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APPENDIX C GENETICS OF ANTIMICROBIAL RESISTANCE George A. Jacoby1 and K. Brooks Low2 NATURE OF ANTIBIOTIC RESISTANCE Resistance to an antibacterial agent may be either natural or acquired. Some bacterial species are by nature uniformly resistant, some uniformly susceptible, and some include both susceptible and resistant strains. Natural resistance is reflected by gaps in the spectrum of activity of an antibiotic. Thus, broad spectrum drugs such as tetracyclines are effective against many bacterial species while narrow spectrum agents such as polymyxins are active against a restricted group of organisms. Natural resistance (sometimes teemed nonsusceptibility) to a particular antibiotic may be caused by a species' impermeability to an agent or by its lack of a target site, which is present in sus- ceptible species. Acquired resistance develops by mutation or by infection with resistance (R) plasmids. A single mutation may produce a high level of resistance to an antibiotic, such as streptomycin. For other drugs serial passage through gradually increasing concentra- tions of an antibiotic is required, and the resulting resistant strain generally carries multiple mutations, each providing a small increment in resistance. Resistance resulting from mutation is usually specific for the selecting agent or closely related drugs. It is inherited, but is rarely, if ever, spread to other bacteria. While some resistant mutants retain parental growth and virulence, other mutants are partially crippled. Mutants of this type are likely to be unstable and to revert or be lost due to a disadvanta- geous growth rate when antibiotic selection is withdrawn. In con- trast, acquisition of an R plasmid generally confers resistance to clinically achievable levels of an antibiotic in a single step. A plasmid may carry resistance to one or to many chemically unre- lated drugs. Furthermore, plasmids are transmissible by conjugation, transduction, or transformation to other bacteria and can thus disperse their resistance genes. They generally do not have dele- terious effects on cell growth or virulence and may in fact carry Infectious Disease Unit, Massachusetts General Hospital, Boston, Mass. 2Radiobiology Laboratories, Yale University School of Medicine, New Haven, Conn. 92
93 genes contributing to v; r'~1 enc*e no w~11 _ _ _ _ ~ _ ~ __ as to antibiotic resistance. Consequently, while study of mutational resistance to antibiotics has revealed much about normal cellular physiology and the actions of antibacterial agents, the major mechanism for resistance in clinical isolates of bacteria is plasmid carriage. Bacteria with either natural or acquired resistance will be selectively favored in humans, animals, or environments in which antibiotics are used. How commonly this occurs depends on the par- ticular antibiotic and on the genetic potential for development of resistance. CHROMOSOMALLY DETERMINED RESISTANCE . Chromosomal mutations conferring resistance to antibiotics occur as spontaneous, random, and relatively rare alterations in the DNA composition of bacteria at frequencies of 10 to 10 10 per cell generation. Table 1 lists some well-studied examples of mutational resistance. Further details can be found in reviews by Benveniste and Davis (1973) and Davies and Smith (1978~. The biochemical basis of resistance usually involves one of four mechanisms: · the target site is altered so that binding of the anti- hiotic is reduced or eliminated, cell, · there is a block in the transport of the drug into the · the antibiotic is detoxified or inactivated, or · the inhibited step in a metabolic pathway is by-passed. Resistance to the aminoglycosides kasugamycin, neamine, strep- tomycin, or spectinomycin can result from alteration of the amino acid in a specific protein of the 30S ribosomal subunit (Funatsu and Wittmann, 1972; Funatsu et al., 1972; Yaguchi et al., 1976; Yoshikawa _ al., 1975~. Coresistance to neomycin and kanamycin probably in- volves the same mechanism but is less well characterized (Apirion and Schlessinger, 1968) . Ribosomal mutants with broad resistance to aminoglycosides, including gentamicin, kanamycin, neomycin, and tobramycin, also occur, but additional nonribosomal mutations are required to achieve a high level of resistance (Bucker et al., 1977~.
94 TABLE 1 Mechanisms of Mutational Resistance to Antibiotics Drug Target Aminoglycosides Ribosome. Inhibition of protein synthesis Erythromycin Ribosome. Inh'bition of protein synthesis Nalidixic ac id J)NA gyra se Novobiocin DNA gyrase Penic illins Cell wall biosynthesis Rifampin RNA polymera se Sulfonami de Folic acid biosynthesis Trimethoprim Folic acid biosynthesis Mechanism of resistance Altered ribosomal pro- teixls (kasugamyc'n, 82a; neamine, S17; streptomy- cin, S12; spectinomycin, S5) Altered ribosomal protein ~ gent amicin, kanamycin, neomycin, tobramycin, and others, L6a) Altered 16S RNA methylation (kasugamycin) Alt ered drug t rans po rt Altered ribosomal proteins (L4, L22) Altered assembly of ribosomal subunits Altered s ubunit of DNA gyras e (nal idixic or oxolinic ac id `Altered drug bans po rt Altered subunit of DNA gyrase (coumermycin A1, novobio- cin) Altered penic illin-binding proteins Increased synthesis of chromo- s omal bet a-fact ama se Alt ered cel 1 envel ope Altered beta subunit of RNA po lymerase Altered dihydropteroate synthase Thymine au~otrophy s paring folic acid requirement Altered rite osomal protein: S = 30S subunit protein; L = 50S subunit protein.
