ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, OCt. 1988, p. 1528-1532 Vol. 32, No. 10 0066-4804/88/101528-05$02.00/0 Copyright C) 1988, American Society for Microbiology

High-Level Resistance to in Clinical Isolates of Streptococcus (Enterococcus) faecium G. M. ELIOPOULOS,1.2* C. WENNERSTEN,' S. ZIGHELBOIM-DAUM,"2 E. REISZNER,1 D. GOLDMANN,2'3 AND R. C. MOELLERING, JR."2 Department of Medicine, New England Deaconess Hospital,' Bacteriology Laboratory, Children's Hospital,3 and Harvard Medical School,2 Boston, Massachusetts 02215 Received 28 March 1988/Accepted 26 July 1988

During a 14-month period beginning in July 1986, three distinct clinical isolates of Streptococcus (Enterococcus)faecium demonstrating high-level resistance (MIC, >2,000 ,Ig/ml) to gentamicin, kanamycin, , and were recovered from individual patients at one institution. Combinations of ampicillin with any of these agents failed to show bactericidal synergism. By filter-mating techniques, high-level gentamicin resistance could be transferred into a susceptible recipient of the same species at frequencies as high as 1 x 10-4; transfer into Streptococcus faecalis JH2-7 occurred at lower frequencies (<2 x 10-7). Aminoglycoside substrate profile analysis of clinical isolates as well as of laboratory-derived cured strains and transconjugants revealed 2"- phosphotransferase and 3'-aminoglycoside phosphotransferase (III) phosphorylating enzymes, AAC-6' acetylating activity above that attributable to the intrinsic activity characteristic of S. faecium, and a streptomycin adenylylating enzyme. All three isolates carried a 51- megadalton . Curing of this plasmid or conjugative transfer into susceptible recipients was associated with the loss or acquisition of high-level gentamicin resistance, respectively. Loss of high-level gentamicin resistance was also observed when curing techniques resulted in a decrease in the size of this plasmid equivalent to a 10-megadalton deletion. Transferable, high-level resistance to gentamicin and other , which was previously recognized in S. faecalis, has now emerged in clinical isolates of S. faecium, with the attendant concerns for possible spread.

High-level resistance to gentamicin among clinical isolates MATERIALS AND METHODS of Streptococcus (Enterococcus) faecalis was first reported Bacterial in 1979 (8). The genetic determinants encoding the amino- strains. Three clinical isolates of S. faecium with high-level resistance to gentamicin were recovered from glycoside-modifying enzymes responsible for high-level gen- individual patients who were hospitalized at Children's tamicin resistance (MIC, >2,000 ,ug/ml) were found to be plasmid-mediated and transmissible in vitro into suitable Hospital Medical Center, Boston, Mass., between July 1986 recipient strains (3, 15). Since that time, isolates of S. and August 1987 (Table 1). Strains were identified to the species level by using the API Rapid Strep system (Analytab faecalis with high-level resistance to gentamicin as well as to Products, Plainview, N.Y.). The species of the organisms other clinically available aminoglycosides have been re- was confirmed by demonstration of -binding pro- worldwide and make a ported (2, 22) currently up significant tein patterns characteristic of S. faecium (26). S. faecium proportion of clinical enterococcal isolates at some centers GE-1, which was used as a in (28). The importance of these observations rests in the fact recipient mating experiments, is a and that high-level aminoglycoside resistance confers resistance rifampin- -resistant laboratory mutant derived from a strain to bactericidal synergism between that aminoglycoside and penicillin-hypersusceptible of S. fae- cell wall-active (3, 15). cium, designated 4379-S (5). The eventual discovery of high-level gentamicin resistance Susceptibility and synergism studies. Antimicrobial agent susceptibilities were determined by a broth macrodilution