Armstrong, D.: Salmonella Oslo: A focal outbreak in a hospital. Amer. J. Med., 60:203, 1976. 17. Citri, N.: The biochemistry and function of f3-lactamase (penicillinase). Advan. Enzymol., 28 :237, 1966. 18. Cohen, S.: Transposable genetic elements and plasmid evolution. Nature, 263 :731, 1976. 19. Cohen, S.N., and Kopecko, D. J.: Structural evolution and bacterial plasmids: role of translocating genetic elements and DNA'sequence insertions. Fed. Proc., 35 :2031, 1976. 20. Costerton, J. W.: Structure and function of the cell envelope of gram negative bacteria. Bact. Rev., 38:87, 1974. 21. Craven, P. C., Jorgensen, J. H., Kasper, R. L., et al.: Amikacin therapy of patients with multiply antibiotic resistant Serratia marcescens infections. Amer. J. Med., 62 :902, 1977. 22. Crosa, J. H., Olarte, J., Mata, L. J., et al.: Characterization of an R-plasmid associated with ampicillin resistance in Shigella dysenteriae type 1 isolated from epidemics. Antimicrob. Agents Chemother, 11 :553, 1977. 23. Dale, J. W., and Smith, J. T.: R-factor mediated f3-lactamases that hydrolyze oxacillin: evidence of2 distinct groups. J. Bact., 119:351,1974. 24. Datta, N.: R-factors in E. coli. Ann. N.Y. Acad. Sci., 182:59,1971. 25. Datta, N.: Transmissible drug resistance in an epidemic strain of Salmonella typhimurium. J. Hyg. (Carob.), 60:301, 1962. 25a. Datta, N., and Kontomichalou, P.: Penicillinase synthesis controlled by infectious R-factors in Enterobacteriaceae. Nature, 208 :239, 1965. 26. Davies, J.: Mechanisms of resistance to aminoglycosides. Amer. J. Med., 62:868, 1977. 27. Davis, C. E., and Anandan, J.: The evolution of R-factors: a study of a preantibiotic community in Borneo. New Engl. J. Med., 282:117,1970. 28. DeGraaff, J., Elwell, L. P., and Falkow, S.: Molecular nature of two f3-lactamase specifying plasmids isolated from Haemophilus inJluenzae type b. J. Bact., 126:439, 1976. 29. DelBene, V. E., and Rogers, M.: Comparison of tetracycline and minocycline transport in E. coli. Antimicrob. Agents Chemother., 7:801, 1975.


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30. Doughty, S. C., Martin, R. R., and Greenberg, S. G.: Treatment of hospital acquired infections with amikacin Amer. J. Med., 62:889, 1977. 31. Eisenach, K. D., Reber, R. M., Eitzman, D. V., et al.: Nosocomial infection due to kanamycin resistant R-factor carrying enteric organisms in an intensive care nursery. Pediatrics, 50:395, 1972. 32. Elwell, L. P., deGraaff, J., Seibert, D., et al.: Plasmid linked ampicillin resistance in H. inJluenzae type b. Infect. Immun., 12 :404, 1975. 33. Elwell, L. P., Roberts, M., Mayer, L. W., et aI.: Plasmid mediated Beta-Iactamase production in Neisseria gonorrheae. Antimicrob. Agents Chemother., 11 :528, 1977. 34. Falkow, S.: In Infectious Drug Resistance. Pion Limited, 1975. 35. Farrar, W. E., and Newsome, J. K.: Diversity of ,B-Iactamase activity among clinical isolate of gram negative bacilli. Amer. J. Clin. Path., 65:570, 1976. 36. Farrar, W. E., and Krause, J. M.: Relationship between ,B-Iactamase activity and resistance of enterob act er to cephalothin. Infect. Immun., 2:610,1970. 37. Finland, M.: Changing patterns of susceptibility of common bacterial pathogens to antimicrobial agents. Ann. Intern. Med., 76:1009, 1972. 38. Finland, M., Frank, P. F., and Wilcox, C.: In vitro susceptibility of pathogenic staphylococci to seven antibiotics. Amer. J. Clin. Path., 20:325,1950. 