95 Some kasugamycin-resistant mutants have altered 16S RNA methyla- tion (Helser et al., 1972~. Lower levels of resistance to aminoglycoside antibiotics can also be attributed to mutations in components of the system by which oxidative energy is coupled to drug transport (Kanner and Gutnick, 1972, Sasarman et al., 1968~. Some aminoglycoside transport mutants are broadly resistant to many other related drugs (Thorbjarnardottir et al., 1978~. Resistance to rifampin develops as a result of an alteration in the beta subunit of its target site, RNA polymerase (Tocchini-Valentini et al., 1968~. Escherichia cold mutants that are resistant to erythromycin can be selected with alterations in specific ribosomal proteins or in the assembly of the two ribosomal subunits (Pardo and Rosset, 1977; Wittmann _ al., 1973~. Penicillin-resistant mutants can result from alterations of penicillin-binding proteins (Sprat", 1978; Suzuki et al., 1978) by increased synthesis of a chromo- somally determined beta-lactamase, which is normally produced at low levels (Eriksson-Grennberg et al., 1965) or by changes in the cell envelope (Nordstrom et al., 1970~. Mutants that are resistant either to nalidixic acid or to novobiocin can result from alterations in components of DNA gyrase activity (Gellert et al., 1977; Higgins et al., 1978~. Resistance to nalidixic acid can also be caused by altered drug permeability (Bourguignon et al., 1973~. Finally, resistance to sulfonamide can develop from decreased binding by dihydropteroate synthase (Ortiz, 1970), and resistance to trimethoprim can result from mutation to auxotrophy for thymine, thus sparing this impor- tant end-product of folic acid activity (Stacey and Simson, 1965~. Considering the variety of mechanisms that cause mutational resistance of bacteria to antibiotics, it is perhaps surprising that the genetic mechanism is not more common in clinical iso- lates. Mutational resistance is clinically important for nali- dixic acid (Ronald et al., 1966), streptomycin (Finland, 1955), rifampin (Eickhoff, 1971), and trimethoprim (Maskell et al., 1976) and may be responsible for broadly aminoglycoside-resistant clinical isolates (Bryan et al., 1976), but for most drugs plas- mids provide the major source of resistance to antibiotics.
96 PLASMIDS Plasmids are extrachromosomal genetic elements composed of circular double-stranded DNA. They vary from a molecular weight of approximately 1 million to over 300 million (Hansen and Olsen, 1978~. Hence, they range from one-thousandth to one-tenth the size of the bacterial chromosome and can accommodate from a few to perhaps 500 genes. Essential genes are concerned with plasmid replication and the partitioning of plasmid DNA to daughter cells at the time of bacterial division. Also in this category are genes that deter- mine the number of plasmid copies per cell chromosome and the plasmid incompatibility behavior. In addition to these require- ments, Plasmids may carry other genes. Many Plasmids are infectious or conjugative and possess the genetic machinery for assuring their transmission on contact among bacterial cells by conjugation, a complex process that, for one well-studied plasmid, requires at least 17 genes (Miki et al., 1978~. Plasmids with a molecular weight less than approximately 20 million lack room for this equipment and are generally transfer-deficient (Tra ~ although some Tra plasmids can be transferred ("mobilized") by another conjugative plasmid that is present in the same cell (Clowes, 1972~. A third category of plasmid genes is involved with ~nterac- tions with other replicons and includes genes that inhibit the propagation of certain bacteriophages or the transfer functions of other plasmids. Finally, Plasmids carry genes that effect the host cell's interaction with the environment. Antibiotic resistance genes are the most familiar, but as shown in Table 2 this category also includes genes determining resistance to metallic compounds (Summers and Silver, 1978) including arsenicals used as feed additives, genes for resistance to physical and chemical agents that damage DNA (Lehrbach et al., 1977), genes for specialized metabolic pathways such as sugar fermentation (Smith et al., 1978) or other catabolic functions (Wheelis, 1975), genes deter- mining bacteriocin production and resistance, and genes involved in pathogenicity such as toxin production or colonization by adherence to mammalian cells. Plasmids can be detected by physical or genetic techniques. Traditionally, plasmid DNA has most often been isolated by cen- trifuging a cell lysate in a cesium chloride density gradient