in isolates was of Streptococcus (Enterococcus) faecium procedure (10) in dextrose phosphate broth (GIBCO Diag- predicted by Chen and Williams (2), who were able to transfer high-level gentamicin resistance determinants from nostics, Madison, Wis.) by using inocula of ca. 2 x 105 to 5 x 105 CFU/ml. Tests of antimicrobial synergism were per- S. faecalis into S. faecium in vitro by conjugation. We report formed as described previously (18). Synergism was defined here the recovery of three distinct clinical isolates of S. as a 2'100-fold killing by the aminoglycoside-penicillin com- faecium which possess resistance to high-level gentamicin. bination at 24 h of incubation compared with the bactericidal We investigated the mechanism of aminoglycoside resis- effect of the penicillin alone. tance in these strains and documented resistance to ampicil- lin-aminoglycoside synergism resulting from the acquisition Curing of resistance determinants. Initial attempts to cure of aminoglycoside resistance determinants. strains of high-level (>2,000 ,ug/ml) resistance to gentamicin with novobiocin (14) were unsuccessful. Therefore, a (This study was presented in part at the 27th Interscience method with ciprofloxacin was used (16). Parent isolates of Conference on Antimicrobial Agents and Chemotherapy [G. M. Eliopoulos, C. Wennersten, S. Zighelboim-Daum, E. S. faecium were incubated in serial twofold dilutions of Reiszner, and R. C. Moellering, Jr., 27th Intersci. Conf. ciprofloxacin in dextrose phosphate broth. After 18 h of incubation, tubes containing the highest subinhibitory con- Antimicrob. Agents Chemother., abstr. no. 1320, 1987].) centration of ciprofloxacin were sampled onto -free plates. Single colonies arising after 24 h of incubation were replica plated onto antibiotic-free and aminoglycoside-con- * Corresponding author. taining agar. Colonies failing to grow on these plates were 1528 VOL. 32, 1988 HIGH-LEVEL GENTAMICIN RESISTANCE IN S. FAECIUM 1529

TABLE 1. Aminoglycoside susceptibilities of clinical enterococcal isolates and laboratory-derived strains MIC (pg/ml) Strain Source Gentamicin Kanamycin Tobramycin Streptomycin S. faecium 87-Pl Wound (July 1986) >16,000 128,000 16,000 64,000 >128,000 S. faecium 87-P2 Urine (February 1987) 8,000 128,000 16,000 1,000 >128,000 S. faecium 87-P4 Urine (August 1987) >8,000 128,000 16,000 4,000 >128,000 S. faecium 87-Cl Cured (strain 87-P2) 32 64,000 500 250 >128,000 S. faecium 87-C2 Cured (strain 87-Pi) 32 64,000 1,000 125 128,000 S. faecium 87-C3 Cured (strain 87-Pl) 500 64,000 16,000 1,000 >128,000 S. faecalis 87-T3 87-Pi x JH2-7 >8,000 64,000 16,000 2,000 128,000 S. faecium 87-T1l 87-Pl x GE-1 8,000 64,000 16,000 >8,000 l125 S. faecium 87-T24 87-P2 x GE-1 >8,000 64,000 16,000 2,000 4,000 S. faecalis JH2-7 Reference 9 32 250 250 250 250 S. faecium GE-1 See text 16 2,000 1,000 125 l125 isolated, reidentified, and subjected to formal susceptibility at rates of ca. 1% (1 of 103 colonies derived from strain testing. 87-Pl; 2 of 256 colonies derived from strain 87-P2). Two of Conjugation experiments. Transfer of high-level gentami- the resulting strains (87-Cl and 87-C2) were inhibited by cin resistance determinants into recipient strains of S. fae- gentamicin at 32 ,ug/ml and also lost high-level tobramycin calis JH2-7 and S. faecium GE-1 was undertaken by two resistance, while a third strain (87-C3) demonstrated an mating techniques. Cross-streak agar plate mating (7) was intermediate level of resistance to gentamicin (MIC, 500 ,ug/ used to maximize detection of low-frequency events. To ml) and retained high-level resistance to tobramycin (Table quantitate transfer frequencies, a filter-mating procedure 1). None of the three strains was cured of high-level resis- was used (21). Donors and recipients were applied in ratios tance to streptomycin or kanamycin or of resistance to of 10:1, 1:1, and 1:10. Selective plates contained gentamicin antibiotics