Outbreak of Vancomycin-, Ampicillin-, and Aminoglycoside- Resistant Enterococcus faecium Bacteremia in an Adult Oncology Unit

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1994, p /94/$4.+ Copyright X) 1994, American Society for Microbiology Vol. 38, No. 6 Outbreak of Vancomycin-, Ampicillin-, and Aminoglycoside- Resistant Enterococcus faecium Bacteremia in an Adult Oncology Unit MARISA A. MONTECALVO,l* HAROLD HOROWITZ,1 CHERYL GEDRIS,2 CAROL CARBONARO,2 FRED C. TENOVER,3 ABDUL ISSAH,1 PERRY COOK,4 AND GARY P. WORMSER' Divisions of Infectious Diseases' and Oncology,4 Department of Medicine, and Department of Clinical Pathology, 2 Westchester County Medical Center, Valhalla, New York; and the Hospital Infection Program, Centers for Disease Control and Prevention, Atlanta, Georgia3 Received 7 December 1993/Returned for modification 7 February 1994/Accepted 4 April 1994 An outbreak of bacteremia caused by Enterococcusfaecium with high-level resistance to vancomycin (MIC of >256,ig/ml), ampicillin (MIC of.64,ug/ml), and gentamicin or streptomycin (MIC of.2,,ug/ml) occurred in an adult oncology unit from June 1991 to May Active surveillance for the presence of this organism in stool or perianal cultures was begun in September Between June 1991 and May 1992, seven patients with bacteremia and 22 noninfected carriers of the organism in stool were identified. The vana gene, tested for by PCR and gene probe, was present in all isolates evaluated. All bacteremic patients also had resistant E. faecium present in a stool or perianal culture; the stool isolates tested were closely related to the respective blood isolates as determined by pulsed-field gel electrophoresis. Antibiotic regimens using high-dose ampicillin and an aminoglycoside were ineffective with four patients. Five patients (71%) had multiple positive blood cultures; four of these patients died. Following a multiple logistic regression analysis, it was found that bacteremic patients received a significantly greater number of total antibiotic days compared with noninfected stool carriers (P =.19). The emergence of E. faecium with high-level resistance to vancomycin, ampicillin, and aminoglycosides underscores the importance of performing susceptibility testing on all clinically significant isolates. In the neutropenic adult oncology patient, bacteremia with this organism is of probable gastrointestinal origin, is often persistent, and is refractory to treatment with ampicillin in combination with an aminoglycoside. Prolonged use of antibiotics may predispose patients with gastrointestinal colonization to develop bacteremia. Enterococci are the third-most-common cause of hospitalacquired bacteremia (29), and at some centers they account for up to 5% of bacteremias that occur in neutropenic patients receiving broad-spectrum antibiotics (2, 25). Over the past 2 years there has been a progressive increase in antimicrobial resistance among enterococci (3, 4, 8, 11, 2). With the emergence of vancomycin resistance in 1986 (15, 3), enterococci can now be multiply resistant to all currently approved antimicrobial agents (5, 9, 1, 14-16, 26). This presents a major therapeutic challenge for clinicians. We report an outbreak of vancomycin-, ampicillin-, and aminoglycoside-resistant Enterococcus faecium (VAAREF) bacteremia in an adult oncology unit. These infections were refractory to antibiotic treatment and resulted in high mortality. Risk factors which may predispose carriers of VAAREF in stool to develop bacteremia were evaluated. MATERIALS AND METHODS Patient population. Westchester County Medical Center is a 65-bed tertiary care teaching hospital. The adult oncology unit is an 11-room, 22-bed discrete geographic area for patients hospitalized with leukemia, lymphoma, or solid tumors. Vancomycin-resistant E. faecium was first recognized at our center in May Active surveillance and control measures were instituted for the oncology unit beginning 1 September * Corresponding author. Mailing address: Division of Infectious Diseases, Westchester County Medical Center, Macy Pavilion 29SE, Valhalla, NY Phone: (914) Fax: (914) , after 3 patients who had vancomycin-resistant E. faecium bacteremia were identified. Surveillance stool cultures were requested weekly for all patients hospitalized in the unit. Beginning in December 1991, perianal cultures were obtained when stool samples were not available. Perianal cultures were obtained by gently swabbing the perianal skin in a circular manner with a rayon-tipped swab moistened in nonbacteriostatic sterile saline. Swabs were transported to the laboratory in