ANTIMICROBIAL AGENTS
AND
CHEMOTHERAPY, Jan. 1991,
p.
Vol. 35, No. 1
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0066-4804/91/010001-04$02.00/0 Copyright C) 1991, American Society for Microbiology
MINIREVIEW
Antimicrobial Resistance
among
Enterococci
DAVID J. HERMAN AND DALE N. GERDING*
Division of Infectious Diseases, Department of Medicine, Veterans Affairs Medical Center and University of Minnesota, Minneapolis, Minnesota 55417
Streptomycin alone killed the organism, but only at a concentration of 1,000 pug/ml, far above the safe range in serum. The combination of penicillin at 10 U/ml and streptomycin at 25 pug/ml, which is a subinhibitory concentration of the aminoglycoside, resulted in synergistic killing of the organism, defined as a greater-than-100-fold decrease in CFU compared with the effect of either agent alone. The same result was obtained when penicillin was replaced by other agents which inhibit various steps in cell wall synthesis. When antibiotics which act by different mechanisms, such as cell membrane disruption or inhibition of ribosomal protein synthesis, were substituted for penicillin, the synergistic effect was lost. In further experimetits using ["4C]streptomycin, Moellering and Weinberg showed that the aminoglycoside uptake was markedly enhanced in enterococci growing in the presence of agents which inhibit the synthesis of bacterial cell walls (39). These investigators felt that disruption of the bacterial cell wall permitted the streptomycin to invade the bacteria, resulting in a bactericidal effect.
INTRODUCTION It has long been recognized that single-agent antibacterial therapy yields poor results in the treatment of serious enterococcal infections (14, 34, 48, 68). In 1946, Hunter reported the first successful use of a combination of antibiotics to treat a patient with enterococcal endocarditis who had failed 2 months of therapy with parenteral penicillin (21). Hunter chose penicillin and streptomycin because the organism appeared to be more susceptible in vitro to the two antibiotics than to either agent alone (22). Although the mechanism accounting for the favorable outcome with these agents was not understood, it soon became the standard of care to treat enterococcal endocarditis with them (8, 14). In recent years, however, increasing numbers of enterococcal isolates have acquired high-level resistance to the aminoglycosides (2, 4, 19, 20, 23, 65). Enterococci for which MICs are above clinically achievable levels of the penicillins and vancomycin in serum have also been reported (19, 31, 42, 43, 57, 59). Serious infections with these multiple-drug-resistant strains may not be curable with the traditional therapy. It is the purpose of this paper to review the various mechanisms of enterococcal antimicrobial resistance to both cell wallactive agents and aminoglycosides.
HIGH-LEVEL AMINOGLYCOSIDE RESISTANCE
Havard et al. (17) in 1959 and Jawetz and Sonne (26) in 1966 recognized that not all strains of enterococci were killed by the combination of penicillin and streptomycin. Standiford et al. in 1970 showed that for enterococci which were highly resistant to streptomycin (MIC, >1,000 ,ug/ml), streptomycin did not exhibit synergism when combined with penicillin (54). In the [14C]streptomycin experiments discussed above, Moellering and Weinberg (39) demonstrated that even in the enterococcal strains with high-level resistance to streptomycin (MIC, >2,000 ,ug/ml) (40), penicillin caused an increase in the [14C]streptomycin uptake, despite the fact that synergism did not occur. Therefore, two types of enterococcal streptomycin resistance were postulated: (i) moderate-level resistance (MIC, 62 to 500 ,ug/ml), which is present in most naturally occurring strains and represents a permeability phenomenon which can be overcome by combination with a cell wall-inhibitory antibiotic, and (ii) highlevel resistance (MIC, >2,000 ,ug/ml), which is not a permeability problem since synergism does not occur even though [14C]streptomycin can be demonstrated to enter the cell in the presence of penicillin. Early studies by Zimmermann et al. found high-level streptomycin resistance in one strain to be ribosomally mediated (67). Studies by Krogstad et al. demonstrated the high-level resistance to be due to a transferable 45-MDa plasmid (30). This plasmid encodes two aminoglycoside-modifying enzymes, streptomycin adenyltransferase and neomycin phosphotransferase, which inactivate the antibiotics (29). Later investigations by Eliopoulos et al. showed that while some clinical strains of enterococci with high-level streptomycin resistance produced inactivat-
TAXONOMY In 1937, Sherman divided the streptococci belonging to serological group D into two physiologically different groups; Streptococcus faecalis and S. faecium were placed in the enterococcus division of the streptococci, whereas S. bovis and S. equinus were placed in the viridans division (51). In 1970, Kalina proposed that S. faecalis and S. faecium should be transferred to the genus Enterococcus (27). Nucleic acid studies have confirmed that S. faecalis and S. faecium are only distantly related to S. bovis and S. equinus (10, 28). Therefore, S. faecalis and S. faecium, as well as eight other species, were reclassified into their own genus, Enterococcus (6, 53).
