presence of high concentrations of antibiotics. The resulting increase in MICs can make an antibiotic useless for chemo- therapy. Antibiotic tolerance is ...
ANTIMICROBIAL AGENTS
AND
CHEMOTHERAPY, OCt. 1986, p. 521-527
Vol. 30, No. 4
0066-4804/86/100521-07$02.00/0 Copyright © 1986, American Society for Microbiology
MINIREVIEW Antibiotic Tolerance
among
Clinical Isolates of Bacteria
ELAINE TUOMANEN,1 DAVID T. DURACK,2 AND ALEXANDER TOMASZ1*
The Rockefeller University, New York, New York 10021,1 and Division of Infectious Diseases, Duke University Medical Center, Durham, North Carolina 277102
Bacteria continue to evolve antibiotic resistance mechanisms which enable them to grow and multiply in the presence of high concentrations of antibiotics. The resulting increase in MICs can make an antibiotic useless for chemotherapy. Antibiotic tolerance is fundamentally different from all other resistance mechanisms in that it does not involve a change in the MIC of the drug. In antibiotic tolerance, bacteria evade only the killing action of an antibiotic. Virtually all studies on this phenomenon have been restricted to tolerance to beta-lactam antibiotics and other cell wall inhibitors, and throughout this review the term refers to this class of antibiotics. Data concerning the phenomenon of tolerance have accrued rapidly during the past several years. In this two-part minireview, based in part on a symposium and roundtable discussion held at the 25th Interscience Conference on Antimicrobial Agents and Chemotherapy in Minneapolis, Minn., in 1985, we have attempted to summarize and critically evaluate this new information. The first part of the minireview considers the mechanisms affecting survival during antibiotic treatment, the phenotypic tolerance of slowly growing and nongrowing bacteria, and antibiotic tolerance in animal models. The second part will deal with problems in the in vitro determination of antibiotic tolerance in clinical isolates of bacteria (J. C. Sherris, Antimicrob. Agents Chemother., in press). A recent critical review (17) of the literature on clinical isolates of tolerant bacteria has already pointed out the multiplicity of definitions and the often inadequate techniques used for the characterization of such isolates. Genotypic versus phenotypic tolerance. Both genetic and physiological factors are known that can selectively suppress susceptibility to the killing action of antibiotics without a change in the MIC, i.e., make bacteria antibiotic tolerant. Genotypic tolerance was first recognized in 1970 in vitro (38); physiological or phenotypic tolerance was described in 1942 by Hobby et al. (19) and named many years later (17, 39). Both forms of tolerance are now known to occur in vivo (17, 39). In terms of chemotherapy, phenotypic tolerance is likely to be a far more frequent obstacle to cure in vivo than genotypic tolerance, because phenotypic tolerance is a property of virtually all strains of bacteria which is manifest in response to just some growth conditions. The most universal example of phenotypic tolerance is that of the nongrowing dormant bacterium (39).
CONTROVERSIES SURROUNDING THE DEFINITION AND MECHANISMS OF GENOTYPIC TOLERANCE Need for standard reference strains. The term antibiotic tolerance was coined in 1970 to describe the peculiar penicillin response of some laboratory strains of pneumococci (38). Whereas the familiar strains rapidly lysed and lost viability during exposure to penicillin concentrations above the MIC, the new strains did not lyse at all and lost viability at a substantially lower rate. By definition, tolerance involves a reduction in the rate of antibiotic-induced killing of a whole bacterial population, relative to some standard culture or by comparison with the behavior of cultures of a strain from which the tolerant strain was derived. One of the obvious problems with the identification of a suspected tolerant strain among clinical isolates is the lack of an adequate standard of comparison. Tolerant bacteria isolated in the laboratory are not totally immune to the bactericidal effect of penicillin but lose viability more slowly than the bacterium from which they were derived. The parent of a suspected tolerant strain detected in clinical specimens is, of course, never known, and therefore a valid comparison strain analogous to the parental cell of a laboratory mutant clearly does not exist. This situation could be considerably improved by the introduction of reference strains with reproducible rates of viability loss or lysis. Autolytic versus tolerant mutants. From the onset, the search for antibiotic tolerance among clinical isolates carried the more or less explicit assumption that such bacteria would be defective in antibiotic-induced autolysis in a manner similar to that of the laboratory isolates of pneumococci described in 1970 (38). However, this need not be the case. The pneumococcal strains and mutants in which antibiotic tolerance was first described and studied were not isolated on the basis of selection for cells that survive treatment by penicillin. They were isolated on the basis of decreased autolysis, i.e., a property directly related to the poor functioning of an enzyme (autolysin). It was during the characterization of these mutants that their peculiar (tolerant) behavior during antibiotic treatment was recognized. Should it be expected, therefore, that mutants isolated by selection for survival during penicillin treatment would also all be autolytic mutants? Certainly not. The reason for this is the complexity of factors, in both the bacterial cell and its environment, that can influence the survival of bacteria during antibiotic treatment. In fact, when survival during penicillin treatment was used in the laboratory for the selection of tolerant mutants of pneumococci or Escherichia coli, the mutants were not only autolysis-defective cells but also included bacteria with normal levels of autolytic activity