Amies media (Difco, Surrey, United Kingdom). Colonized or infected patients were placed on contact isolation. Cohorting was allowed. Body substance isolation, which included stool precautions (17), was already in use for all patients in the unit. The outbreak was considered over on 8 May 1992, 11 weeks after the last time a patient had bacteremia and after there had been two consecutive weeks in which 1% of the patients hospitalized in the unit had stool or perianal cultures done and no stool carriers were identified. Hospital records of bacteremic patients and stool carriers were reviewed retrospectively. Microbiologic methods. Blood isolates were subcultured from Bactec medium NR6A/7A (Becton Dickinson, Towson, Md.) onto Trypticase soy agar containing 5% sheep blood (TSBA) (BBL, Cockeysville, Md.). Stool or perianal swab samples were plated onto Columbia CNA agar containing 5% sheep blood and incubated at 35 C for 24 h. Beginning 1 January 1992, samples were also plated onto enterococcus agar (Difco, Detroit, Mich.) supplemented with 5 plg of vancomycin per ml. On the basis of the growth observed, at least five isolates with colonial morphology consistent with enterococci were selected at random from either medium. A lawn of each was spread onto TSBA, incubated at 35 C for 24 h, and 1363

2 1364 MONTECALVO ET AL. screened for vancomycin-resistant colonies by using a 3-,ig vancomycin disk. Identification of isolates from blood, stool, and perianal cultures as E. faecium was based upon standard techniques (7). Resistance to vancomycin (MIC of >16,ug/ml) and ampicillin (MIC of >8,ug/ml) and high-level resistance to gentamicin or streptomycin (MIC of >2,,ug/ml) were determined initially by using the MicroScan Positive MIC Type 6 susceptibility panel (Baxter Diagnostics, Inc., W. Sacramento, Calif.) according to the National Committee for Clinical Laboratory Standards guidelines (24). P-lactamase production was assayed by using a nitrocefin disk (Cefinase; BBL). MICs of penicillin, ampicillin, vancomycin, teicoplanin (Merrell Dow Pharmaceuticals, Inc., Cincinnati, Ohio), gentamicin, and streptomycin were determined by broth macrodilution and microdilution methods (23, 28). To detect the presence of the vana resistance determinant in the enterococcal isolates, a PCR assay and a vana gene probe were used as previously described (6). Pulsed-field gel electrophoresis (PFGE) was performed to determine the molecular relatedness of six blood isolates and three stool isolates from five bacteremic patients and of three stool isolates from three nonbacteremic stool carriers. Organisms were lysed as previously described (6), and the DNA was digested in situ with the restriction endonuclease SmaI (GIBCO Bethesda Research Laboratories). Electrophoresis and gel interpretation were performed as described by Murray et al. (21, 22). Control strains were E. faecium A256, E. faecium D366, Enterococcus faecalis V583, E. faecium 228, and Enterococcus gallinarum CDC-NJ4. Definitions. Bacteremia was defined as the presence of VAAREF in at least 1 blood culture. Stool carriers were defined as patients with VAAREF present in stool or perianal culture but who manifested no evidence of infection with this organism during the entire period of their hospitalization. Neutropenia was defined as an absolute neutrophil count of '19 neutrophils per liter, and fever was defined as an oral temperature of.1.4f. Antibiotic treatment is described in terms of "duration of antibiotic therapy," which is defined as the number of days a patient received at least one intravenous antibiotic, and "total antibiotic days," which is defined as the summation of the numbers of days each antibiotic was given. Statistical analysis. Differences for continuous variables were analyzed by using a Wilcoxon rank sum test. Differences for categorical variables were analyzed by using Fisher's exact test or chi-square analysis. A multiple logistic regression analysis was performed to determine the best subset of variables that predicted only VAAREF bowel colonization and not bacteremia with this organism. Odds ratios and exact 95% confidence intervals were computed by using regression coefficients. All P values are results of two-tailed tests performed at type I error of.5 (12, 31). RESULTS The outbreak Seven (1.7%) of the 413 oncology patients hospitalized in the unit from 1 June 1991, the day the first patient developed bacteremia, until 8 May 1992, the day the outbreak was considered over, developed VAAREF bacteremia (Fig. 1). During the period of active surveillance, 1 September 1991 to 8 May 1992, stool or perianal cultures were obtained from 167 (48%) of the 347 nonbacteremic patients hospitalized in the unit and 22 (13%) of the 167 patients were identified as stool carriers. Blood cultures were obtained from 15 (68%) of the stool carriers while they were neutropenic and -E X 2 2 ~'5.T 'a15- IO- U 25T a. 5- D c ANTiMICROB. AGENTS CHEMOTHER. 