PENICILLIN-AMINOGLYCOSIDE SYNERGY Although several investigators confirmed the synergistic activity of penicillin and streptomycin against the enterococcus both in vitro (22, 25, 47) and in vivo (21, 22, 47, 56, 58), it was not until 1971 that the mechanism by which this combination exhibited synergy was elucidated. Moellering et al. demonstrated that penicillin alone in concentrations ranging from 1 to 1,000 U/ml inhibited growth but did not produce significant killing of an E. faecalis strain (41). *
Corresponding author. 1
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MINIREVIEW
ing enzymes, others possessed a ribosomally mediated mechanism of streptomycin resistance (9). The enzymatically mediated inactivation could be overcome at high streptomycin concentrations (up to 16,000 ,ug/ml), while the ribosomally mediated resistance was absolute since it was not overcome even at streptomycin concentrations up to 128,000 ,ug/ml. The ability to predict in vivo penicillin-aminoglycoside synergy based on the lack of high-level aminoglycoside resistance in vitro is applicable only to studies of E. faecalis. E. faecium strains are usually more resistant to both penicillin and the aminoglycosides (57). For these strains, the lack of high-level aminoglycoside resistance is predictive of a synergistic effect only for gentamicin and streptomycin, because E. faecium produces a 6'-acetyltransferase with activity against kanamycin, tobramycin, and netilmicin (61). All studies involving the combination of penicillin with amikacin, tobramycin, kanamycin, sisomicin, or netilmicin have failed to demonstrate synergism even when the aminoglycoside MICs are less than 2,000 ,ug/ml (37). Therefore, unless the species of an enterococcal isolate will be identified, gentamicin or streptomycin is the aminoglycoside of choice when a synergistic antimicrobial combination is needed. Among 203 enterococcal strains isolated from patients at the Massachusetts General Hospital from 1969 to 1976, 45% demonstrated high-level resistance to streptomycin, 38% demonstrated high-level resistance to kanamycin, and 0% demonstrated high-level resistance to gentamicin (4). In 1979, Horodniceanu et al. in Paris reported the isolation of three strains of E. faecalis harboring plasmids which carried markers for resistance to gentamicin (20). Resistance was mediated by two aminoglycoside-modifying enzymes. The first gentamicin-resistant strains were detected in the United States in 1981 (36). These strains were the first reported strains to be resistant to all aminoglycosides, since the French isolates were susceptible to high-level streptomycin. From 1981 to 1984, the frequency of isolation of E. faecalis strains resistant to high-level gentamicin at the University of Michigan Hospital rose from 0.04 to 3.7% and, by 1987, to 16% of all enterococcal isolates (65, 66). Among 159 enterococcal isolates collected from eight U.S. tertiary-care hospitals in six geographic regions between July 1988 and March 1989, 24.5% demonstrated high-level gentamicin resistance (15). Thus, enterococci with high-level gentamicin resistance are increasing in prevalence and are generally resistant to all aminoglycosides, with the occasional exception of streptomycin (50, 53), which is inactivated by an enzyme distinct from the other aminoglycoside-inactivating enzymes (5). One other aminoglycoside resistance mechanism in a single organism was reported by Moellering et al. in 1980 (38). A patient with E. faecalis endocarditis relapsed after repeated adequate courses with ampicillin plus gentamicin but was cured with ampicillin plus tobramycin. Gentamicin resistance appeared to be secondary to an abnormality in transport through the cell membrane or to impaired binding to intracellular target sites. BETA-LACTAM RESISTANCE
Because [14C]streptomycin permeated enterococci in the presence of a cell wall-inhibitory antibiotic, it was initially felt that any combination of a cell wall-inhibitory antibiotic and an aminoglycoside would cause cell death as long as the enterococcus did not possess high-level resistance to the aminoglycoside (39, 54). However, studies using either
ANTIMICROB. AGENTS CHEMOTHER.