or . (250 pLg/ml), rifampin (100 ,ug/ml), and fusidic acid (25 ,ug/ Mating experiments. Transfer of high-level gentamicin re- ml). sistance to S.faecalis JH2-7 occurred at low frequencies (<2 Plasmid detection. Plasmid DNA was prepared by the X 10-7 per recipient) but was detected by the cross-streak method of Birnboim and Doly (1) with the following modifi- mating technique. Resistance was transferred to the S. cations. Cells were grown at 35°C in dextrose phosphate faecium recipient at frequencies of 6 x 10-6 to 1 x 10-4 per broth (12 ml) to the mid-log phase, at which time glycine was recipient, depending on the ratio of donors to recipients added to a final concentration of 5% and incubation was applied in the mating mixture. Properties of the resulting continued for 1 h (23); lysozyme (5 mg/ml), sodium hydrox- transconjugants are given in Table 1. ide (0.3 M), and sodium dodecyl sulfate (3%) were used for Synergism studies. When ampicillin was combined with alkali lysis. Separation of plasmid bands was accomplished any one of several aminoglycosides, no bactericidal syner- by horizontal agarose (0.7%) gel electrophoresis in 0.089 M gism was seen against any of the clinical isolates. Curing of Tris borate buffer (13). Digestion of plasmid DNA with high-level resistance to gentamicin restored synergism be- restriction endonucleases EcoRI and HindIII was performed under the conditions recommended by the manufacturer tween ampicillin and gentamicin, but not kanamycin, tobra- (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). mycin, or amikacin. Transfer of high-level aminoglycoside resistance into S. or S. faecium GE-1 was Aminoglycoside-modifying enzymes. Aminoglycoside-mod- faecalis J142-7 ifying enzyme activity in sonic extracts of bacterial cells was accompanied by a loss of synergism between that aminogly- determined by previously described methods (4, 11, 20). coside and penicillin or ampicillin. Among the aminoglycoside analogs used were , Aminoglycoside-modifying enzymes. Aminoglycoside-mod- 6'-ethylnetilmicin, gentamicin C1 and Cla, , and ifying enzyme activities were studied in the first two clinical 2"-deoxysisomicin, which were generously provided by isolates and in their laboratory-derived strains (Table 2). In George Miller, Schering Corp., Bloomfield, N.J. both clinical strains, 2"-aminoglycoside phosphotransferase (APH-2"), 3'-aminoglycoside phosphotransferase (APH-3'), and AAC-6' activities were detected, as were streptomycin- RESULTS adenylylating enzymes. 6'-Acetylating activity was recorded Strain characteristics and antimicrobial agent susceptibili- as positive only when it was greatly in excess of the ties. The three high-level gentamicin-resistant clinical iso- low-level, chromosomally mediated enzyme activity charac- lates resembled typical S. faecium strains in colony size and teristic of S. faecium (25) (Table 2). Substrate profiles of the morphology and had strong alpha-hemolysis on horse blood phosphorylating and acetylating enzymes of representative agar. Sources of isolation and aminoglycoside susceptibili- strains are given in Table 3. The fact that netilmicin is a ties are given in Table 1. MICs of ampicillin against strains relatively poor substrate for the AAC-6' enzyme, when 87-Pi, 87-P2, and 87-P4 were 32, 32, and 128 ,ug/ml, respec- compared with kanamycin (15), partially obscures the fact tively. All strains were resistant to (MIC, that acetylating activities of strains 87-Pi and 87-C3 against >256 ,uglml), (MIC, >256 ,ug/ml), and tetracy- netilmicin were approximately 200 and 25 times, respec- cline (MIC, .16 pug/ml). tively, those against 6'-ethylnetilmicin, indicating that 6'- Curing experiments. Determinants of high-level resistance acetylating activity existed in both strains. These enzymes to gentamicin were cured after treatment with ciprofloxacin were also found in strain 87-P4. 1530 ELIOPOULOS ET AL. ANTIMICROB. AGENTS CHEMOTHER.