39. Finland, M.: Changes in the susceptibility of selected pathogenic bacteria to widely used antibiotics. Ann. N.Y. Acad. Sci., 182:5, 1971. 40. Finland, M.: Treatment of pneumonia and other serious infections. New EngI. J. Med., 263 :207, 1960. 41. Forbes, I., Gray, A., Hurse, A., et al.: The emergence of gentamicin resistant klebsiella in a large general hospital. Med. J. Austr., 1 :14, 1977. 42. Foster, T. J., and Shaw, W. V.: Chloramphenicol acetyltransferases specified by fiR factor. Antimicrob. Agents Chemother., 3:99, 1973. 43. Franco, J., Eitzman, D. V., Baer, H., et al.: Antibiotic usage and microbial resistance in an intensive care nursery. Amer. J. Dis. Child., 126:318, 1973. 43a. Franklin, T. J., and Godfrey, A.: Resistance of E. coli to tetracycline. Biochem. J., 94:54, 1965. 44. Gangarosa, E. J.: From the C.D.C.: An epidemic association episome? J. Infect. Dis., 126:215, 1972. 45. Gardner, P., Smith, D. H., Moellering, R. C., Jr., et aI.: Recovery of R-factors from a drug free community. Lancet 2:774, 1969. 46. Gardner, P., and Smith, D. H.: Studies on the epidemiology of resistance eR) factors. Ann. Intern. Med., 71 :1, 1969. 47. Gibson, C. D., and Thompson, W. C.: Response of burn wound staphylococci to alternating programs of antibiotic therapy. Antibiot. Ann. 1955-56:32, 1956. 48. Glew, R. H., Moellering, R. C., Jr., and Kunz, L. J.: Infections with Acinetobacter calcoaceticus (Here Ilea vaginicola): clinical and laboratory studies. Medicine, 56:79, 1977. 49. Glew, R. H., Moellering, R. C., Jr., and Wennersten, C.: Comparative studies of activity of nafcillin, oxacillin and methicillin in combination with gentamicin against enterococci. Antimicrob. Agents Chemother., 7:828, 1975. 50. Gottesman, M. M., and Rosner, J. L.: Acquisition of a determinant for chloramphenicol resistance by Coliphage lambda. Proc. Nat!. Acad. Sci., 72:5041, 1975. 51. Grant, R. B., Bannatyne, R. M., and Shapley, A. J.: Resistance to chloramphenicol and ampicillin of Salmonella typhimurium in Ontario, Canada. J. Infect. Dis., 134:354, 1976. 52. Greene, W. H., Moody, M., Schimpff, S., et al.: Pseudomonas aeruginosa resistant to carbenicillin and gentamicin. Ann. Intern. Med., 79:684, 1973. 53. Guerrant, R. L., Strausbaugh, L. J., Wenzel, R. P., et al.: Nosocomial bloodstream infections caused by gentamicin resistant gram negative bacilli. Amer. J. Med., 62:894, 1977. 54. Hable, K. A., Matsen, J. M., Wheeler, D. J., et al.: Klebsiella type 33 septicemia in an infant intensive care unit. J. Pediat., 80:920, 1972. 55. Hallander, H. 0.: Epidemiological and clinical aspects of methicillin resistance and enterotoxin production in S. aureus. Ann. N .Y. Acad. Sci., 182 :98, 1971. 56. Hedges, R. W., Vialard, J. L., Pearson, N. J., et al.: R plasmids from Asian strains of Vibrio cholerae. Antimicrob. Agents Chemother., 11 :585, 1977. 57. Hedges, R. W., Datta, N., Kontomichalou, P., et al.: Molecular specificities of Rfactor determined ,B-Iactamases: correlation with plasmid compatability. J. Bact., 117 :56, 1974. 58. Heffron, F., Rubens, C., and Falkow, S.: Translocation of plasmid DNA sequence which mediates ampicillin resistance: molecular nature and specificity of insertion. Proc. NatI. Acad. Sci., 72:3623, 1975. 59. Heffron, F., Sublett, R., Hedges, R. W., et al.: Origin of the TEM ,B-lactamase gene found on plasmids. J. Bact., 122 :250, 1975.

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