1 _O * * June July Aug Sept Oct Nov Dec Jan Feb Mar Apr May +t MONTHS FIG. 1. Incidence of new patients (number) with VAAREF bacteremia per month and prevalence of noninfected stool carriers (percent) per month in the adult oncology unit from 1 June 1991 until 8 May The arrow indicates the beginning of active surveillance in the unit. after VAAREF had been identified in a stool or perianal culture, but none of the blood cultures grew VAAREF. Antibiotic resistance patterns of E. faecium. The E. faecium blood and stool isolates from the seven bacteremic patients and the stool or perianal isolates from the 22 stool carriers were resistant to vancomycin and ampicillin and had high-level gentamicin or streptomycin resistance as indicated by the MicroScan susceptibility panel. MICs were determined for E. faecium blood and stool isolates in the case of three bacteremic patients, for blood isolates only in the case of two bacteremic patients, for stool isolates only in the case of two bacteremic patients, and for stool or perianal isolates in the case of 18 of 22 stool carriers. MICs of vancomycin were between 256 and 512,ug/ml, MICs of teicoplanin were between 32 and 512,ig/ml, MICs of ampicillin were between 64 and 128,ug/ml, MICs of penicillin were >256,ug/ml, and high-level resistance to gentamicin (MIC of >2,,ug/ml) was present in 6 of 7 isolates (86%) from bacteremic patients and in 12 of 18 isolates (67%) from stool carriers. High-level resistance to streptomycin (MIC of >2,,ug/ml) was present in 5 of 7 isolates (71%) from bacteremic patients and in 16 of 18 isolates (88%) from stool carriers. None of the isolates produced 1-lactamase. The presence of the vana gene in 5 blood isolates and 3 stool or perianal isolates tested was confirmed by using a PCR assay and a vana gene probe specific for this resistance determinant. Clinical features of bacteremic patients. Five patients had acute leukemia, one had lymphoblastic lymphoma, and one had chronic myelogenous leukemia with lymphoblastic transformation. At the time of VAAREF bacteremia these seven patients had been hospitalized for a median of 24 days (range, 15 to 52 days) and had had neutropenia due to chemotherapy or persistent leukemia for a median of 24 days (range, 9 to 52 days). On the day of the initial positive blood culture for VAAREF all seven patients had fever. The source of infection was not apparent for any of the seven patients. Urine cultures were sterile for two of the patients; urinalyses were normal for four. None of the patients showed definite evidence of an intraab-

3 VOL. 38, 1994 OUTBREAK OF VANCOMYCIN-RESISTANT E. FAECIUM BACTEREMIA 1365 CO) 5 4, EZZII+II+ Death A,A Death Death A,T/A,A A,G/A,A Ez- 1 A,T t Survived Death kb I~~~~~~~~~~~~~~~~~~ TIME IN DAYS FIG. 2. Days on which VAAREF was present (+) in blood culture or absent (-) from blood culture beginning on the day of the first positive blood culture (day 1) and continuing until death or discharge of the five patients with multiple positive blood cultures. The arrow indicates the resolution of neutropenia, which only occurred in patient 2. The boxes indicate the time periods in which the patients were receiving ampicillin plus an aminoglycoside. A, G, ampicillin plus gentamicin; A, T, ampicillin plus tobramycin; A, A, ampicillin plus amikacin. FIG. 3. PFGE of E. faecium stool (S) and blood (B) isolates from three bacteremic patients (1, 3, and 4), blood isolates only from two bacteremic patients (2 and 5), and stool isolates from three nonbacteremic stool carriers (SCI, SC2, and SC3). Lanes from left to right are stool and blood isolates from patient 1 (Si and B1), stool and blood isolates from patient 3 (S3 and B3), stool and blood isolates from patient 4 (S4 and B4), a blood isolate from patient 5 (B5), blood isolates from patient 2 (B2R and B2s), and stool isolates from nonbacteremic stool carriers (SC1, SC2, and SC3). The molecular size standard is lambda concatemer. dominal infection. All 7 patients had a central venous catheter in place at the time of bacteremia, but none had pain or erythema at the insertion site. Catheter tip cultures, performed by using the roll plate technique of Maki et al. (18), were negative for four patients. VAAREF was present in a sputum culture of one