97 TABLE 2 Plasmid-~)etermined Properties Other Than Antibiotic Resistance a Plasmid carrier Gram-negat ive Gram-posit ive Property bacteria bacteria Resistance to metallic compounds Antimony + Arsenic + + Bi smut t, + Boron + Cadmium + Chromiu~r ~+ + Cobalt + Lead + Me rcury + + Nickel + Silver + Tellur ium + Zinc + Res i s tance to age nt s the t damage DNA Alkylating agents + ~ -Irradiat ion + Ultraviolet irradiation + Me t abolic f unct ions Catabolism of camphor, + naphthalene, nicotine, octane, salicylate, or to luene Citrate ut ilization + Fe rmentat ion of lactose, + + raf finose, or sucrose Hemolys in product ion + + Hydrogen sulf ide product ion + Nuclease product ion + Protea se prod~ ct ion + Urease product ion + Bacter~ocin production and + + res i stance
98 TABLE 2 CONT INUED Property - Toxin product ion Ent ero toxin Exfoliat ive t ox' n Other factors af fecting virulence Colonization factors K88, K9 9, and ot he rs Colicin V Vir pla smid a + + From Jacoby and Swartz, in press, with permission. _lasmi d carrier Gram-negat ive bacteria Gram-posit ive bacteria +
99 containing an intercalating dye such as ethidium bromide that binds differentially to plasmid and chromosomal DNA to allow their separation (Helinski and Clewell, 1971~. Recently, agarose gel electrophoresis has been adapted to allow simpler and more rapid visualization of extrachromosomal DNA (Meyers et al., 1976~. The presence of many plasmids can be demonstrated physically and their molecular weights identified within a day by modifications of this technique (Eckhardt, 1978~. Conjugative plasmids can be detected by testing for transfer of the property they determine to a suitable recipient by mating. Transmission of nonconjugative plasmids can be accomplished by transduction with bacteriophage, transformation with purified plasmid DNA, or mobilization with another plasmid. Finally, certain agents, curing compounds, or physical techniques promote the loss of plasmids or select against plasmid-containing cells. Consequently, they can be used to provide presumptive evidence for the presence of a plasmid- determined characteristic. Further physical and genetic characterization of plasmids is generally necessary to identify and assess the spread of plasmids in epidemiological investigations. Basic physical characteristics are plasmid size, DNA composition as expressed by percent guanine plus cytosine, and the fragmentation pattern produced by restric- tion endonucleases, which are enzymes that recognize specific DNA sequences as cleavage sites (Roberts, 1976~. After endonuclease treatment, the fragments that are produced are separated by agarose gel electrophoresis to produce a characteristic pattern for a par- ticular plasmid that is usually, but not always, independent of the host (Causey and Brown, 1978~. Incompatibility behavior is often used to classify plasmids further. Compatible plasmids can coexist in the same host cell while incompatible plasmids cannot and tend to displace each other. By this test plasmids found in enterobacteria can be divided into more than 30 groups (Chabbert et al., 1972; Datta, 1975; Grindley _ al., 1973; Jacob et al., 1977; see Table 3~. Similar incompa- tibility schemes allow Pseudomonas plasmids to be classified into 11 or more groups (Jacoby and Matthew, 1979) and Staphylococcus plasmids to be assigned to at least seven groups (Novick et al., 1977~. Although only a few genes are involved in incompatibility specificity (Palchaudhuri and Maas, 1977), plasmids belonging to the same incompatibility (Inc) group usually share much greater TUNA homology than do plasmids of different Inc groups, thereby reflecting other similarities in gene function (Falkow et al.,
100 TABLE 3 IncomDatihilT,v Groups for Enteric Plasmids ~ Inc _ tibility ~_ r-specific group designation Syno _ s phage susceptibility Examples A RA4 8 locO, ComlO R16, Co1Ia-K9 C locA-C, Com6 R57b, D fd R711b FI fd, MS2, otbers F. R386, ColV, Ent FII fd, MS2, otbers R1, ColB2, Ent FIII fd, others ColB-~98, MIP240 (Hly), Vir FIV fd, MS2, others R124 FV fd F ~ FVI MS2 Hly-P212 G MS2 Rms149 H1 R27 H2 tncS TP116,pWR23 H3 MIP233 I1 IncI~, IF , Ifl R64,ColIb-~9 Com1 I2 Ifl IPI14, MIP241 (Hly) I3 tnCY If1 R621a, ColIb-[M1420 I4 lncI6 Ifl R721 J R391 R387
101 TABLE 3 CO NT INUEI) Incompatibility Donor-specific group Synonyms phage susceptibility Examples L R94 bb M IncL, Com7 RIP6 9 N Com2 Il~e, PRDl R4 6 P Com4 PRR1, PRI)1, Ike, R~1 others Q R300B T PilE/81 Rtsl V R753 W PRD 1 R388 X R6K y Phage P1 9 Com9 RIP7 1 From Jacoby and Swartz, in press, wi th pennission. Based on tl~e compila- tion of Jacob _ al. (1977), R. ~. ~rledges (Royal Postgraduate Medical School, London, personal communication, 1979), and other sour`:es. Abbr evi a t io ns: In c, inc ompa t ib i li t y; Com, compat ibili ty . Abbreviations: Col, colicinogenic plasrnid; Ent, enterotoxigenic plasmid; F. fertility plasmid; Rly, hemolysin-producing plasmid; R. resistance plasmid; Vir, the Vi r plasmirl. Some of the plasmids li s ted are me tabolic plasmid.s carryillg genes for sugar utilization, e. g., Plac, FOlac, pWR23, and MIP233.