TABLE 2. Relationship between high-level resistance and aminoglycoside-modifying enzymes in clinical isolates of S. faecium and laboratory-derived strains High-level resistance to": Enzyme activity Strain Gentamicin Kanamycin Tobramycin Streptomycin APH-2" APH-3' AAC6 b ANT-SM' 87-PI + + + + + + + + 87-P2 + + + + + + + +

87-Cl - + - + - + - + 87-C2 - + - + - + - + 87-C3 _+ + + + + +

87-T3 + + + + + + + + 87-T1l + + + - + - + - 87-T24 + + + + + + + + a MIC, >2,000 ±Lg/ml for gentamicin, kanamycin, tobramycin, and streptomycin. b Enzyme activity above low-level intrinsic activity of S. faecium (25). c ANT-SM, Streptomycin adenylyltransferase. d MIC, 500 ,ug/ml.

Plasmid analysis. Clinical isolates 87-Pl and 87-P2, which DISCUSSION were isolated 7 months'apart, demonstrated identical plas- At a time when the increasing prevalence of highly genta- mid patterns consisting of a high-molecular-mass band of micin-resistant S. faecalis isolates has become widely appre- approximately 51 megadaltons (MDa) and two lower-molec- ciated (15, 28), we have documented here the emergence of ular-mass bands of 10 and 6 MDa. Isolate 87-P4 also carried high-level gentamicin resistance among clinical isolates of S. a 51-MDa plasmid but demonstrated a si'ngle 8-MDa low- faecium as well. High-level resistance to aminoglycosides in molecular-mass band.' Comparison of restriction fragments these S. faecium strains could be attributed to the presence from plasmid DNA of the three clinical isolates sugges'ted a of aminoglycoside-modifying enzymes comparable to those high degree of genetic relatedness among the high-molecu- seen in highly resistant S. faecalis strains (15). The presence lar-mass of these isolates. Curing of high-level of APH-3' (III) in cured derivatives was deduced from gentamicin resistance from 87-P-2 to yield 87-Cl was associ- differences in phosphorylation of kanamycin and tobramycin ated with a decreas'e in size of the high-molecular-mass (3'-deoxykanamycin B) and by phosphorylation of amikacin plasmid to 41 MDa, which was consistent with a deletion of 10 MDa (Fig. 1). In cured strains 87-C2 and 87-C3, the high-molecular-mass plasmid seen in the parent strain was no longer evident. The smaller plasmid bands remained unchanged in each of the cured strains. Transfer of high- level gentamicin resistance by conjugation was associated with the acquisition of high-molecular-mass bands in strains 87-T1l and 87-T24. In strain 87-T24, the 51-MDa plasmid was transferred unchanged, while in strain 87-T1l, the apparent molecular mass of the acquired plasmid was mea- sured at approximately 58 MDa. Despite multiple attempts, plasmid DNA could not be demonstrated in strain 87-T3.