nafcillin-gentamicin (33) or cephalothin-gentamicin (1) combinations in experimental enterococcal endocarditis were unsuccessful unless prohibitively high dosages were used. Further studies implicated penicillin-binding proteins (PBPs) as a cause of the failure of some cell wall-inhibitory agents against enterococci (11). PBPs with a low affinity for penicillin were found in different species of enterococci but were absent from strains of group A, B, C, and G streptococci and Streptococcus pneumoniae (12). Penicillin resistance was directly proportional to the amount or penicillin affinity of PBP 5 and inversely proportional to the amount of PBP 5* produced by the bacteria (62, 64). Penicillinase production in an enterococcus was first reported in 1983 by Murray and Mederski-Samoraj (43). This strain also carried high-level resistance to all aminoglycosides. It is not surprising to find both penicillinase production and gentamicin resistance in the same bacterium, since both can be encoded on transferrable plasmids (42, 43). Although penicillinase-producing enterococci appear to be rare, a few other strains have been isolated (24, 45, 46). Genetic probe studies suggest that the penicillinase was acquired from Staphylococcus aureus (44). E. faecium strains are more resistant to both the cell wall-inhibitory agents and the aminoglycosides than are E. faecalis strains (57). Regardless of the aminoglycoside used, higher concentrations of penicillin are required to produce synergism against E. faecium than against E. faecalis. Concentrations of penicillin less than one-half of the MIC appear to be ineffective in producing synergism in vitro against E. faecium (37). Bush et al. found 14% of all enterococcal clinical isolates to be E. faecium; one third of them were resistant to high-level (>200-pRg/ml) penicillin G, resulting in loss of bactericidal activity when an aminoglycoside was combined with penicillin (3). These E. faecium isolates were not P-lactamase producers. A mechanism for the greater antimicrobial resistance of E. faecium compared with that of E. faecalis is explained by their PBPs. Fontana et al. showed that the loss of the ability of an E. faecium strain to synthesize PBP 5 caused this highly resistant strain to become hypersusceptible not only to penicillin but also to cephaloridine, cephalexin, and ceftazidime, with MICs decreasing from 64 to 0.015 U/ml for penicillin and from 64, 2,048, and 4,096 to 0.06, 1, and 4 jig/ml for the three cephalosporins, respectively (13). The susceptibilities to rifampin and minocycline were not affected. MICs of penicillin for a limited number of the other rarely isolated enterococcal species have been published (35, 63), but generalizations about antibiotic susceptibilities and synergistic effects of cell wall-active antibiotics and aminoglycosides cannot be made.
GLYCOPEPTIDES Only occasional vancomycin-resistant enterococci had been reported in the literature through 1973, and none of them involved clinically significant infections (16, 57, 60). Since 1988, a number of reports have documented vancomycin resistance among enterococci, particularly E. faecium (32, 52, 59). Vancomycin resistance is not as prevalent as penicillin, ampicillin, or aminoglycoside resistance but may be underreported due to problems in detection, particularly when the disk diffusion test is used (49, 55). The topic of vancomycin resistance has recently been the subject of a minireview in this journal (7) and is not discussed further here. Enterococci may no longer be assumed to be susceptible
VOL. 35, 1991
to a combination of a cell wall-active antibiotic and an aminoglycoside. Many strains have acquired resistance to either one or both of these agents. Microbiological screening for enterococci and treatment of serious enterococcal infections may require special considerations, which will be
discussed in part 2 of this article (18). REFERENCES 1. Abrutyn, E., L. Lincoln, M. Gallagher, and A. J. Weinstein. 1978. Cephalothin-gentamicin synergism in experimental enterococcal endocarditis. J. Antimicrob. Chemother. 4:153-158. 2. Basker, M. J., B. Slocombe, and R. Sutherland. 1977. Aminoglycoside-resistant enterococci. J. Clin. Pathol. 30:375-380. 3. Bush, L. M., J. C. Calmon, C. L. Cherney, M. Wendelar, P. Pitsakis, J. Poupard, M. E. Levison, and C. C. Johnson. 1989. High-level penicillin resistance among isolates of enterococci. Ann. Intern. Med. 110:515-520. 4. Calderwood, S. A., C. Wennersten, R. C. Moellering, Jr., L. J. Kunz, and D. J. Krogstad. 1977. Resistance to six aminoglycosidic aminocyclitol antibiotics among enterococci: prevalence, evolution, and relationship to synergism with penicillin. Antimicrob. Agents Chemother. 12:401-405. 5. Chen, H. Y., and J. D. Williams. 1985. Transferable resistance and aminoglycoside-modifying enzymes in enterococci. J. Med. Microbiol. 20:187-196. 