102 1974; Grindley _ al., 1973~. Plasmids that are transmissible to _ cold have also been broadly divided into those that inhibit the fertility factor (Fi ~ and those that do not (Fi ~ (Meynell et al., 1968~. Bacteriophage interactions can also be used for plasmid classification. Certain phases, termed donor-specific or male phages (see Table 3), adsorb specifically to surface struc- tures, such as thread-like pill, which are part of the conjugation apparatus for transfer-positive (Tra+) plasmids. Other plasmids characteristically interfere with the propagation of certain phages so that both phage susceptibility and resistance can be used for plasmid classification. PLASMID TRANSFER In the laboratory Tra+ plasmids vary in their transfer fre- quencies from barely detectable values of 10 to 10 to virtually 100X efficiency. Typical average values are 10 to 10 5 per donor. The transfer mechanism of some plasmids is naturally repressed, but is relieved from repression when the plasmid enters a new host (Meynell _ al., 1968~. Other plasmids transfer much more effi- ciently on solid rather than in liquid media. For some plasmids transfer is sensitive to temperature, occurring much more readily at 22 C than at 37°C (Rodriguez-Lemoine et al., 1975~. Such plasmids have been responsible for outbreaks of resistant typhoid fever but could have been overlooked had mating experiments been performed at the conventional temperature (Smith et al, 1978~. _ -viva transfer of plasmids appears to be even less efficient than under laboratory conditions, although it has been observed in the gut of both humans and laboratory animals (Smith, 1969 ~ 1970) e A number of factors have been incriminated. Anderson (1974) found that strains containing R plasmids survive less well in the human intestine than do their R counterparts. The anaerobic environment also reduces the fertility of some plasmids (Burman, 1977) as does the presence of bile salts (Wiedemann, 1972) and the metabolic activity of other gut bacteria, especially Bacteroides (Anderson, 1975~. Finally, smooth colony types are less efficient as plasmid recipients than are the rough variants used for laboratory experi- ments (Jarolmen an] Kemp, 1969~. After a volunteer drank a suspen- sion containing 10 donor organisms, Smith (1969) found that few ingested Rob Escherichia cold strains were able to colonize the intestine, that transfer to other E. cold occurred rarely, and that these strains persisted for only a few days. In similar studies with even larger inocula, Anderson et al. (1973a,b) were unable to detect transfer of R plasmids unless specific antibiotics were also
103 administered, but under these conditions they found that plasmid transfer occurred at high frequency and that R+ organisms could persist for several months. Similar results were obtained by Burton _ al. (1974) who could only detect transfer of a plasmid determining resistance to tetracycline (among other antibiotics) when 1 g of tetracycline was given daily for 9 days. However, high-level shedding of resistant bacteria persisted from 2 to 43 days in the absence of continued administration of tetracycline. Since plasmid-containing strains, like those sensitive to anti- biotics, have variable but finite persistence in the gut flora, continuous administration of antibiotics is more likely to promote plasmid carriage than would intermittent use (Lipton et al., 1975~. PLASMID HOST RANGE An important biological limitation to transfer is plasmid host range, which correlates with plasmid Inc specificity. Trans- fer of intact plasmids between Gram-positive and Gram-negative bacteria has not been accomplished (Mahler and Halvorson, 1977), and transmission of plasmids between facultative and strict Gr~ra- negative anaerobes has proven difficult to demonstrate (Burt and Woods, 1976; Del Bene _ al., 1976~. Many plasmids transfer effi- ciently to closely related bacteria, for example, among E. coli, Salmonella, and Shigella (Datta and Hedges, 1972~. Some, such as certain plasmids found in P. aeruginosa, transfer only to other Pseudomonas species (Shahrabadi et al., 1975~. Hence, it is impor- tant to use a related organism as a recipient when testing for transmission of a potentially plasmid-determined characteristic. Other plasmids are much more promiscuous. Members of the IncP group have been transferred to virtually every Gram-negative host tested as recipient. Not surprisingly, P-type plasmids have been found in a variety of natural hosts. Other plasmid Inc groups tend to be associated more often with particular bacterial species, but the number of plasmids examined for host specificity is still small. The question of whether R plasmids of human and animal origin differ has been examined by Anderson et al. (1975~. They found a high degree of DNA homology between plasmids of the same Inc group inde- pendent of their origin, suggesting a common pool of R factors in humans and animals.
104 PLASMID DISTRIBUTION Plasmids have been found in practically all bacteria that have been carefully investigated. Not all are R plasmids. In- deed, some have no established function and are termed cryptic plasmids. Table 4 lists the antibiotic resistances that have been found on plasmids in particular pathogenic organisms. Gram-negative pathogens have been studied most intensively. R plasmids have been observed in Enterobacteriaceae including Enterobacter, Escherichia, Klebsiella, Proteus, Providencia, Salmonella, Serratia, and Shigella and in other Gram-negative pathogens including Acinetobacter, Bor- detella, Haemophilus, Neisseria gonorrhoeae, Pasteurella, Pseudomonas, Vibrio, and Yersinia. Among the Gram-positive pathogens, plasmids determining resistance are well known in Staphylococcus aureus and S. epidermidis and have also been found in streptococci of groups A, B. and D. Some streptococcal plasmids are even conjugative (Jacob and Hobbs, 1974~. Plasmids have been found in S. pneumonias as well (Young and Mayer, 1979) but have not yet been associated with anti- biotic resistance. Recently, R plasmids have also been found in anaerobes including Clostridium perfringens (Brefort et al., 1977) and Bacteroides fragilis (Mancini and Behme, 1977; Privitera et al., 1979; Tally et al., 1979~. Evidently, plasmids provide a general mechanism for carrying a potentially advantageous but dispensable function in some members of a population in a form that can be spread to other bacteria under selective conditions (Clowes, 1972~. PLASMID PREVALENCE Studies of primitive societies little exposed to antibiotics and of bacterial isolates from the preantibiotic era indicate that R plasmids preexisted contemporary antibiotic usage. Mare (1968) found resistant Gram-negative bacteria in 10% of over 500 fecal specimens from African bushmen and wild animals, but none of the bacteria could transfer their resistance patterns. In contrast, Gardner et al. (1969) reported the existence of R factors in 2 of 40 stool specimens from humans in a remote section of the Solomon Islands, and Davis and Anandan (1970) found six R+ strains of _ cold in 4 of 128 stool specimens from natives living in North Borneo. These low frequencies provide a reasonable estimate of plasmid prevalence in the absence of obvious antibiotic selection.
