JouRNat_oF Clinica. Micn« 0095-1137877804.00+0 Copyright © 1997, American Society for Microbiology ‘oc; Dee, 1997, p, 3026-3031 Vol. 35, No. Fecal Carriage of Vancomycin-Resistant Enterococci in Hospitalized Patients and Those Living in the Community in The Netherlands HUBERT P, ENDTZ,! NICOLE vaN sx BRAK}! ALEX van BELKUM,'* JAN A. J. W. KLUYTMANS,* JOHANNES G. M, KOELEMAN."* LODEWLIK SPANJAARD,?® ANDREAS VOSS," ANNEMARIE J. L. WEERSINK:” CHRISTINA M. I. . VANDENBROUCKE-GRAULS,* ANTON G. M. BUITING, ANNERIET van DUIN,’ ao HENRI A. VERBRUGH'* Erasmus University Medical Center and Zuidercickenhuis? Rotterdam, Working Party Hospital Infection Epidemiology Netherlands (WHEN)? and Jgnatlus Hospital,® Breda, Univesity Hospital Vrije Universiteit and Academie Medical Center| Amsterdam, Radboud University Hospital, Nipmegen,° University Hospital, Utrech,” and Elisabeth Hospital, Tilburg The Netherlands Received 17 March 1997/Retumed for modiifation 25 June 1997/Accepted 10 September 1997 In order to determine the prevalence of vancomycin-resistant enterococci (VRE) in The Netherlands, 624 hospitalized patients from intensive care units or hemato-oncology wards in nine hospitals and 200 patients living in the community were screened for VRE colonization. Enterococci were found in 49% of the hospitalized patients and in 80% of the patients living in the community. Of these strains, 43 and 32%, respectively, were Enterococcus faecium. VRE were isolated from 12 of 624 (2%) and 4 of 200 (2%) hospitalized patients and patients living in the community, respectively. PCR analysis ofthese 16 strains and 11 additional clinical VRE Isolates from one of the participating hospitals revealed 24 yan gene-containing, 1 vanB gene-containing, and 2 vanCl gene-containing strains. All strains were cross-resistant to avoparcin but were sensitive to the novel lycopeptide antibiotic LY333328, Genotyping of the strains by arbitrarily primed PCR and pulsed-field gel electrophoresis revealed a high degree of genetic heterogeneity. This underscores a lack of hospital-driven endemicity of VRE clones. It is suggested that the VRE in hospitalized patients have originated from unknown sources in the comm Enterococeus spp. have recently emerged as important nos- ‘ocomial pathogens (35). According to the data from the Na- tional Nosocomial Infections Surveillance System, enterococci are the fourth leading cause of nosocomial infections in the United States (12). Enterococeal infections that have fre- ‘quently been reported include urinary tract infections, bacte- emia, endocarditis, intra-abdominal infections, and surgical ‘wound infections (27). Enterococcus faecalis is commonly iso- lated from the human gastrointestinal tract, whereas Entero- coccus faecium is less frequently isolated from that site (31), This latter species, however, is noted for its antimicrobial re- sistance. Vancomycin-resistant £, faecium (VREF) strains hhave emerged in a setting of increasing high-level resistance of enterococci to penicillins and aminoglycosides (28). During the last few years, nosocomial outbreaks due to VREF have been described (17, 25). The emergence of VREF has raised serious concerns (28), and in response, the Hospital Infections Control Practices Advisory Committee (HICPAC), in collaboration with the Centers for Disease Control and Prevention, has de- veloped recommendations for preventing the spread of vanco- mycin-resistant enterococci (VRE) (18). Given the concera that vancomycin resistance genes may transfer from entero- ‘oct to Staphylococcus aureus, a phenomenon that has been observed in vitro (31), control measures have already been proposed, should vancomyein-resistant S. aureus strains even- ‘wally arise (10). * Corresponding author. Mailing address: Department of Medical Microbiology & Infectious Diseases, Erasmus University Medical Cen- ter, Dr Molewaterplein 4), 3015 GD Rotterdam, The Netherlands Phone: (31)104635813, Fax: (31)104638875, E-mail: vanbelkum@bac! sa. ‘The microbiology laboratory has an important role in the detection, reporting, and control of VRE. The HICPAC doc- tument emphasizes the need for routine susceptibility testing of all enterococci isolated from clinical specimens. Furthermore, in hospitals where VRE have not yet been detected, periodie ‘culture surveys of stools or rectal swabs of patients at high risk for VRE infection or colonization are indicated (18). In The Netherlands, no systematic study has been done to evaluate the prevalence of VRE infection or colonization in hospitalized patients or patients living in the