patient. This was considered to represent colonization because a sputum Gram stain showed no organisms and the patient's respiratory status was improving. None of the patients had clinical signs of endocarditis; one patient had a normal transthoracic echocardiogram. All seven bacteremic patients had VAAREF present in a stool or perianal culture. VAAREF were detected in stool or perianal culture either prior to (by 18, 7, or 7 days) the day of the first positive blood culture, on the day of the first positive blood culture, or following (by 4, by 4, or 11 days) the day of the first positive blood culture. None of these last three patients had stool or perianal cultures performed prior to or on the day of the first positive blood culture. Three patients had polymicrobic bacteremia, including one patient who was infected by two strains of E. faecium. This patient had a VAAREF strain with high-level gentamicin resistance and a strain of E. faecium that was only resistant to ampicillin (MIC of 128,ug/ml). Five patients had multiple positive blood cultures for VAAREF over periods ranging from 7 to 63 days. Four of these patients died during their hospitalization, all of whom remained neutropenic because of persistent leukemia and had positive blood cultures for VAAREF just prior to death (Fig. 2). Two of the patients who died had a recurrence of VAAREF bacteremia after having had multiple negative blood cultures for VAAREF over periods of 56 days and 13 days, respectively. Of note, one patient with a recurrence of bacteremia (Fig. 2, patient 1) had VAAREF present in five of six stool cultures obtained during the 6 weeks in which the blood cultures were sterile. The three patients who survived all had their neutropenia resolve during the period of their hospitalization (9 to 3 days following the first blood culture with VAAREF). Two of the surviving patients had VAAREF present in only one blood culture; each of these patients had two subsequent blood cultures without VAAREF. The other patient had VAAREF present in five of seven blood cultures obtained over a 9-day period, but during the subsequent 1 days, while neutropenia was resolving, another seven blood cultures were sterile (Fig. 2, patient 2). High-dose ampicillin (2 g every 4 h) plus an aminoglycoside was given to five patients. Ampicillin was used with these patients because at the time only the susceptibility determined by the MicroScan system (MIC of >8,ug/ml) was known. Since the organisms did not produce 1-lactamase, it was expected that the MIC of ampicillin for the organisms would be between 16 and 32,ug/ml, as is usually the case for E. faecium (19, 2), and therefore high-dose ampicillin could have some activity against the organism. One patient was not treated, because she was improving clinically, and another patient died prior to receiving ampicillin. Of the five patients who received ampicillin and an aminoglycoside, four had VAAREF bacteremia either during treatment with the ampicillin-containing regimen or after ampicillin treatment was discontinued (Fig. 2, patients 1, 2, 3, and 5). The other patient had VAAREF in only a single blood culture and did not have VAAREF isolated from any subsequent blood cultures either during or following treatment with ampicillin and an aminoglycoside. PFGE indicated that the VAAREF isolates from the stool (S) and blood (B) of patient 1 (Si and B1) (Fig. 3) were identical, while the paired stool and blood isolates from patient 3 (S3 and B3) were closely related, differing by only a single band. The stool and blood isolates from patient 4 (S4 and B4) also were closely related, differing by a single band, but one different from that which differed in the isolates from patient 3 (S3 and B3). The stool and blood isolates from patients 3 (S3 and B3) and 4 (S4 and B4) were also highly related to one another, to the blood isolate from patient 5 (B5), and to two of the three isolates from nonbacteremic stool carriers (SC1 and SC3) that were analyzed. The two blood isolates from patient 2, (B2R and B2s), one of which was resistant to vancomycin and gentamicin (B2R) and the other of which was susceptible to both drugs (B2s), differed only by the presence of a single additional high-molecular-weight band in the fragment pattern of the vancomycin-resistant isolate (Fig. 3). Bacteremic patients compared with stool carriers. When bacteremic patients at the time of first positive blood culture for VAAREF were compared with noninfected carriers of this organism in the stool at the time of first positive stool or perianal culture, several variables were significantly associated