105 TABLE 4 Detection of Plasmid-Determined Antibiotic' Resistance in Pathogens a b Antibiotic _ Organism Beta-lactam Cm Gm Km MLS Sm Su Tc Tm Tp Gram-negative Enterobacteriaceae + + + + + + + + + Haemophi lus + + + + inf luenzee Neisseria gonorrhoeae Pseudomonas aerug ino sa Gram-posi tive + + + + + + + + Stachvlococcus + + + + + + + + aureus Staphylococcus + epidermidis . Streptococcus . . Group A Group B + + + Group D + + + + Anaerobes Clostridium perf ringers Bacteroides f ragilis + + + + ~ From Jacoby and Swartz, in press, with permission. + b Abbreviations: Beta-lactam, penicillin and cephalosporin; Cm, chlo~ amphenicol; Gm, gentamicin; Km, kanamycin; MLS, macrolide (erythro- mycin), lincomycin, and streptogramin B-type antibiotics; Sm. streptomycin; Su, sulfonamide; Tc, tetracycline; Tm, tobramycin; Tp, tr ime tho pr im.
106 The incidence of R factors in intestinal bacteria of healthy people in industrial societies is much higher. For example, Linton et al. (1972) reported that 53% of 193 healthy adults and children in Bristol, England, who had not received antibiotics or had not been recently hospitalized, carried antibiotic-resistant coliform bacteria in their feces. Transmissible R plasmids could be demonstrated in 61% of the resistant strains. Antibiotic usage exerts a strong selective pressure favoring R plasmid carriage. Smith (1971) found sensitive E. cold in the fecal flora of 59% of pigs, 73% of chickens, and 90X of calves that had not been fed antibiotics, but in less than 2% of pigs and chickens that had been fed tetracycline. Datta et al. (1971) reported that 30Z of the women admitted to hospitals in England excreted resistant _ cold but that 18 of 18 given tetracycline had resistant _ cold in their stools and that 66% of these strains contained transmissible R plasmids. Many studies have shown a higher frequency of antibiotic-resistant bacteria among hospitalized patients than among people outside the hospital. There is a higher incidence of antibiotic resistance in colifonms from hospital sewers than from domestic sewers, and multiple resistance is more common (Lipton et al., 1974~. The incidence of plasmids in hospital isolates is very high. For example, using agarose gel electrophoresis Laufs and Kleimann (1978) found plasmid DNA in 76% of E. colt, 31% of Proteus, 95% of Klebsiella pneumonias, 24% of Pseudomonas aeruginosa, 40% of Staphylococcus aureus, and 36X of group D streptococci from clin- ical sources. Multiple plasmids were often present, and 45X of the plasmid-containing enteric strains could transfer antibiotic resistance to an E. cold recipient. In some cases, the presence of multiple plasmids leads to higher levels of antibiotic resist- ance than is observed with a single plasmid (Pinney and Smith, 1974). In some pathogenic strains multiple resistance is becoming more common. In Japan, where plasmid-determined resistance to chloramphenicol, streptomycin, sulfonamide, and tetracycline appeared in Shigella during the 1950's, this pattern of quadruple resistance has remained by far the most common (Tanaka et al., 1975~. In Great Britain, on the other hand, plasmid-determined resistance to streptomycin and sulfonamide appeared in Salmonella typhimurium in 1963. As antibiotic usage, especially in animal feed, increased, resistance to tetracycline, ampicillin, and kanamycin as well as to streptomycin and sulfonamide became the predominant pattern (Anderson, 1968~. In this case the strains
107 acquired additional plasmids. In the last decade, the introduc- tion of newer aminoglycosides has been followed by the appearance and spread of plasmid-determined resistance to amikacin, genta- micin, and tobramycin. In some cases, multiple resistance seems to be acquired as new resistance genes are incorporated into existing plasmids (Smith et al., 1975~. PLASMID-DETERMINED ANTIBIOTIC RESISTANCE Plasmids determine antibiotic resistance by the same general biochemical strategies discussed previously, but the mechanisms are usually different from those produced by chromosomal mutations. Some specific plasmid-determined mechanisms of resistance, which are listed in Table 5, have been reviewed by Davies and Smith (1978~. Penicillins and cephalosporins are hydrolyzed by beta-lactamases, of which 11 types produced by plasmids of Gram-negative bacteria can be differentiated (Matthew, 1979~. Chloramphenicol is also detoxified, but by acetylation. The aminoglycosides are attacked by a variety of enzymes that attach acetyl, nucleotidyl (adenylyl), or phosphate groups to key hydroxyl