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Patients most at risk of acquiring vancomycin-resistant enterococcal (VRE) infections frequently have underlying medical conditions that require long-term care and the use of multiple antibiotics (2, 17). Genetic fingerprinting has been useful in the investigation of nosocomial VRE transmission (2, 13, 16). However, the presence of multiple strains in individual patients may complicate its use. Since the use of multiple antibiotics can alter microbial flora, it is important to systematically examine multiple isolates from single patients in order to better understand the emergence and carriage of VRE in these patients (2, 4, 14, 23). In this study, 63 enterococcal isolates from two patients were analyzed by pulsed-field gel electrophoresis (PFGE), plasmid profile, vanA and vanB PCR, and hybridization with vanA and vanB probes.

(This work was presented in part at the 96th Annual Meeting of the American Society for Microbiology, New Orleans, La., 19 to 23 May 1996 [21a].)

VRE were isolated from two patients admitted to a long-term-care unit in a Veterans Affairs Medical Center in upstate New York. At approximately 2-week intervals, isolates were obtained from stool specimens on Campylobacter blood agar that had been overlaid with a clindamycin solution (7). VRE and vancomycin-sensitive enterococci were isolated from other clinical specimens by standard microbiological techniques. Up to five colonies of each morphologically distinct enterococcal colony type were subcultured to sheep blood agar for further analysis. Isolates were identified to species level by conventional methods (9). MICs of vancomycin and teicoplanin were determined by the agar dilution method (19).

Genomic DNA for PFGE was prepared as previously described (18) and digested with SmaI. DNA fragments were separated by PFGE with a CHEF Dr II (Bio-Rad Laboratories, Richmond, Calif.). The pulse time was increased linearly from 5 to 35 s over 22 h at 6 V/cm. PFGE DNA fingerprint types were assigned by using published guidelines (22). Plasmid DNA was purified by a modification of a previously described alkaline lysis method (10). Plasmid DNA fingerprint types were assigned by using a Dice coefficient of similarity (CS) (5). Isolates with a CS of 1.0 (identical) were assigned to a single type, isolates with a CS of 0.7 but <1.0 were considered subtypes, and isolates with a CS of <0.7 were assigned to different types. The vanA and vanB genes were amplified by PCR with a modification of a previously published procedure (3). Cycle parameters were as follows: 95°C for 10 min; 30 cycles of 94°C for 30 s, 50°C for 1 min, 72°C for 1 min; and 72°C for 10 min. Positive PCR controls consisted of Enterococcus faecium A256 (vanA) and Enterococcus faecalis V583 (vanB) (8). PCR products were analyzed by agarose gel electrophoresis. For hybridization studies, DNA was transferred to nylon membranes. The vanA probe was a 698-bp fragment cloned from E. faecium 228 (11), and the vanB probe was a 433-bp PCR product from E. faecalis V583 (8). Probes were labeled with [³²P]ATP and hybridized at moderate stringency. Autoradiography was performed with enhancement screens at 70°C.

Patient A was a 75-year-old diabetic male with severe peripheral vascular disease and a foot infection. He was initially hospitalized for 39 days, received intravenous antibiotic therapy, and underwent a bypass graft procedure to improve the blood supply to his foot. Sixteen days after his initial discharge, he was readmitted. Isolates were obtained from a foot wound, urine, and feces over a period of 4 months (Table 1; Fig. 1a). VRE were detected initially as part of a mixed flora in a foot infection. The MICs for the vancomycin-resistant strains were 512 µg/ml for vancomycin and 128 µg/ml for teicoplanin. For the five vancomycin-sensitive E. faecalis strains, the MICs were 2 µg/ml for vancomycin and 0.5 µg/ml for teicoplanin. Six PFGE types and six subtypes were present among the VRE (Fig. 2a). Plasmid profiles showed five types and two subtypes. PFGE and plasmid profile combined detected 13 strains. The sensitive E. faecalis isolates obtained from a single fecal specimen 2 months after the cessation of antibiotic therapy had identical PFGE and plasmid profiles, which were distinct from those of the resistant E. faecium isolates. All of the VRE were positive for the vanA gene and negative for the vanB gene by PCR. The sensitive E. faecalis strains were negative for both. Hybridization showed that the vanA gene was located on a large plasmid in all resistant strains. There was no hybridization to the sensitive E. faecalis isolates. The patient was negative for VRE approximately 6 weeks after termination of antibacterial therapy (Fig. 1a).

View larger version (36K): [in this window] [in a new window]   FIG. 1.   Timelines of antimicrobial administration and isolation of enterococci from longitudinal patients A (a) and B (b). V, VRE isolated; E, vancomycin-sensitive enterococci isolated; S, negative screen for VRE. Dates are given as (month)/(day); MRSA, methicillin-resistant Staphylococcus aureus.