6. Collins, M. D., D. Jones, J. A. E. Farrow, R. Kilpper-Balz, and K. H. Schleifer. 1984. Enterococcus avium nom. rev., comb. nov.; E. casseliflavus nom. rev., comb. nov.; E. durans nom. rev., comb. nov.; E. gallinarum comb. nov.; and E. malodoratus sp. nov. Int. J. Syst. Bacteriol. 34:220-223. 7. Courvalin, P. 1990. Resistance of enterococci to glycopeptides. Antimicrob. Agents Chemother. 34:2291-2296. 8. Dowling, H. F. 1965. Present status of therapy with combinations of antibiotics. Am. J. Med. 39:796-803. 9. Eliopoulos, G. M., B. F. Farber, B. E. Murray, C. Wennersten, and R. C. Moeliering, Jr. 1984. Ribosomal resistance of clinical enterococcal to streptomycin isolates [sic]. Antimicrob. Agents Chemother. 25:398-399. 10. Farrow, J. A. E., D. Jones, B. A. Phillips, and M. D. Collins. 1983. Taxonomic studies on some group D streptococci. J. Gen. Microbiol. 129:1423-1432. 11. Fontana, R., G. Bertoloni, G. Amalfitano, and P. Canepari. 1984. Characterization of penicillin-resistant Streptococcus faecium mutants. FEMS Microbiol. Lett. 25:21-25. 12. Fontana, R., R. Cerini, P. Longoni, A. Grossato, and P. Canepari. 1983. Identification of a streptococcal penicillin-binding protein that reacts very slowly with penicillin. J. Bacteriol. 155:1343-1350. 13. Fontana, R., A. Grossato, L. Rossi, Y. R. Cheng, and G. Satta. 1985. Transition from resistance to hypersusceptibility to ,-lactam antibiotics associated with loss of a low-affinity penicillinbinding protein in a Streptococcus faecium mutant highly resistant to penicillin. Antimicrob. Agents Chemother. 28:678-683. 14. Geraci, J. E., and W. J. Martin. 1954. Antibiotic therapy of bacterial endocarditis. VI. Subacute enterococcal endocarditis: clinical, pathologic, and therapeutic consideration of 33 cases. Circulation 10:173-194. 15. Gordon, S., J. Swenson, R. Facklam, N. Pigott, B. Hill, R. Cooksey, W. Jarvis, C. Thornsberry, and the NNIS Enterococcal Study Group. 1989. Program Abstr. 29th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 790. 16. Harwick, H. J., G. M. Kalmanson, and L. B. Guze. 1973. In vitro activity of ampicillin or vancomycin combined with gentamicin or streptomycin against enterococci. Antimicrob. Agents Chemother. 4:383-387. 17. Havard, C. W. H., L. P. Garrod, and P. M. Waterworth. 1959. Deaf or dead? A case of subacute bacterial endocarditis treated with penicillin and neomycin. Br. Med. J. 1:688-689. 18. Herman, D. J., and D. N. Gerding. Antimicrob. Agents Chemother., in press. 19. Hoffman, S. A., and R. C. Moellering, Jr. 1987. The enterococcus: "putting the bug in our ears." Ann. Intern. Med. 106:757-
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761. 20. Horodniceanu, T., L. Bougueleret, N. El-Solh, G. Bieth, and F. Delbos. 1979. High-level, plasmid-borne resistance to gentamicin in Streptococcus faecalis subsp. zymogenes. Antimicrob. Agents Chemother. 16:686-689. 21. Hunter, T. H. 1946. The treatment of subacute bacterial endocarditis with antibiotics. Am. J. Med. 1:83-92. 22. Hunter, T. H. 1947. Use of streptomycin in the treatment of bacterial endocarditis. Am. J. Med. 2:436-442. 23. Ikeda, D. P., A. L. Barry, and S. G. Andersen. 1984. Emergence of Streptococcus faecalis isolates with high-level resistance to multiple aminocyclitol aminoglycosides. Diagn. Microbiol. Infect. Dis. 2:171-177. 24. Ingerman, M., P. G. Pitsakis, A. Rosenberg, M. T. Hessen, E. Abrutyn, B. E. Murray, and M. E. Levison. 1987. Beta-lactamase production in experimental endocarditis due to aminoglycoside-resistant Streptococcus faecalis. J. Infect. Dis. 6:12261232. 25. Jawetz, E., J. B. Gunnison, and V. R. Coleman. 1950. The combined action of penicillin with streptomycin or chloromycetin on enterococci in vitro. Science 111:254-256. 26. Jawetz, E., and M. Sonne. 1966. Penicillin-streptomycin treatment of enterococcal endocarditis. N. Engl. J. Med. 274:710715. 27. Kalina, A. P. 1970. The taxonomy and nomenclature of enterococci. Int. J. Syst. Bacteriol. 20:185-189. 28. Kilpper-Balz, R., G. Fischer, and K. H. Schleifer. 1982. Nucleic acid hybridization of group D streptococci. Curr. Microbiol. 7:245-250. 29. Krogstad, D. J., T. R. Korfhagen, R. C. Moellering, Jr., S. Perzynski, and J. Davies. 1978. Aminoglycoside-inactivating enzymes in clinical isolates of Streptococcus faecalis. An explanation for resistance to antibiotic synergism. J. Clin. Invest. 62:480-486. 30. Krogstad, D. J., T. R. Korflbagen, R. C. Moellering, Jr., C. Wennersten, and M. N. Swartz. 1978. Plasmid-mediated resistance to antibiotic synergism in enterococci. J. Clin. Invest. 61:1645-1653. 31. Leclercq, R., E. Derlot, J. Duval, and P. Courvalin. 1988. Plasmid-mediated resistance to vancomycin and teicoplanin in Enterococcusfaecium. N. Engl. J. Med. 319:157-161. 32. Leclercq, R., E. Derlot, M. Weber, J. Duval, and P. Courvalin. 1989. Transferable vancomycin and teicoplanin resistance in Enterococcus faecium. Antimicrob. Agents Chemother. 33:1015. 