*Corresponding author. 521
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MINIREVIEW
which nevertheless could not be lysed by penicillin (21, 44). There is a wide variety of conceivable mechanisms for the increased survival of bacteria during antibiotic treatment, and specific illustrations of this point are provided in a recent review of antibiotic tolerance (17). In conclusion, although an autolytic defect often leads to antibiotic tolerance, tolerance need not be caused only by an autolytic defect. Mechanisms of improved survival in clinical isolates. At least four mechanisms have already been described for clinical isolates of bacteria that contribute to superior survival during treatment with penicillin or other cell wall inhibitory antibiotics, and it would be confusing to refer to all of these by the term tolerance. (i) Recent reports suggest that staphylococcal (14) or "Streptococcus viridans" (M. Glauser, personal communication) isolates differ strikingly and in a reproducible, strain-specific manner with respect to degree of survival after overnight treatment with antibiotic. This seems to be independent of the antibiotic concentration. Based on the description of these isolates, it is not clear whether strains with high degrees of survival also die at slower rates during antibiotic exposure than strains with low degrees of survival. If final survival is independent of the kill rate, then those strains represent what may be called persister mutants. Persisters may be cell divisional mutants in which the average cell spends a longer time in a phase of the cell cycle in which autolytic or killing events are inhibited (28). (ii) Isolates of several bacterial pathogens, including staphylococci, group A and D streptococci, and "S. viridans," exhibit the paradoxical effect in which bactericidal activity does not occur above a certain concentration of the antibiotic (17). (iii) The South African strains of pneumococci (23) and some of the tolerant staphylococci exhibit a drug-specific tolerance reminiscent of the LYT+ TOL+ laboratory mutants of pneumococci (44). (iv) Many strains of Streptococcus sanguis, some strains of Listeria monocytogenes (43), and probably some strains of enterococci, Streptococcus bovis, and lactobacilli (22) may have more general autolytic defects similar to those of the lysis-defective (tolerant) laboratory mutants of pneumococci and other bacteria. The common feature of these four types of isolates is that their unique behavior does not result from a change in drug susceptibility (i.e., MIC) but from some secondary mechanism involving cidal and lytic effects. We suggest that such mechanisms collectively be referred to as survivor mutations. Antibiotic-induced lysis versus antibiotic-induced death. Another confusion often encountered in the literature on clinical isolates of tolerant bacteria has to do with the notion that antibiotic-induced loss of viability and lysis are synonymous. Although most autolysin-defective laboratory mutants lose viability more slowly than the parent cells (implying that one mechanism contributing to penicillin-induced killing involves the autolytic enzyme), such bacteria are not immune to penicillin-induced death (38). For instance, after overnight incubation with 5 to 10 times the MIC of penicillin, the decrease in viable titer of tolerant pneumococci is very significant (105), although it is still 3 orders of magnitude less than that observed in the parental (LYT+) cells (108). The major difference between mutant and parent is in the early rates of viability loss. The arbitrariness of the criteria often used in the clinical literature to define tolerance (namely, an MBC/MIC ratio higher than 32) is shown by the fact that the pneumococcal mutant(s) in which antibiotic tolerance was first defined would not be classified as a tolerant bacterium by that test. The mechanism of the residual killing observed in the autolysis-defective mutants is not understood, but it
may be related to the residual activity of autolysin in these
cells. Are tolerant bacteria selected for by antibiotic pressure? In 1974, Best and his colleagues tested the response to oxacillin of a number of clinical isolates of Staphylococcus aureus and identified one strain (strain Evans) as a candidate for the first genotypically tolerant clinical isolate (2): strain Evans had about the same oxacillin MIC (0.8 ,ug/ml) as the rest of the strains, but it did not undergo the rapid lysis characteristic of most of the strains when treated with higher concentrations of the drug. Using similar criteria, Mayhall et al. in 1976 (26) and Sabath et al. in 1977 (33) reported a surprisingly high proportion of tolerant strains among staphylococcal clinical isolates. By 1984, the number of bacterial strains that were claimed to exhibit a tolerant response to penicillin treatment had increased to include more than 20 species (17). Whether these strains were all selected for by antibiotic treatment is far from clear. A case for selection could be made for the recently described tolerance in the South African strains of pneumococci (23). Certainly, in the laboratory, tolerant mutants can be consistently selected by cycles of exposure to killing doses of penicillin or other antibiotics, and it would be surprising if this does not occur in in vivo environments as well. It is possible, of course, that in the in vivo environments, contraselecting forces also exist. For instance, S. sanguis cultures which are highly tolerant to penicillin and other wall inhibitors can be rapidly lysed by the supplementation of the antibiotic-containing medium with human lysozyme (20). Some tolerant bacteria can become more sensitized to phagocytosis by antibiotic treatment. It may be significant in this respect that the most abundant sources of tolerant bacteria have been infection sites not easily accessible to the immune system (valvular endocarditis, meningi-
tis, deep-seated infections).