View larger version (51K): [in this window] [in a new window]   FIG. 2.   (a) PFGE of vancomycin-resistant E. faecium isolates from longitudinal patient A. Lane 1,  concatemers (48.5 kb); lane 2, A1; lane 3, A2; lane 4, A4; lane 5, A5; lane 6, A3; lane 7, A6; lane 8, A7; lane 9, A8; lane 10, A9; lane 11, A10; lane 12, A11; lane 13, A12; lane 14, A13; lane 15, A14; lane 16, A15; lane 17, A16; lane 18, A17; lane 19, A18. (b) PFGE of vancomycin-resistant E. faecium isolates from longitudinal patient B. Lane 1,  concatemers (48.5 kb); lane 2, B1; lane 3, B2; lane 4, B6; lane 5, B7; lane 6, B11; lane 7, B12; lane 8, B17; lane 9, B18; lane 10, B24; lane 11, B25; lane 12, B26; lane 13, B27; lane 14, B30; lane 15, B34; lane 16, B32; lane 17, B36; lane 18, B37; lane 19, B40; lane 20, B38; lane 21, B39.

Patient B was a 62-year-old male who had previously had an abdominal aneurysm resected and who was admitted for drainage of a retroperitoneal abscess. Isolates from this patient were obtained from an abdominal wound, left and right abdominal drainage tubes, urine, and feces over a period of 6 weeks (Table 2; Fig. 1b). The presence of purulent material in his wound and abdominal drainage and cloudiness in his urine prompted microbiological evaluation. Initially, numerous vancomycin-sensitive enterococci, as well as smaller numbers of yeast, were present. Eleven days later, VRE were isolated from all of these sites and from feces. The MICs of vancomycin were >512 µg/ml, while those of teicoplanin were either 64 or 128 µg/ml. Four PFGE types and five subtypes were present (Fig. 2b). There were three plasmid types and one subtype. PFGE and plasmid profile combined detected 12 strains. All of the VRE were positive for the vanA gene and negative for the vanB gene by PCR. A vanA probe hybridized to a large plasmid in all of the PFGE type 2 strains tested. However, none of the type 1, 3, and 4 strains, with the exception of one type 1 strain with a different plasmid profile (strain B10, in which the vanA probe hybridized to a large plasmid), hybridized with the vanA probe. This suggests that the vanA genes in these strains differ somewhat in DNA sequence from the probe. Since several variants of vanC (6, 20) and a vanD gene (21), all of which are related to vanA and vanB, have already been described, it would not be surprising if there are multiple forms of other vancomycin resistance genes. A recent study supports this hypothesis (24). The specimens obtained from the patient's abdominal wound, drainage tubes, and urine became negative for VRE successively in the 4 weeks following cessation of antibacterial therapy, but fecal specimens were still VRE positive at the time of discharge from the hospital (Fig. 1b).

Both patients had multiple procedures, received multiple antibiotics, and had extended hospital stays. Both were colonized and/or infected with multiple strains of VRE that emerged rapidly after antimicrobial therapy was initiated. Emergence of VRE in such patients increases the probability that they may serve as reservoirs of VRE for other patients (12, 13, 16).

Although plasmid analysis is not the method of choice for fingerprinting, it is useful for determining the location of resistance genes and therefore, to some degree, their potential for transmission. For strains in this study in which DNA hybridization was positive, the vanA genes were located on plasmids large enough to be self-transmissible. Resistance genes located on such plasmids are generally presumed to be readily transferred, especially in environments such as the gastrointestinal tract, where there are numerous potential recipient strains (15). The degree of heterogeneity within individual PFGE types was variable. The presence of multiple subtypes within some PFGE types (e.g., type 1 in patient A and type 2 in patient B) and the lack of multiple subtypes in others may indicate that certain strains more readily exchange and rearrange DNA.

It is important to note that patient A became negative for VRE and patient B became negative from all sites except feces following cessation of antibacterial therapy. Patient A had VRE (all E. faecium) in the feces at 1 month after cessation of antibacterial therapy, but 2 months after antibacterial therapy was stopped VRE were no longer detected. After another month, feces were still negative for VRE and all five enterococcal isolates analyzed were vancomycin-sensitive E. faecalis. These results suggest that E. faecium was the dominant enterococcal species when selective pressure existed due to the use of antibacterial drugs but that E. faecalis became predominant in the absence of antibacterial therapy. However, the importance of other factors, such as the patient's underlying disease, cannot be excluded.

In summary, the emergence of multiple strains of VRE during the hospitalization of these patients is significant. It demonstrates that a lack of genetic relatedness among outbreak strains when only a single isolate from each specimen is examined does not exclude the possibility that VRE have spread from common sources. In this study, multiple PFGE types were present simultaneously in both patients. However, a recent study found that multiple isolates from individual patients showed little genetic variation (1). Careful epidemiologic investigation, therefore, sometimes requires examination of multiple strains from each source. The rapid emergence of VRE strains after initiation of antibiotic therapy complicates the clinical management of patients, because multiple genotypes, which emerge under these conditions, can vary in virulence and antibiotic susceptibilities. Disappearance of VRE in these patients upon cessation of antibiotic therapy further illustrates the importance of judicious use of antibiotics in the control of nosocomial VRE.