33. Lincoln, L. J., A. J. Weinstein, M. Gallagher, and E. Abrutyn. 1977. Penicillinase-resistant penicillins plus gentamicin in experimental enterococcal endocarditis. Antimicrob. Agents Chemother. 12:484-489. 34. Loewe, L., S. Candel, and H. B. Eiber. 1951. Therapy of subacute enterococcus (Streptococcus faecalis) endocarditis. Ann. Intern. Med. 34:717-736. 35. Mackowiak, P. A. 1989. The enterococci: evidence of speciesspecific clinical and microbiologic heterogeneity. Am. J. Med. Sci. 297:238-243. 36. Mederski-Samorai, B. D., and B. E. Murray. 1983. High-level resistance to gentamicin in clinical isolates of enterococci. J. Infect. Dis. 147:751-757. 37. Moellering, R. C., Jr., 0. M. Korzeniowski, M. A. Sande, and C. B. Wennersten. 1979. Species-specific resistance to antimicrobial synergism in Streptococcus faecium and Streptococcus faecalis. J. Infect. Dis. 140:203-208. 38. Moellering, R. C., Jr., B. E. Murray, S. C. Schoenbaum, J. A. Adler, and C. B. Wennersten. 1980. A novel mechanism of resistance to penicillin-gentamicin synergism in Streptococcus faecalis. J. Infect. Dis. 141:81-86. 39. Moellering, R. C., Jr., and A. N. Weinberg. 1971. Studies on antibiotic synergism against enterococci. Il. Effect of various antibiotics on the uptake of 14C-labeled streptomycin by enterococci. J. Clin. Invest. 50:2580-2584. 40. Moellering, R. C., Jr., C. Wennersten, T. Medrek, and A. N. Weinberg. 1971. Prevalence of high-level resistance to aminoglycosides in clinical isolates of enterococci, p. 335-340. Anti-
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microb. Agents Chemother. 1970. 41. Moeliering, R. C., Jr., C. Wennersten, and A. N. Weinberg. 1971. Studies on antibiotic synergism against enterococci. I. Bacteriologic studies. J. Lab. Clin. Med. 77:821-828. 42. Murray, B. E., D. A. Church, A. Wanger, K. Zscheck, M. E. Levison, M. J. Ingerman, E. Abrutyn, and B. MederskiSamoraj. 1986. Comparison of two ,B-lactamase-producing strains of Streptococcus faecalis. Antimicrob. Agents Chemother. 30:861-864. 43. Murray, B. E., and B. Mederski-Samoraj. 1983. Transferable beta-lactamase. A new mechanism for in vitro penicillin resistance in Streptococcusfaecalis. J. Clin. Invest. 72:1168-1171. 44. Murray, B. E., B. Mederski-Samoraj, S. K. Foster, J. L. Brunton, and P. Harford. 1986. In vitro studies of plasmid-mediated penicillinase from Streptococcusfaecalis suggest a staphylococcal origin. J. Clin. Invest. 77:289-293. 45. Patterson, J. E., S. M. Colodny, and M. J. Zervos. 1988. Serious infection due to beta-lactamase-producing Streptococcusfaecalis with high-level resistance to gentamicin. J. Infect. Dis. 158:1144-1145. 46. Rhinehart, E., C. Wennersten, E. Gorss, G. Eliopoulos, R. Moellering, N. Smith, and D. Goldmann. 1988. Program Abstr. 28th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1073. 47. Robbins, W. C. 1949. The summation of penicillin and streptomycin activity in vitro and the treatment of subacute bacterial endocarditis. J. Clin. Invest. 28:806. 48. Robbins, W. C., and R. Tompsett. 1951. Treatment of enterococcal endocarditis and bacteremia. Results of combined therapy with penicillin and streptomycin. Am. J. Med. 10:278-299. 49. Sahm, D. F., J. Kissinger, M. S. Gilmore, P. R. Murray, R. Mulder, J. Solliday, and B. Clarke. 1989. In vitro susceptibility studies of vancomycin-resistant Enterococcusfaecalis. Antimicrob. Agents Chemother. 33:1588-1591. 50. Sahm, D. F., and C. Torres. 1988. Effects of medium and inoculum variations on screening for high-level aminoglycoside resistance in Enterococcus faecalis. J. Clin. Microbiol. 26:250256. 51. Sherman, J. M. 1937. The streptococci. Bacteriol. Rev. 1:3-97. 52. Shlaes, D. M., A. Bouvet, C. Devine, J. H. ShIaes, S. Al-Obeid, and R. Williamson. 1989. Inducible, transferable resistance to vancomycin in Enterococcusfaecalis A256. Antimicrob. Agents Chemother. 33:198-203. 53. Spiegel, C. A., and M. Huycke. 1989. Endocarditis due to streptomycin-susceptible Enterococcus faecalis with high-level gentamicin resistance. Arch. Intern. Med. 149:1873-1875. 54. Standiford, H. D., J. B. deMaine, and W. M. M. Kirby. 1970. Antibiotic synergism of enterococci. Arch. Intern. Med. 126: 255-259. 55. Swenson, J. M., B. C. Hill, and C. Thornsberry. 1989. Problems