community, Therefore, the present study was started to determine the prevalence of fecal ‘carriage of VRE in hospitalized patients with an increased risk for infection or colonization with VRE and in patients living in the community. We determined the susceptibility of VRE to vancomycin, teicoplanin, avoparcin (a glycopeptide available throughout Europe as an additive in animal feed [15]), and 1333328 (a new glycopeptide antibiotic (37). In order to determine the genetic basis of the glycopeptide resistance phe- notype, PCR assays aimed at the various resistance genes were performed. Moreover, the VRE were typed by pulsed-field gel electrophoresis (PFGE), arbitrarily primed PCR (AP-PCR), and ribotyping to determine the degree of genetic relatedness of this group of resistant microorganisms. MATERIALS AND METHODS Prevalence sted. Fio Due sniversiy hospitals In Rotterdam, Uireeht Nijmegen and Amsterdam and fou regional teching hospitals fn Breda and “Tiburgpartpated in the study. Su hundred sweayyTour peas Who wete ‘hogpaid in ho floving wards were sereene for getritestnal arige of VRE: medial and surge! inlensve caret (ICU). thors surge 1CU, ‘ourologicl and neuonugieal TCU, pediatric ICU either surgical, neonetl, ‘senealpediatc), and homatoncalgy wards. The prevalence study was cir ‘ed out in Noveraber 1985 and Febranry 1856. In addtion, 200 ouipatents ‘leading gonerelprocioners or dares were sexed. Pot ths ter group 3026wor anulyaed. Four of thse 6 salas mete tld at osm Unicrsty sail em alte sin ot feceot Sy top yaaa ce ies Plea A cer wana ae bog atte a ea ay a ee sme ep omtrnctig ecm a at Oa we Fe rete et eetert art weer mepaeaal crf ete Be tech ae Sil iy Ei 6 fee Sie ete Tce Se iertig sreh ate ere ee pee (aL a eae ot cone Gane faa Papeete penalised Gai ere oes Se tei rk i ota Se Age spin cen thn toni nic reer aT, satin, eu eons Ga enero Seth eae BB Saianteetis a etre See mek eH ae i es Gime halen ecrgara es a Hoke oe ete iri a a Bache race pe eS FASTER ag OT 07 9 a A ga A PREVALENCE OF VANCOMYCIN-RESISTANT ENTEROCOCCI 3027 Amplileaion of DNA wx pecormed in w Biomed adel 60 thermocyeler by ‘sig predensuration a 94°C for ln, followed by 40 eees of Tin a 9, Tinin at 25°C. and 2 min at 74°C. Banding pattems were isualaed afer electrophoresis #19 aparose gel containing elbidum bromide i tho pres fence of wtp halle. Banding patters were tstoxpreted by wil inspetion, Ditrent ines were Montifed on te Dass of even a single diferent iting DNA fimgmeut. Diforenses in ethidium bromide saninginlonsily wre ignored. 'PFGE. Ten colores ot an everaight culture yrown on blood agar were sus pended in 100g of EET (100 mM sodium EDTA, 10 mM EOTA, 1 mM {rseHIC pl AO), This suspension was mixed with 100 jl of 1% agarose (eect, gross; FMC Bioprodvets Com, Roekarl, Maine) athe miture vs ans Fevred into sample plug molds (inal agurose concentration, 0.8%). The plugs ‘sere incubated Tor 4h 37°C in 1 ml of BET buller combining 10 mg ot Iysozyme (Sigma Chemical Co. St. Lows, Mo.). This sis soltion was replaced bya al EET hufer solution coniaining 1 mg of proteltase Kand Ue soatam soice sulfate for a further overnight incubation at 37°C. The plugs wece \washod stimes (0 min each ime at room tempers) in TH slut (10 mM ‘Tes-FGi[pH 0}, 1 mM EDTA), To digest the DNA, 4 Samm slice othe sample plug vas placed in a TE solution (10 oN THis HCI [pA AG}, Ml mM EDTA) wi 401 of Snot Boctringsr GmbH), and the mincure ws incubated overnight 25. The plugs were loaded onto 2 1% agarose 1 (SsaKem GTG sparse FMC) la 5 TBE (Tris orate, EDTA) (32)- Ek stophoreis was pr With CHEF DR I apparats (Bio-Rad, Rishmon |, Calc) progrommed inthe fuipalgorthm mode (Boek 1, run pe of 8 bard witch time of 0.5 to 135, Block 2 tine ut IO ands ne a 18 ta 3¢ 3). The gos were stained wih chau romide for 1S mia and were destained fv istillee water for Ih before PPhologrphy. Al gels were Inspected vsialy ty two diferent investigators. Profle wee designate by 4 diferent eapltal baer ay tine that a dt pester (Jference of four or moe bands) vas olained. Isolates wit ie profiles were asigned the same Teter. Isolates that vifered by one 0 three ‘consistent wih a single genatic event, were assigned to a sublype (38) ‘Staisal anal. Fisher's Woralled tz wis used to asses dierences between frequencies of isolation af emterrenect In the two diferent patient populations RESULTS Three hundred six (49%) of the 624 hospitalized patients and 161 (80%) of 200 diartheic patients living in the commu- nity carried enterococei in their gastrointestinal tracts (P< 0,01). OF the 306 enterococe! isolated