PHENOTYPIC TOLERANCE: SLOWLY GROWING AND NONGROWING BACTERIA Phenotypic tolerance in vitro. Several recent reports have addressed the variety of environmental factors that can influence bacterial survival during treatment with antibiotics. The interested reader is referred to references 5 to 7, 17, 39, and 41. Bacteria deprived of virtually any essential nutrient are phenotypically tolerant to beta-lactam antibiotics when growth stops. This universal rule of bacterial physiology forms the basis of penicillin selection techniques for auxotrophic mutants. Recent studies with chemostatgrown cultures indicate that phenotypic tolerance extends even farther (6, 41). It is not only a property of completely dormant, nongrowing bacteria but also of bacterial cells which are multiplying slowly. In fact, it appears that the rate of antibiotic-induced killing decreases in strict proportion to the decrease in the rate of bacterial growth (41). Thus, enhanced survival during antibiotic therapy can be modulated by the enormous variety of factors which influence the bacterial growth rate. It is interesting that whereas the antibacterial effects of antibiotics include a postantibiotic effect in the case of growing cells (27), phenotypically tolerant cells do not exhibit a delay in the onset of growth when the drug concentration falls below the MIC (40). If the antibiotic is removed from a nongrowing culture and growth conditions are restored, e.g., by the addition of a missing amino acid, bacterial growth resumes without delay. Nongrowing cells are also tolerant to the sensitizing activity of penicillin which promotes lysis by exogenous hydrolytic enzymes, such as
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lysozyme or pneumococcal autolysin (20). Thus, nongrowing bacteria evade a wide variety of direct and synergistic bactericidal effects of antibiotics. Phenotypic tolerance in vivo. Because many common pathogens are multiple heterotrophs, it is not surprising that sporadic or slow multiplication has been frequently observed when bacteria grow at infection sites in vivo (45). Nongrowing bacteria survive virtually indefinitely in the presence of antibiotics and thus represent a reservoir of viable pathogens capable of maintaining infection. Recently, the use of the rabbit meningitis model has allowed us, for the first time, to observe by direct experimentation the influence of the bacterial growth rate on the efficacy of antibiotic treatment in vivo (39). The source of the bacterial inoculum in meningitis is most likely a bacteremia. When pneumococci grown in rabbit serum are transferred into cerebrospinal fluid, the bacteria experience a nutritional "step-down," as evidenced by a prolonged lag before multiplication resumes (Fig. 1). A challenge of such bacteria with penicillin during the growth lag results in only poor killing; rapid and extensive bactericidal activity reappears once the cells start to multiply (Fig. 1). Thus, changes in the growth rate both within one site of infection and between different body locales may constantly create obstacles to rapid bactericidal activity during chemotherapy. Antibiotics which kill phenotypically tolerant bacteria. Despite the general nature of phenotypic tolerance in all nongrowing or slowly growing bacteria, observations in the literature suggest that it may be possible to overcome this mechanism (39, 42). Unusual beta-lactam compounds were recently found which retain various degrees of bactericidal activity against slowly growing and even nongrowing bacteria. In Table 1, several beta-lactam antibiotics are compared in terms of two parameters which have been suggested to provide an index of efficacy of nongrowth killing: MnBC and tolerance window (39). The MnBC is similar to the MBC except for nongrowing cells, i.e., it is the concentration of drug which achieves a 1 log kill in 24 h of cells starved for 10 min before the addition of antibiotic. The tolerance window is a new parameter with no direct equivalent in growing cells. It reflects the finding that bacterial killing becomes progressively more difficult as bacteria are starved for longer
o
(15
in vitro
41*
2-
0
3
6
12
9
15
HRS
FIG. 1. Phenotypic tolerance of nongrowing pneumococci to penicillin in vivo. Pneumococci grow rapidly in serum (dotted line). Upon transfer to cerebrospinal fluid in vivo (solid line) or in vitro (dashed line), growth stops for approximately 3 h and then resumes. No killing follows the addition of penicillin (Pen) to nongrowing cells (A); in contrast, growing cells (0) are killed rapidly, demonstrating that pneumococci are transiently phenotypically tolerant in vivo. Replotted from reference 39.
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TABLE 1. Relative efficacy of compounds in killing nongrowing E. colia
MnBCb for cells Compound
Imipenem CGP 14233 Nocardicin MT 141 Penicillin
MIC
(,ug/ml) 0.8 0.8 32 0.5 5
10
starved for:
30
min
min
2 5 5 5 50
10 10 20 20 >100
Tolerance windowc
45 30 20 20