or amino moieties on the antibiotic. Modifi- cation of aminoglycoside occurs at a faster rate than the uptake of aminoglycoside so that no active drug is available to block protein synthesis. Tetracycline resistance is not due to drug degradation or modification but rather to a combination of diminished tetracy- cline uptake and an intracellular inhibition of tetracycline activity (Levy and McI4urry, 1978~. Plasmid effects on antibiotic uptake have also been suggested for chloramphenicol, kanamycin, and penicillin. Plasmid-determined resistance to the MLS group of drugs (macrolide, lincosamide, and streptogramin B-type antibiotics) involves methylation of adenine residues in 23S RNA of the larger ribosomal subunit so that binding of these drugs to their target is prevented. Resistance to three drugs used in animal feed, oleandomycin, tylosin, and virginiamycin, develops in this way. Finally, resistance to sulfonamides and tri- methoprim involves plasmid-detenmined folic acid biosynthetic enzymes that bypass the metabolic block produced by these chemotherapeutic agents. It is noteworthy that whenever antibiotic degradation is not involved, the drug can remain to exert a continued selective pressure for resistant organisms. For a few antibiotics plasmid-determined resistance has not yet been found. Specifically, this mechanism of resistance is not known to occur for bacitracin, bambermycin, colistin, monensin, nalidixic
108 TABLE 5 Mechanisms of Plasmid-Determined Antibiotic Resistancea Drug - b Ami noglycos ides Amikac in Gentamicin Kanamycin Str ep tomyci n Spe ct inomyci n To bramyci n Beta- lactams Penic~ llins Chloramphenicol Erythromyc'n L· e 1ncomycln Sulfo namide Tetracy clin e Trime tho prim . aFrom Jacoby and Swartz Mechanism of resistance 6 ~ -_-Ace tyltr ens fe rase 3-_- and 6'-N-Acetyltransferases 2 "-O-Adenyl yltrans ferase 6'-N-Acetyltransferase 4'-0-Adenylyltransferase 3'-0-Phosphotransferase 3"-_-Adenylyl tr ens f e rase 3 "-O-Phos photrans ferase 3"-_-Adenylyltr ens fe rase 3-N- and 6 '-N-Acetyltrans ferase 2 "-O-Adenylyltr ens ferase Various beta-lactamases (TEM-1, TEM-2, OXA-1, 0XA-2, OXA-3, PSE-1, PSE-2, PSE-3, PSE-4, HMS-1, SHY-1) 3-0-Ace t yl tra ns fe r as e Methylation of 23S RNA lIethylation of 23S RNA Subs titute dihydropteroate synthase Diminished uptake Inh ibi tion of intracel lular act ion Substitute di hydrofolate reductase in press, with permission. bAdditional modifying enzymes for aminoglycosides found in Gram-positive bacteria are reviewed by Da~7ies and Smith ( 19 78~.
109 acid, nitrofuran, novobiocin, polymyxin, rifampin, or vancomycin, although resistance to each of these agents can occur intrinsically or by chr~mosomal mutation. PLASMID STRUCTURE AND REARRANGEMENTS For some plasmid systems the resistance and transfer genes occur on separate molecules, each of which is capable of independ- ent replication. Transfer of such an aggregate or multimolecular planted is achieved by mobilization of the Tra R-plasmid by its Tra partner. In other plasmids the resistance genes and transfer functions are combined in a single molecule which may or may not be able to dissociate into separate replicons (Clowes, 1972~. In a cointegrate plasmid the resistance genes are often clustered to- gether on the it-determinant segment of the plasmid, while the genes for transfer and other basic plasmid functions are clustered on the resistance transfer factor (RTF) segment. These two segments may be linked by specific repeated segments of DNA known as insertion sequences since the insertion of such a DNA segment into a struc- tural gene results in its inactivation. Interactions between paired insertion sequences allow plas- mids with this structure to dissociate into two component replicons. The resistance genes may also transpose to another replicon as a unit or duplicate to higher degrees of multiplicity in the presence of an antibiotic to amplify the level of resistance (Ptashne and Cohen, 1975; Rownd et al., 1975; Yagi and Clewell, 1976~. Individ- ual resistance genes or groups of genes may also be located between repeated segments of DNA, which allow them to be transposed to another replicon such as another plasmid, a phage genome, or the bacterial chromosome. Such a transposable genetic element has been termed a transposon (Hedges and Jacob, 1974~. Transposons, some of which are listed in Table 6, comprise virtually all types of plasmid-determined antibiotic resistance, including resistance to beta-lactam antibiotics, chloramphenicol, erythromycin, gentamicin, kanamycin, streptomycin, sulfonamide, tetracycline, and trimethoprim. Plasmids must thus be thought of as assemblages of potentially transposable resistance genes. Transposition if not a common event. It generally occurs at fre- quencies of 10 or less, even under the most favorable conditions (Kleckner, 1977~. However, where the selection pressure is high, the results of transposition events can be dramatic. Multire- sistant R plasmids have evolved in this way (Rubens et al., 1979~. Furthermore, a resistance gene that becomes prevalent in one group