TABLE 3. Substrate profiles of aminoglycoside-modifying enzymes from selected strains

Phosphorylation (t)" Acetylation Aminoglycoside 87-Pl 87-Tll 87-C3 87-Pl 87-C3 Kanamycinb ioo 100 100 100 100 Gentamicin Complex 318 364 5 Cia 65 36 FIG. 1. Agarose gel electrophoresis of plasmid DNA from clini- C1. <1 <1 cal isolates and laboratory-derived strains. S. faecium 87-Pl (lane Tobramycin 108 90 2 3), 87-P2 (lane 8), and 87-P4 (lane 11) were clinical isolates. Vertical Amikacip 201 80 230 69 28 arrows indicate the positions of high-molecular-mass plasmid bands. 113 5 390 Horizontal arrows indicate low-molecular-mass bands in 87-Pl, Lividomycin 137 2 449 87-P2, and derivative strains. Cured derivatives 87-C2 (lane 4) and Sisomicin 465 328 3 87-C3 (lane 5) were obtained from 87-Pl, while 87-Cl (lane 9) was 2"-Deoxysisomycin 2 2 <1 derived from 87-P2. Strains 87-T3 (lane 6), 87-Tll (lane 7), and Netilmicin 9 3 87-T24 (lane 10) represent transconjugants that were selected for the 6'-Ethylnetilmicin <1 <1 acquisition of high-level resistance to gentamicin. Molecular mass standards were as follows: Plac (101 MDa), Ri (62 MDa), RP4 (36 a Activity relative to kanamycin, which was equal to 100%. MDa), and R6K (26 MDa) (lanes 1 and 12); pDR1 (45 MDa) (lane 2); b , 96 to 99% (4). and Col El (4.2 MDa). which is not shown on this gel. VOL. 32, 1988 HIGH-LEVEL GENTAMICIN RESISTANCE IN S. FAECIUM 15311 and lividomycin (3, 24). Phosphorylation of sisomicin but not should remain alert to the worrisome possibility that highly 2"-deoxysisomycin pointed to the presence of an APH-2" gentamicin-resistant strains of S. faecium may emerge as an enzyme in parent strains. In two of three cured derivatives increasing clinical problem. and in all three transconjugants, the presence or absence of APH-2" activity predicted coexisting AAC-6'. This observa- ACKNOWLEDGMENTS tion is consistent with the recent characterization that APH- We thank Ann Macone and the technologists of the Children's 2"-AAC-6' activities of S. faecalis arise from a single bifunc- Hospital Bacteriology Laboratory for their assistance in identifying tional enzyme (6). Interestingly, strain 87-C3 demonstrated these clinical bacterial isolates. considerable acetylating activity in the absence of notable APH-2" phosphorylation. It is not known whether this situ- LITERATURE CITED ation was the result of an event leading to hyperproduction 1. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction of the intrinsic acetylating enzyme associated with curing of procedure for screening recombinant plasmid DNA. Nucleic the bifunctional enzyme or to the dissociation of acquired Acids Res. 7:1513-1523. AAC-6' and APH-2" activities, which are presumably fused 2. Chen, H. Y., and J. D. Williams. 1985. Transferable resistance under normal circumstances (6). As has been the case with and aminoglycoside-modifying enzymes in enterococci. J. Med. Microbiol. 20:187-196. highly gentamicin-resistant S. faecalis, there was no evi- 3. Combes, T., C. Carlier, and P. Courvalin. 1983. Aminoglyco- dence of aminoglycoside adenylyltransferase-2" (ANT-2") side-modifying enzyme content of a multiply resistant strain of activity in these strains of S. faecium (3, 15). Adenylylating Streptococcusfaecalis. J. Antimicrob. Chemother. 11:41-47. activity against streptomycin was found in clinical isolates. 4. Courvalin, P., and C. Carlier. 1981. Resistance towards amino- Characterization of this activity as ANT-6 was inferred by glycoside- antibiotics in . J. Antimicrob. the lack of effective adenylylation of (data not Chemother. 8(Suppl. A):57-69. shown) (3, 15). 5. Eliopoulos, G. M., C. Wennersten, and R. C. Moellering, Jr. Loss of high-level resistance to gentamicin was associated 1982. Resistance to ,B-lactam antibiotics in Streptococcus fae- with curing of a high-molecular-mass plasmid without a cium. Antimicrob. Agents Chemother. 22:295-301. 6. Ferretti, J. J., K. S. Gilmore, and P. Courvalin. 