JOURNAL OF CLINICAL MICROBIOLOGY, 0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3379–3381.2001 Sept. 2001, p. 3379–3381 Vol. 39, No. 9 Copyright © 2001, American Society for Microbiology. All Rights Reserved. Heteroresistance to Vancomycin in Enterococcus faecium M. RABIUL ALAM, 1,2 SUSAN DONABEDIAN, 2 WILLIAM BROWN, 3,4 JAMES GORDON, 1,4 JOSEPH W. CHOW, 1,4,5 MARCUS J. ZERVOS, 1,2,4 * AND ELLIE HERSHBERGER 2 Division of Infectious Disease, Department of Medicine, 1 and DMC University Laboratories, 3 William Beaumont Hospital, 2 Royal Oak, and Veterans Administration Medical Center, 5 and Department of Medicine, Wayne State University, 4 Detroit, Michigan Received 9 April 2001/Returned for modification 26 May 2001/Accepted 15 June 2001 This study presents the first report of vancomycin heteroresistance in an Enterococcus faecium isolate from a patient. The original isolate was susceptible in vitro to vancomycin. E-tests showed growth of subcolonies in a zone of inhibition with a vancomycin MIC of >256 g/ml. Both the susceptible and resistant colonies were from the same strain as determined by PFGE, and both contained the vanA gene as determined by PCR. Enterococci are recognized to be important human patho- gens that are responsible for serious nosocomial infections, including bacteremia, endocarditis, and intra-abdominal and urinary tract infections (5, 9). Recent data suggest that 50 to 90% of Enterococcus faecium isolates are resistant to vanco- mycin (1, 6). Treatment of vancomycin-resistant enterococci (VRE) has become a clinical challenge, since E. faecium is resistant to multiple antimicrobial agents. Resistance to vancomycin among enterococci is known to be homogenous within a culture. However, heteroresistance to vancomycin has been previously observed in staphylococci (10). This is the first report of a hetero-vancomycin-resistant E. faecium (hetero-VREF) isolate; importantly, it was isolated from a patient with endocarditis. This isolate was reported to be susceptible to vancomycin, in vitro; however, E-test (AB Biodisk, Solna, Sweden) results showed a subpopulation of isolates resistant to vancomycin. Standardized automated quantitative testing methods may not detect the presence of resistant subpopulations. Therefore, a subsequent adverse out- come is possible when an inappropriate use of vancomycin is combined with quantitative methods based on broth microdi- lution, especially if a rapid reading of results is performed. The patient in the present study was a 31-year-old female with a history of intravenous drug abuse who was transferred to William Beaumont Hospital in March 2000; for tricuspid valve replacement surgery after recurrent VRE tricuspid valve en- docarditis. In October of 1999, the patient was hospitalized and received 6 weeks of intravenous vancomycin treatment for tricuspid valve endocarditis caused by methicillin-resistant Staphylococcus aureus. In December, she was readmitted and treated for 4 weeks with quinupristin-dalfopristin plus rifampin for both VRE bacteremia and possible endocarditis. She was hospitalized again in February 2000, for recurrent fever, night sweats, and buttock abscess. The blood culture collected on February 20 2000 grew vancomycin-susceptible E. faecium (VSEF; vancomycin MIC, 2 g/ml by microtiter broth dilu- tion), and quinupristin-dalfopristin therapy was restarted sub- sequently with no improvement. Follow-up blood cultures 2 days later grew vancomycin-resistant E. faecium (VREF; van- comycin MIC, 16 g/ml), and treatment with quinupristin- dalfopristin plus rifampin was continued. Subsequently, the patient was treated with tricuspid valve replacement and 6 weeks of quinupristin-dalfopristin plus rifampin, which cured the endocarditis. Although the original isolate was susceptible in vitro when tested by microtiter broth dilution, follow-up E-tests showed subcolonies present in the clear zone of inhibition with a van- comycin MIC of 256 g/ml (Fig. 1). Isolates. Two clinical isolates of E. faecium collected from the blood of the patient on 20 February 2000 (VSEF isolate WBH22608) and 22 February 2000 (VREF isolate WBH22609) were evaluated. Susceptibility testing. The MIC of vancomycin (Eli Lilly & Co.) was determined by broth microdilution according to Na- tional Committee for Clinical Laboratory Standards guidelines (8). Vancomycin MIC determinations were also repeated by E-test according to the manufacturer’s specification. E. faecalis strain ATCC 29212 was used as the control for the in-vitro susceptibility studies. The MICs for both the original isolates and the subcolonies from within the E-test zone of inhibition were evaluated. The MIC of the VSEF isolate (WBH22608) was reassessed after serial passages in broth containing a sub- inhibitory concentration (0.125 g/ml) of vancomycin. Strain typing by PFGE. Genomic DNA was prepared in agarose plugs, and digested with the enzyme SmaI (New En- gland BioLabs, Beverly, Mass.), and pulsed-field gel electro- phoresis (PFGE) was performed using a CHEF-DRIII appa- ratus (Bio-Rad Laboratories, Richmond, Calif.) as previously described (3). Interpretation of gels was performed by visual inspection using the criteria of Tenover et al. (11). Detection of vancomycin resistance genes by (PCR). The vancomycin resistance gene content of each strain type was determined by PCR, using methods previously described (4, 12). The oligonucleotide primers used for amplification of vanA and vanB genes were described by Clark et al. (2). The oligonucleotide primers used to amplify the vanRS, vanSH, vanHAX, vanXY, and vanYZ regions of Tn1546 were described previously (7). By broth microdilution, the MICs of vancomycin for isolates WBH22609 and WBH22608 were 256 and 0.25 g/ml, respec- * Corresponding author. Mailing address: William Beaumont Hos- pital, 3601 West 13 Mile Rd., Royal Oak, MI 48073. Phone: (248) 551-0419. Fax: (248) 551-5069. E-mail: Mzervos@Beaumont.edu. 3379 on February 2, 2016 by guest http://jcm.asm.org/ Downloaded from