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with the disk diffusion test for detection of vancomycin resistance in enterococci. J. Clin. Microbiol. 27:2140-2142. 56. Toala, P., A. McDonald, C. Wilcox, and M. Finland. 1969. Susceptibility of group D Streptococcus (Enterococcus) to 21 antibiotics in vitro, with special reference to species differences. Am. J. Med. Sci. 258:416-430. 57. Tompsett, R., and W. McDermott. 1949. Recent advances in streptomycin therapy. Am. J. Med. 7:371-381. 58. Tompsett, R., and M. Pizette. 1962. Enterococcal endocarditis. Lack of correlation between therapeutic results and antibiotic sensitivity tests. Arch. Intern. Med. 109:146-150. 59. Uttley, A. H. C., C. H. Collins, J. Naidoo, and R. C. George. 1988. Vancomycin-resistant enterococci. Lancet i:57-58. 60. Watanakunakorn, C., and C. Bakie. 1973. Synergism of vancomycin-gentamicin and vancomycin-streptomycin against enterococci. Antimicrob. Agents Chemother. 4:120-124. 61. Wennersten, C. B., and R. C. Moellering, Jr. 1980. Mechanism of resistance to penicillin-aminoglycoside synergism in Streptococcusfaecium, p. 710-712. In J. D. Nelson and C. Grassi (ed.), Current chemotherapy and infectious disease. Proceedings of the 11th International Congress of Chemotherapy and the 19th Interscience Conference on Antimicrobial Agents and Chemotherapy, vol. I. American Society for Microbiology, Washington, D.C. 62. Williamson, R., S. B. Calderwood, R. C. Moellering, Jr., and A. Tomasz. 1983. Studies on the mechanism of intrinsic resistance to beta-lactam antibiotics in group D streptococci. J. Gen. Microbiol. 129:813-822. 63. Williamson, R., L. Gutmann, T. Horaud, F. Delbos, and J. F. Acar. 1986. Use of penicillin-binding proteins for the identification of enterococci. J. Gen. Microbiol. 132:1929-1937. 64. Williamson, R., C. LeBouguenec, L. Gutmann, and T. Horaud. 1985. One or two low affinity penicillin-binding proteins may be responsible for the range of susceptibility of Enterococcus faecium to benzylpenicillin. J. Gen. Microbiol. 131:1933-1940. 65. Zervos, M. J., S. Dembinski, T. Mikesell, and D. R. Schaberg. 1986. High-level resistance to gentamicin in Streptococcus faecalis: risk factors and evidence for exogenous acquisition of infection. J. Infect. Dis. 153:1075-1083. 66. Zervos, M. J., C. A. Kauffman, P. M. Therasse, A. G. Bergman, T. S. Mikesell, and D. R. Schaberg. 1987. Nosocomial infection by gentamicin-resistant Streptococcus faecalis. An epidemiologic study. Ann. Intern. Med. 106:687-691. 67. Zimmermann, R. A., R. C. Moellering, Jr., and A. N. Weinberg. 1971. Mechanism of resistance to antibiotic synergism in enterococci. J. Bacteriol. 105:873-879. 68. Zweifler, B., E. Sar, and I. Feder. 1951. Subacute bacterial endocarditis due to Streptococcus faecalis. Ann. Intern. Med. 34:217-223.
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0066-4804/91/010001-04$02.00/0 Copyright C) 1991, American Society for Microbiology
MINIREVIEW
Antimicrobial Resistance
among
Enterococci
DAVID J. HERMAN AND DALE N. GERDING*
Division of Infectious Diseases, Department of Medicine, Veterans Affairs Medical Center and University of Minnesota, Minneapolis, Minnesota 55417
Streptomycin alone killed the organism, but only at a concentration of 1,000 pug/ml, far above the safe range in serum. The combination of penicillin at 10 U/ml and streptomycin at 25 pug/ml, which is a subinhibitory concentration of the aminoglycoside, resulted in synergistic killing of the organism, defined as a greater-than-100-fold decrease in CFU compared with the effect of either agent alone. The same result was obtained when penicillin was replaced by other agents which inhibit various steps in cell wall synthesis. When antibiotics which act by different mechanisms, such as cell membrane disruption or inhibition of ribosomal protein synthesis, were substituted for penicillin, the synergistic effect was lost. In further experimetits using ["4C]streptomycin, Moellering and Weinberg showed that the aminoglycoside uptake was markedly enhanced in enterococci growing in the presence of agents which inhibit the synthesis of bacterial cell walls (39). These investigators felt that disruption of the bacterial cell wall permitted the streptomycin to invade the bacteria, resulting in a bactericidal effect.
INTRODUCTION It has long been recognized that single-agent antibacterial therapy yields poor results in the treatment of serious enterococcal infections (14, 34, 48, 68). In 1946, Hunter reported the first successful use of a combination of antibiotics to treat a patient with enterococcal endocarditis who had failed 2 months of therapy with parenteral penicillin (21). Hunter chose penicillin and streptomycin because the organism appeared to be more susceptible in vitro to the two antibiotics than to either agent alone (22). Although the mechanism accounting for the favorable outcome with these agents was not understood, it soon became the standard of care to treat enterococcal endocarditis with them (8, 14). In recent years, however, increasing numbers of enterococcal isolates have acquired high-level resistance to the aminoglycosides (2, 4, 19, 20, 23, 65). Enterococci for which MICs are above clinically achievable levels of the penicillins and vancomycin in serum have also been reported (19, 31, 42, 43, 57, 59). Serious infections with these multiple-drug-resistant strains may not be curable with the traditional therapy. It is the purpose of this paper to review the various mechanisms of enterococcal antimicrobial resistance to both cell wallactive agents and aminoglycosides.