from hospitalized pa- tients, 132 (43%) were identified as E: faecium. OF 161 entero- cocci from patients outside the hospital, 52 (32%) were iden- tified as E. faecium (P < 0.05). Thus, E: faeciton was isolated from 132 of 624 (21%) of the hospitalized patients and 52 of 200 (26%) of the patients living in the community (P > 0.05), ‘VRE were isolated from 12 (2%) of the 624 hospitalized pa- tients and 4 (2%) of the 200 patients living in the community. Fifteen VRE were identified as E. faecium; one was identified as E. faecalis. Filteen (8%) of 184 E. faecium strains isolated in the prevalence study were vancomycin resistant. In addition, 11 strains of VRE were isolated in hospital A at times separate from the period of the prevalence study. Nine were identified as E. faecium, and two were identified as E. gallinanon. Thus, 27 strains of VRE were available for further studies. ‘The susceptibilities of the 27 strains of VRE to vancomycin, ‘cicoplanin, avoparcin, and LY333328 and the resistance ge- notype are presented in Table 1. Complete cross-resistance between vancomycin and avoparcin was found. LY333328, however, was 250- to >1,000-fold more active than vancomycin against wna VRE, Major discrepancies were observed be- ‘tween the MICs of LY333328 that were determined by the agar dilution method and those determined by the broth dilution method: on agar, the MIC of LY333328 at which 90% of isolates ave inhibited (MICiy) for vad VRE was 4 mg/liter (range, 0.25 10 4 mgfliter), whereas in broth the MICiq was 0.5 mgfliter (range, 0.125 to 1 mgfliter). We did not observe such differences with the other glycopeptide agents tested. Twenty- four of the 27 strains of VRE, including all VRE from the prevalence study, had the vand genotype; 1 had the vanB genotype, and 2 had the vanC? genotype. For all van E, Jaeciunt isolates vancomycin MICs were >256 mgfiter and teicoplanin MICs were >64 mg/liter. For the vanB and vaiCl3028 ENDTZ ET AL. J. CLin. MICROBIOL. TABLE 1. In vitro activities of four glycopeptide agents against 27 strains of VRE* MIC (melliter)’ Strain group and MIC (meiliter) no. Species _ ‘Avo LY333328 enna Clinical isolates* 10-a E, faecium l vanA 10-b E, faecium 0.5 vanA 10-c . faecium 0.25 vanA 10-d faecium 0.5 van 10-e . faecium 0.5 vanA 10-£ . faecium 0.5 vanA 1 . faecium 0.25 vanA 10-h E. faecium 0.5 vanA 10-i E, gallinarum 0.25 vanCl 10-) E. faecium 0.25 vanB 10-k E. gallinarum 0.25 vanCl Survey isolates Ll E. faecium 0.25 vanA 12-m* E. faecium 0.125 vanA 12-n° E. faecalis 0.25 vanA 12-0° E. faecium 0.125 vanA 21-p E. faecium 0.125 vanA E. faecium 1 vanA E, faecium 0.25 vanA E. faecium 0.25 vanA E. faecium 0.25 vanA E, faecium 0.125 vanA E. faecium 0.125 vanA E. faecium 0.125 vanA E. faecium 0.125 vanA E. faecium 0.25 vanA E. faecium 0.25 vanA E. faecium 0.5 vanA * In vitro activities were determined by a standard NCCLS broth dilution method. ’ Van, vancomycin; Tei, teicoplanin; Avo, avoparcin. ‘The breakpoints for vancomycin and teicoplanin defined by NCCLS are 4 and 8 mg/liter, respectively. Breukpoints have not yet been defined for avoparcin and LY33 © Strains isolated in hospital A. strains the vancomycin MIC was 8 mg/liter and the teicoplanin inatory power of AP-PCR with primers AP-7 and ERIC-1 was MIC was 0.5 mg/liter. low compared to that with primers AP-1 and ERIC-2. There- The restriction endonuclease patterns obtained by PFGE fore, only the results of AP-PCR with primers AP-1 and with Smal for the 27 strains of VRE are presented in Fig. 1. An ERIC-2 are presented in Table 2 (see also Fig. 2). Analysis of overview of all typing results is given in Table 2. The discrim- all 27 strains of VRE revealed 23 different patterns by PFGE, Location 111111111111111222233456666 Screening 000000000001222122212222222 Strain ABCDE FGH!I1J KLMNOPQRSTUVWXYZa FIG riction endonuclease patterns obtained by PFGE with Smal for 27 strains of VRE isolated from hospitalized patients and patients living in the The Netherlands. From left to right, the strains appear in the lanes in the same order in which they are listed in Tables 1 and 2. Each strain has a two-digit, one-letter code corresponding to the location (1, Rotterdam; 2, Amsterdam; 3, Breda; 4, Utrecht; 5, Nijmegen; 6, community), the screening (0, routine isolates from hospital A; 1, prevalence study of November 1996; 2, prevalence study of February 1997), and strain letter code corresponding to the order of the strains listed in Tables 1 and 2. A 50-kb ladder (Bio-Rad, Veenendaal, The Netherlands) is shown in the lane on the right as a molecular size standard.