110 TABLE 6 Selected Drug-Resistance Transposons a Molecular 6 Transposon Drug resistance weight (x 10 Tnl Ampicillin 3.2 Tn4 Ampicillin, streptomycin, sulfonamide 13. 6 Tn5 Kanamycin 3. 5 Tn7 Streptomycin, trimethoprim 8.5 Tn9 Chloramphenicol 1. 7 TnlO Tetracycline 6.2 Tn551 Erythromycin, lincomycin 3.5 Tnl 6 96 Chl oramphen ic al, ge ntamic in, s tr ep to - 9. 1 mycin, sulfonamide . From Jacoby and Swartz, in press, with permission
111 of organisms, such as the transposon responsible for ampicillin resistance in enteric bacteria (Matthew and Hedges, 1976), seems to have crossed genetic boundaries to appear in unrelated patho- gens and to have caused resistance to penicillin in gonococci (Elwell et al., 1977) and to ampicillin in Raemophilus influenzas (Elwell _ al., 1975) by inserting into plasmids that are ind- igenous to these organisms. Thus, when one takes into account all the known modes of al- teration and transfer of genes in bacteria, it is clear that there is a broad spectrum of factors that can influence the activities and spread of genetic determinants leading to antibiotic resistance. These factors are: Mutation: creation or loss of enzymatic activity by changes in DNA base sequence (see Table 1) Derepression: increase in production of enzyme causing re- sistance in response to an external stimulus (Franklin, 1967) Amplification: tandem multiplication of it-determinants per plasmid or increased number of plasmid copies per cell (Uhlin and Nordstrom, 1977) Multiple plasmids: total level of antibiotic resistance sometimes higher than that due to individual plasmids Genetic rearrangements: - Transposition: insertion of R-detenminants from one region of DNA into a new location Fusion: recombination between two plasmids to produce a composite plasmid Disassociation: separation of a plasmid into its component replicons Microevolution: small deletions, insertions, or duplications (Timmis et al., 1978) Gene transfer (reviewed by Low and Porter, 1978~: Conjugation: transfer through cell-to-cell con- nection of either plasmid or chromosomal TUNA
112 Hormone-stimulated conjugation: intercellular signalling leading to cell aggregation and high frequency trans- fer (Dunny et al. 197 9 ~ Transduction: infection by viral particles that carry drug resistance genera) from previous host cell Transformation: uptake of naked DNA from surrounding medium Loss of function: Genetic deletion Insertional inactivation Plasmid loss or curing PLASMIDS AND VIRULENCE Other genes of importance for bacterial infection are also carried on plasmids. Production of enterotoxin in both Staphylo- coccus aureus (Shalita et al., 1977) and E. cold (Smith and Linggood, 1971b) may be determined by plasmids. Two types of E. cold enterotoxin are produced: a small, heat-stable polypeptide and a larger, heat-labile protein toxin that has a structure and mechanism of action similar to that of cholera toxin (Dallas and Falkow, 1979~. The production of toxin alone is not sufficient for _ cold to cause disease in animals. Enteropathogenic strains also carry a plasmid that codes for a surface antigen that facili- tates adherence to the gut wall (Smith and Linggood, 1971a). Species-specific antigens have been described for pig strains (the K88 antigen), calf and lamb strains ~ the K99 antigen), and strains of human origin (Evans et al., 1975; McNeish et al., 1975~. Com- monly, enterotoxin-producing E. cold also carry R plasmids, and transfer of resistance often results in concurrent transfer of enterotoxin production (Echeverria et al. , 1978~. Genes for anti- biotic resistance and enterotoxin production can even be linked on the same plasmid (Gyles et al., 1977), and there is evidence that the genes for toxin production are transposable (So et al. , 1979~. Whether plasmid carriage has other effects on virulence has long been debated. Watanabe (1971) initially found smooth R+ strains of Salmonella typhimurium less virulent for mice than . their R parents, but he was unable to repeat the observation.