HIGH-LEVEL AMINOGLYCOSIDE RESISTANCE
Havard et al. (17) in 1959 and Jawetz and Sonne (26) in 1966 recognized that not all strains of enterococci were killed by the combination of penicillin and streptomycin. Standiford et al. in 1970 showed that for enterococci which were highly resistant to streptomycin (MIC, >1,000 ,ug/ml), streptomycin did not exhibit synergism when combined with penicillin (54). In the [14C]streptomycin experiments discussed above, Moellering and Weinberg (39) demonstrated that even in the enterococcal strains with high-level resistance to streptomycin (MIC, >2,000 ,ug/ml) (40), penicillin caused an increase in the [14C]streptomycin uptake, despite the fact that synergism did not occur. Therefore, two types of enterococcal streptomycin resistance were postulated: (i) moderate-level resistance (MIC, 62 to 500 ,ug/ml), which is present in most naturally occurring strains and represents a permeability phenomenon which can be overcome by combination with a cell wall-inhibitory antibiotic, and (ii) highlevel resistance (MIC, >2,000 ,ug/ml), which is not a permeability problem since synergism does not occur even though [14C]streptomycin can be demonstrated to enter the cell in the presence of penicillin. Early studies by Zimmermann et al. found high-level streptomycin resistance in one strain to be ribosomally mediated (67). Studies by Krogstad et al. demonstrated the high-level resistance to be due to a transferable 45-MDa plasmid (30). This plasmid encodes two aminoglycoside-modifying enzymes, streptomycin adenyltransferase and neomycin phosphotransferase, which inactivate the antibiotics (29). Later investigations by Eliopoulos et al. showed that while some clinical strains of enterococci with high-level streptomycin resistance produced inactivat-
TAXONOMY In 1937, Sherman divided the streptococci belonging to serological group D into two physiologically different groups; Streptococcus faecalis and S. faecium were placed in the enterococcus division of the streptococci, whereas S. bovis and S. equinus were placed in the viridans division (51). In 1970, Kalina proposed that S. faecalis and S. faecium should be transferred to the genus Enterococcus (27). Nucleic acid studies have confirmed that S. faecalis and S. faecium are only distantly related to S. bovis and S. equinus (10, 28). Therefore, S. faecalis and S. faecium, as well as eight other species, were reclassified into their own genus, Enterococcus (6, 53).
PENICILLIN-AMINOGLYCOSIDE SYNERGY Although several investigators confirmed the synergistic activity of penicillin and streptomycin against the enterococcus both in vitro (22, 25, 47) and in vivo (21, 22, 47, 56, 58), it was not until 1971 that the mechanism by which this combination exhibited synergy was elucidated. Moellering et al. demonstrated that penicillin alone in concentrations ranging from 1 to 1,000 U/ml inhibited growth but did not produce significant killing of an E. faecalis strain (41). *
Corresponding author. 1
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ing enzymes, others possessed a ribosomally mediated mechanism of streptomycin resistance (9). The enzymatically mediated inactivation could be overcome at high streptomycin concentrations (up to 16,000 ,ug/ml), while the ribosomally mediated resistance was absolute since it was not overcome even at streptomycin concentrations up to 128,000 ,ug/ml. The ability to predict in vivo penicillin-aminoglycoside synergy based on the lack of high-level aminoglycoside resistance in vitro is applicable only to studies of E. faecalis. E. faecium strains are usually more resistant to both penicillin and the aminoglycosides (57). For these strains, the lack of high-level aminoglycoside resistance is predictive of a synergistic effect only for gentamicin and streptomycin, because E. faecium produces a 6'-acetyltransferase with activity against kanamycin, tobramycin, and netilmicin (61). All studies involving the combination of penicillin with amikacin, tobramycin, kanamycin, sisomicin, or netilmicin have failed to demonstrate synergism even when the aminoglycoside MICs are less than 2,000 ,ug/ml (37). Therefore, unless the species of an enterococcal isolate will be identified, gentamicin or streptomycin is the aminoglycoside of choice when a synergistic antimicrobial combination is needed. Among 203 enterococcal strains isolated from patients at the Massachusetts General Hospital from 1969 to 1976, 45% demonstrated high-level resistance to streptomycin, 38% demonstrated high-level resistance to kanamycin, and 0% demonstrated high-level resistance to gentamicin (4). In 1979, Horodniceanu et al. in Paris reported the isolation of three strains of E. faecalis harboring plasmids which carried markers for resistance to gentamicin (20). Resistance was mediated by two aminoglycoside-modifying enzymes. The first gentamicin-resistant strains were detected in the United States in 1981 (36). These strains were the first reported strains to be resistant to all aminoglycosides, since the French isolates were susceptible to high-level streptomycin. From 1981 to 1984, the frequency of isolation of E. faecalis strains resistant to high-level gentamicin at the University of Michigan Hospital rose from 0.04 to 3.7% and, by 1987, to 16% of