4 1366 MONTECALVO ET AL. TABLE 1. Characteristics of patients with VAAREF bacteremia compared with those of nonbacteremic stool carriers No. of subjects or value for characteristic Characteristic Bacteremic Stool carriers patients (n = 7) (n = 22) P value Age in years 36 (2-48)a 58.5 (2-8).6 Male 4 (57)b 14 (64) >.5 Race = white 6 (86) 18 (82) >.5 Diagnosis of acute 6 (86) 17 (77) >.5 leukemia Days hospitalized 24 (15-52) 21.5 (4-7).295 Patients 7 (1) 13 (59).66 neutropenic Days neutropenic 24 (9-52) 16 (-64).43 Neutrophil count 2 (1-3) 96 (1-12,741).34 Total antibiotic 84 (47-112) 4.5 (-13).7 days Duration of 24 (16-51) 16.5 (-48).23 antibiotic treatment Intravenous 12 (6-38) 6.5 (-35).55 vancomycin days Ceftazidime days 24 (-37) 1.5 (-35).23 Patients receiving 2 (29) 1 (.5).67 oral vancomycin a Median value for characteristic followed by range in parentheses. b Number of subjects with characteristic followed by percentage in parentheses. with bacteremia by univariate analysis (Table 1). However, following a multiple logistic regression analysis, only total antibiotic days was significantly associated with the development of bacteremia (P =.19). DISCUSSION ANTIMICROB. AGENTS CHEMOTHER. Although enterococci are an increasing cause of infection among oncology patients (2, 13, 25, 27), this is the first report concerning adult oncology patients in which the organisms were resistant to vancomycin, ampicillin, and aminoglycosides and the first to document persistent or relapsing bacteremia. The patient's endogenous gastrointestinal flora appeared to be the reservoir for this organism on the basis of the presence of VAAREF in a stool or perianal skin culture for all bacteremic patients, the presence of an isolate from a stool or perianal skin culture that was nearly identical to the blood isolate for the three patients studied, as determined by PFGE, and the absence of an identifiable alternative source of infection. The presence of multiple positive blood cultures for VAAREF in the case of five patients was a striking and unique feature of our outbreak which was absent from other reports of VAAREF infections (9, 1, 14, 16). Sustained neutropenia due to leukemia was likely an important factor contributing to the bacteremia. Although the pattern of bacteremia raised concern about a possible endovascular site of infection, none was apparent clinically. The lack of a bactericidal regimen for the treatment of VAAREF may have also contributed to the persistence of bacteremia. Since there is currently no clearly effective antimicrobial therapy for VAAREF bacteremia, with neutropenic patients the use of colony-stimulating factors or granulocyte transfusions (1) should be considered a possible therapeutic option. Twenty-two patients were found to be stool carriers. We suspect that this may be an underestimate of the actual number of stool carriers because surveillance cultures were only obtained one day per week, a selective medium for primary isolation of the organism was not initially used, and only 48% of the nonbacteremic patients had stool or perianal cultures done. By obtaining stool or perianal cultures on one day each week, we also may have selected for stool carriers who were hospitalized for at least 7 days. Since even among this highly vulnerable group of oncology patients the majority of patients from whom VAAREF was isolated were noninfected stool carriers, we examined variables that may have predisposed stool carriers to develop bacteremia. Following a multiple logistic regression analysis, only total antibiotic days was significantly associated with bacteremia. We hypothesize that in the neutropenic leukemia or lymphoma patient, the more prolonged the use of multiple antibiotics, the greater the selective pressure on the bowel flora which may promote the growth of large numbers of resistant E. faecium organisms. Particularly in the setting of an intestinal epithelium damaged because of chemotherapy, this selective pressure on the bowel flora may result in a greater propensity to develop bacteremia. Factors associated with transmission and control of VAAREF were not studied during this outbreak. The PFGE indicated that the isolates had closely related genetic material and were likely to be clonally related (21, 22). We suspect, as others have (1, 14, 16, 26), that the use of contact isolation measures may have contributed to the control of the outbreak. However, the efficacy of contact isolation as a measure to reduce or eliminate the spread of this organism needs to be established. 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