113 Smith (1972) found that a few R plasmids caused a marked reduc- tion in virulence of S. typhimurium for chicks, but that most had only a slight or no effect. The high prevalence of plasmids in hospital isolates of a variety of pathogenic bacteria suggests that most R+ strains are fully virulent. In particular, Br~,mfitt _ al. (1971) found that the incidence of resistant fecal E. cold was the same in women with or without urinary tract infection, that those women with resistant organisms in the urine carried the same organism in the stool, and that R plasmid carriage neither hindered nor facilitated infection of the urinary tract by intestinal E. colt. _ cold that cause extraintestinal infections in humans are not likely to be toxigenic or invasive (Wachsmuth et al., 1975~. Whether they have other virulence properties is a subject of ac- tive current investigation. Smith (1974) found that an E. cold strain causing bacteremia in a lamb carried a virulence plasmid (dir) that determined an exotoxin, which was lethal to other animals when intravenously injected. Only one of 190 E. cold isolates had the same property and it also came from a lamb with bacteremia. In the same study, Smith (1974) also found an E. cold strain isolated from an outbreak of bacteremia in chickens that could transfer increased animal lethality to other _ colt. Increased survival in blood and peritoneal fluids rather than toxin produc- tion was associated with this virulence factor, which proved to be identical with the Col V plasmid (Smith and Huggins, 1976~. The prevalence of Col V is high in invasive E. cold strains from ani- mals and humans. The production of colicin per se is not essential for enhanced virulence (Quackenbush and Falkow, 1979~. Rather, increased virulence correlates with serum resistance and, hence, with decreased sensitivity to host defense mechansims that depend on antibody and complement (Binns et al., 1979~. Col V plasmids can carry antibiotic resistance genes. Therefore, as in the case of enterotoxin production, factors that increase the prevalence of R plasmids can also increase the prevalence of virulence factors. ATTACK ON R FACTOR REPLICATION OR TRANSFER A wide variety of physical and chemical agents, including some antibiotics and at least one drug used in animal feed, bam- benmycin, can block pleased transfer or enhance loss In vitro (Brinton, 1971~. Conjugation, which is probably the predominant
114 mode of plasmid transfer in nature, requires several conditions. The formation of sex pill, which is essential for mating pair formation, requires a certain temperature ranged For the well studied F factor, this range is approximately 30 C to 43 C. The recipient cell surface must also contain certain components such as outer membrane proteins, the lack-of which results in conjugative deficiency (Skurray et al., 1974~. In the labora- tory, plasmid transfer can be blocked by the addition of a small amount of sodium dodecylsulfate (Yokota and Akiba, 1961), which presumably disrupts either the recipient or donor cell surface. Rifampin (M`ndi and Btl~di, 1974), levallorphan Closer et al., 1971), phenylethyl alcohol (Brinton, 1971), donor-specific phases, or the concomitant presence of an Fi+ plasmid prevent formation or function of sex pill as do mutations in the plasmid genes that confer transmissibility (tra genes). Nalidixic acid also blocks DNA transfer during conjugation (Hane, 1971), presumably by its action on DNA gyrase. In many studies these agents have been tested against only a limited number of plasmids. Consequently, whether their effects apply to all plasmids is not known. Their effectiveness in block- ing transfer _ viva has not been studied. It is possible that mutants resistant to their action could develop. Further studies are needed to determine the potential value of this approach. APPLICATIONS AND FUTURE PROSPECTS The variety of mechanisms that bacteria have evolved to deal with antibiotics is impressive, and there is no assurance that new ones will not appear. Plasmids provide microorganisms with a remarkably versatile system for packaging resistance genes, toxin determinants, and other virulence factors in a fond that can be both transmitted from cell to cell and transposed from plasmid to plasmid. While there appears to be!a natural barrier to the trans- fer of plasmids between Gram-negative and Gram-positive organisms, there is considerable potential for the flow of genetic information within each group. Antibiotic usage leads to antibiotic-resistant organisms in both animals and humans. Studies not reviewed here leave little doubt that resistant organisms from animals are, on occasion, a cause of human disease. Even if certain organisms lack the ability to infect both humans and animals, resistance plasmids that can be carried by bacteria from animals or humans share no such specificity. Hence, antibiotic resistance arising in animals from subtherapeutic antibiotic usage is undoubtedly a potential cause of
115 resistance of bacteria to antimicrobials used to treat infections in humans. The relative importance of an animal reservoir of resistant pathogens is, however, not known. One could assume that selec- tion of resistant bacteria should be proportional to antibiotic usage for animals or humans. Therefore, since approximately 36% of U.S. antibiotic production in 1974 was devoted to animal feeds or other nonmedicinal uses (Food and Drug Administration, 1978), one might assume that a similar percentage of the total selection pressure for resistant bacteria arose from the use of antibiotics in animal feeds. In one attempt to study the issue directly, Richmond and Lint on (1980) conducted a survey of tetracycline usage in the Avon area near Bristol, England. They found that most tetracy- cline was prescribed by general practitioners in their offices rather than by physicians in hospitals. From earlier studies of the effect of the antibiotic on fecal flora, they calculated that 0.75% of Avon's population would be expected to excrete tetracy- cline-resistant organisms because of tetracycline therapy. "On the basis of the data reported here, they concluded, 'there hardly seems a need to postulate a veterinary source for the resistant colifonms encountered in the human population.' However, the observed frequency of tetracycline-resistant coliforms in Bristol sewage in 1974 was 3% (Lipton et al., 1974~. It can be argued that the use of other antibiotics to treat humans and the linkage of other antibiotic resistance genes to tetracycline resistance on plasmids account for the discrepancy between 0.757 and 3% or, al- ternatively, that only 25% of the observed tetracycline resistance can be attributed to the use of antibiotics in humans. Further surveys of this sort are needed. Inasmuch as some antibiotics suited for use in animal feed do not select for transmissible resistance to drugs used in humans, studies sould be conducted to document the potential economic and therapeutic benefits of their subtherapeutic use. More extensive epidemiological studies of nosocomial infection caused by plasmids in bacteria are also desirable. Many invest- igators have documented the prevalence of antibiotic resistance in hospital pathogens. Combining detailed classification of resistant organisms and molecular and genetic characterization of the plasmids they contain should help elucidate the relative roles of cross-infec- tion by resistant organisms as opposed to spread of resistance by
116 particular plasmids. The epidemiology of plasmids in resistant Gram-positive pathogens has been particularly neglected. Finally, while the search continues for better antibiotics and combinations that are effective against plasmid-containing strains, attention should also be directed to a search for natu- rally occurring agents that might directly attack R factor replication or maintenance.
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