all enterococcal isolates (65, 66). Among 159 enterococcal isolates collected from eight U.S. tertiary-care hospitals in six geographic regions between July 1988 and March 1989, 24.5% demonstrated high-level gentamicin resistance (15). Thus, enterococci with high-level gentamicin resistance are increasing in prevalence and are generally resistant to all aminoglycosides, with the occasional exception of streptomycin (50, 53), which is inactivated by an enzyme distinct from the other aminoglycoside-inactivating enzymes (5). One other aminoglycoside resistance mechanism in a single organism was reported by Moellering et al. in 1980 (38). A patient with E. faecalis endocarditis relapsed after repeated adequate courses with ampicillin plus gentamicin but was cured with ampicillin plus tobramycin. Gentamicin resistance appeared to be secondary to an abnormality in transport through the cell membrane or to impaired binding to intracellular target sites. BETA-LACTAM RESISTANCE
Because [14C]streptomycin permeated enterococci in the presence of a cell wall-inhibitory antibiotic, it was initially felt that any combination of a cell wall-inhibitory antibiotic and an aminoglycoside would cause cell death as long as the enterococcus did not possess high-level resistance to the aminoglycoside (39, 54). However, studies using either
ANTIMICROB. AGENTS CHEMOTHER.
nafcillin-gentamicin (33) or cephalothin-gentamicin (1) combinations in experimental enterococcal endocarditis were unsuccessful unless prohibitively high dosages were used. Further studies implicated penicillin-binding proteins (PBPs) as a cause of the failure of some cell wall-inhibitory agents against enterococci (11). PBPs with a low affinity for penicillin were found in different species of enterococci but were absent from strains of group A, B, C, and G streptococci and Streptococcus pneumoniae (12). Penicillin resistance was directly proportional to the amount or penicillin affinity of PBP 5 and inversely proportional to the amount of PBP 5* produced by the bacteria (62, 64). Penicillinase production in an enterococcus was first reported in 1983 by Murray and Mederski-Samoraj (43). This strain also carried high-level resistance to all aminoglycosides. It is not surprising to find both penicillinase production and gentamicin resistance in the same bacterium, since both can be encoded on transferrable plasmids (42, 43). Although penicillinase-producing enterococci appear to be rare, a few other strains have been isolated (24, 45, 46). Genetic probe studies suggest that the penicillinase was acquired from Staphylococcus aureus (44). E. faecium strains are more resistant to both the cell wall-inhibitory agents and the aminoglycosides than are E. faecalis strains (57). Regardless of the aminoglycoside used, higher concentrations of penicillin are required to produce synergism against E. faecium than against E. faecalis. Concentrations of penicillin less than one-half of the MIC appear to be ineffective in producing synergism in vitro against E. faecium (37). Bush et al. found 14% of all enterococcal clinical isolates to be E. faecium; one third of them were resistant to high-level (>200-pRg/ml) penicillin G, resulting in loss of bactericidal activity when an aminoglycoside was combined with penicillin (3). These E. faecium isolates were not P-lactamase producers. A mechanism for the greater antimicrobial resistance of E. faecium compared with that of E. faecalis is explained by their PBPs. Fontana et al. showed that the loss of the ability of an E. faecium strain to synthesize PBP 5 caused this highly resistant strain to become hypersusceptible not only to penicillin but also to cephaloridine, cephalexin, and ceftazidime, with MICs decreasing from 64 to 0.015 U/ml for penicillin and from 64, 2,048, and 4,096 to 0.06, 1, and 4 jig/ml for the three cephalosporins, respectively (13). The susceptibilities to rifampin and minocycline were not affected. MICs of penicillin for a limited number of the other rarely isolated enterococcal species have been published (35, 63), but generalizations about antibiotic susceptibilities and synergistic effects of cell wall-active antibiotics and aminoglycosides cannot be made.
GLYCOPEPTIDES Only occasional vancomycin-resistant enterococci had been reported in the literature through 1973, and none of them involved clinically significant infections (16, 57, 60). Since 1988, a number of reports have documented vancomycin resistance among enterococci, particularly E. faecium (32, 52, 59). Vancomycin resistance is not as prevalent as penicillin, ampicillin, or aminoglycoside resistance but may be underreported due to problems in detection, particularly when the disk diffusion test is used (49, 55). The topic of vancomycin resistance has recently been the subject of a minireview in this journal (7) and is not discussed further here. Enterococci may no longer be assumed to be susceptible
VOL. 35, 1991
to a combination of a cell wall-active antibiotic and an aminoglycoside. Many strains have acquired resistance to either one or both of these agents. Microbiological screening for enterococci and treatment of serious enterococcal infections may require special considerations, which will be
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