1986. Nucleo- change in the content of smaller plasmid bands. In previous tide sequence analysis of the gene specifying the bifunctional studies in S. faecalis, determinants for high-level gentamicin 6'-aminoglycoside acetyltransferase 2"-aminoglycoside phos- resistance have been localized to plasmids of approximately photransferase enzyme in Streptococcusfaecalis and identifica- 45 to 60 MDa (8, 19, 27), which is comparable in size to the tion and cloning of gene regions specifying the two activities. J. high-molecular-mass plasmids in our strains. That genetic Bacteriol. 167:631-638. determinants mediating production of APH-3' and ANT-6 7. Franke, A. E., and D. B. Clewell. 1981. Evidence for a chromo- enzymes were not located on the high-molecular-mass plas- some-borne resistance transposon (Tn916) in Streptococcus mid was supported by experiments which demonstrated faecalis that is capable of "conjugal transfer" in the absence of curing of the 51-MDa plasmid without loss of APH-3' and a conjugative plasmid. J. Bacteriol. 145:494-502. 8. Horodniceanu, T., L. Bougueleret, N. El-Solh, G. Bieth, and F. ANT-6 activities (strain 87-C2) and conjugative acquisition Delbos. 1979. High-level, plasmid-borne resistance to gentami- of the 58-MDa plasmid without acquisition of these enzymes cin in Streptococcus faecalis subsp. zymogenes. Antimicrob. (strain 87-Til). Previous investigations of transferable resis- Agents Chemother. 16:686-689. tance markers in S. faecium have associated conjugative 9. Jacob, A. E., and S. J. Hobbs. 1974. Conjugal transfer of transfer of high-level resistance to streptomycin and kana- plasmid-borne multiple antibiotic resistance in Streptococcus mycin with plasmids of various sizes, but such transfer has faecalis var. zvmogenes. J. B3acteriol. 117:360-372. also occurred in the absence of detectable extrachromo- 10. Jones, R. N., A. L. Barry, T. L. Gavan, and J. A. Washington II. somal DNA in either donor or recipient strains (12). 1985. Susceptibility tests: microdilution and macrodilution Because of the species-specific resistance of S. faecium to broth procedures, p. 972-977. In E. H. Lennette, A. Balows, W. J. Hausler, Jr., and H. J. Shadomy, Manual of clinical synergistic killing by combinations of with ami- microbiology, 4th ed. American Society for Microbiology, noglycosides that are susceptible to acetylation at the 6' Washington, D.C. position (17) and the frequent occurrence of APH-3' (III) in 11. Krogstad, D. J., T. R. Korfhagen, R. C. Moellering, Jr., C. these isolates, gentamicin is often the only clinically avail- Wennersten, M. N. Swartz, S. Perzynski, and J. Davies. 1978. able aminoglycoside that is capable of synergistically killing Aminogylcoside-inactivating enzymes in clinical isolates of these bacteria when combined with a cell wall-active antibi- Streptococcusfaecalis. J. Clin. Invest. 62:480-486. otic. The clinical significance of high-level resistance to 12. LeBouguenec, C., and T. Horodniceanu. 1982. Conjugative R gentamicin is, therefore, that synergism between gentamicin plasmids in Streptococcus faecium (group D). Antimicrob. and cell wall-active antimicrobial agents against such strains Agents Chemother. 21:698-705. 13. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular of S. faecium is precluded. Although strains of S. faecium cloning: a laboratory manual, p. 89-91. Cold Spring Harbor possessing high-level resistance to gentamicin appear to be Laboratory, Cold Spring Harbor, N.Y. extremely uncommon, at least one clinical isolate, in addi- 14. McHugh, G. L., and M. N. Swartz. 1977. Elimination of plas- tion to those described here, has been referred to the Centers mids from several bacterial species with novobiocin. Antimi- for Disease Control (B. Metchock, R. Cooksey, B. Hill, and crob. Agents Chemother. 12:423-426. C. Thornsberry, 27th ICAAC, abstr. no. 1321, 1987). In 15. Mederski-Samoraj, B. D., and B. E. Murray. 1983. High-level assessing the potential importance of these observations, resistance to gentamicin in clinical isolates of enterococci. J. one can note that frequencies of conjugative transfer of Infect. Dis. 147:751-757. high-level gentamicin resistance from the strains studied 16. Michel-Briand, Y., V. Uccelli, J.-M. Laporte, and P. Plesiat. 1986. 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