ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2008, p. 1195–1197 0066-4804/08/$08.00⫹0 doi:10.1128/AAC.00531-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 52, No. 3
High Rates of Fecal Carriage of Nonenterococcal vanB in both Children and Adults䌤 M. Graham,1 S. A. Ballard,1 E. A. Grabsch,2 P. D. R. Johnson,1,3 and M. L. Grayson1,3,4* Infectious Diseases1 and Microbiology2 Departments, Austin Health, Heidelberg, and Department of Medicine, University of Melbourne,3 and Department of Epidemiology and Preventive Medicine, Monash University,4 Melbourne, Australia Received 22 April 2007/Returned for modification 2 July 2007/Accepted 21 December 2007
We examined the rate of fecal carriage of vanB in the absence of cultivable vancomycin-resistant enterococci in three distinct populations (children, community adults, and hemodialysis patients). Nonenterococcal vanB carriage was similarly high in hemodialysis patients (45%) and community adults (63%; P ⴝ 0.066) and significantly more common among community adults than children (27%; P ⴝ 0.001). required to complete a questionnaire assessing demographic information including age, sex, antibiotics during the previous 2 weeks, hospital admission during the previous 3 and 12 months, and contact with a health-care worker (participant, parent of participant, or household member working in a hospital). Hemodialysis patients were also assessed for the presence of diabetes, an organ transplant, and/or treatment with immunosuppressive medication. Fecal specimens were collected from all participants and stored at ⫺80°C. A sterile 10-l loop was inserted into the center of the fecal specimen, and a sample (⬃30 mg [wet weight]) was removed and used to inoculate 6 ml anaerobe basal broth (Oxoid, Australia). Fecal broth cultures were then incubated anaerobically at 35°C for 36 to 48 h. Control cultures of Enterococcus faecalis ATCC 51299 (vanB positive) and E. faecalis ATCC 29212 (vanB negative) and uninoculated anaerobic basal broth were also prepared. VRE were isolated as previously described (14). In brief, a sterile 10-l loop full of fecal broth culture was plated onto Enterococcosel agar (BBL, BD, Sparks, MD) containing vancomycin at 6 g/ml (EVA) and incubated at 35°C for 48 to 72 h. All isolates of different esculin-positive colony morphologies were investigated. Isolates provisionally identified as Enterococcus faecium or E. faecalis were confirmed by multiplex PCR for the D-Ala-D-Ala ligase gene (7). The glycopeptide resistance genotype was determined by multiplex PCR for vanA, vanB, and vanC (3). To screen for vanB carriage, DNA was extracted from a 500-l volume of fecal broth culture as described by Stinear et al. (18). DNA samples were frozen at ⫺20°C prior to testing by PCR for vanB using the primers vanBP1 (5⬘-TTGCATGGAC AAATCACTGG-3⬘) and vanBP2 (5⬘-GCTCGTTTTCCTGA TGGATG-3⬘) of Stinear et al. (19). DNA samples were also tested by PCR for the 16S rRNA gene (rrs) using the universal bacterial primers U340 forward primer (5⬘-TCCTACGGGAG GCAGCAGT-3⬘) and U806 reverse primer (5⬘-GGACTACC AGGGTATCTAATCCTGTT-3⬘) to control for the presence of PCR inhibition (13). DNA extracted from control cultures of E. faecalis ATCC 51299, E. faecalis ATCC 29212, and anaerobic basal broth as well as multiple sterile water samples was included in each PCR run to control for cross-contamination. PCR for vanB and rrs was performed in separate 50-l reaction mixes each containing 5 l template DNA; 10 mM
The emergence of vancomycin-resistant enterococci (VRE) is a serious infection control issue in hospitals worldwide (14, 16). Recently the vanB genotype has been described in several nonenterococcal (NE) organisms comprising part of the human bowel flora (2, 5, 6, 15). As both in vitro and in vivo transfer of vanB from these organisms to enterococci has been demonstrated, the presence of NE vanB organisms may contribute to VRE emergence (4, 11). We and others have recently reported high rates of vanB carriage (17 to 85%) in the absence of cultivable VRE in fecal/rectal samples during studies of direct detection of VRE and have attributed this in part to the presence of anaerobic gram-positive bacilli carrying vanB Tn5382/Tn1549 (1, 2, 5, 10, 17, 21). As the presence of NE vanB organisms within stool specimens can impact significantly on the specificity of direct detection for VRE, it is important to better understand the epidemiology of both the vanB gene and VRE genotypes in the local population. Studies to investigate the epidemiology of NE van gene carriage are rare, except for that of Domingo et al. (6), who found a rectal carriage rate of 4.8% for non-VRE vanB compared with 1.6% for vanB VRE in hospitalized patients. With this in mind, we sought to investigate the epidemiology of NE vanB carriage in various populations to assess the hypothesis that patient age and comorbidities may affect carriage of these elements. (This work was presented in part at the 46th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 2006 [7a].) Participants comprised 56 children aged ⬍5 years (sourced from eight local preschools), 97 adults recruited from the community, and 50 hemodialysis outpatients with no previous history of colonization or infection with VRE (Austin Hospital, Melbourne, Australia). Community adults were recruited via advertisements, via contact with local social clubs, and from Australians (aged 55 to 74 years) participating in a regular preventive bowel cancer screening program. Participants were * Corresponding author. Mailing address: Infectious Diseases Department, Austin Hospital, Austin Health, Studley Rd., Heidelberg, VIC, Australia 3084. Phone: (613) 9496-6676. Fax: (613) 9496-6677. E-mail:
[email protected]. 䌤 Published ahead of print on 7 January 2008. 1195
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NOTES
ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Prevalence of vanB and VRE in three study populations
Study population
Preschool children (n ⫽ 56) vanB positive vanB negative
No. VRE positive
No. with inhibited PCR
0
0
No. (%) with noninhibited PCR
56 15 (27)b,c 41 (73)b,c
P (vanB positive vs vanB negative) Community adults (n ⫽ 97) vanB positive vanB negative
1a
5
91 57 (63)b,d 34 (37)b,d
P (vanB positive vs vanB negative) Hemodialysis adults (n ⫽ 50) vanB positive vanB negative
1a
0
49 22 (45)c,d 27 (55)c,d
P (vanB positive vs vanB negative)
Mean age ⫾ SD (median; range) (yr)
4.6 ⫾ 0.9 (5.0; 2.2–5.2) 3.8 ⫾ 1.4 (4.1; 0.2–5.6)
No. male/no. total
26/56 7 19
0.045
0.78
65.7 ⫾ 9.5 (67.8; 42.4–82.1) 62.2 ⫾ 12.3 (62.8; 30.2–81.9)
61/91 40 21
0.13
0.55
73.4 ⫾ 8.5 (73.9; 49.8–84.2) 67.4 ⫾ 13.1 (70.0; 31.4–83.8)
32/49 9 23
0.07
0.003
a
Both isolates of VRE were vanB Enterococcus faecium. Significance of difference between preschool children and community adults: P ⫽ 0.001. c Significance of difference between preschool children and hemodialysis adults: P ⫽ 0.083. d Significance of difference between community adults and hemodialysis adults: P ⫽ 0.066. b
Tris-HCl (pH 8.3); 50 mM KCl; 2 mM MgCl2; 200 M (each) of dATP, dTTP, dGTP, and dCTP; 0.25 M of each primer, and 0.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems, by Roche Molecular Systems, Inc., Branchburg, NJ) and cycled on a GeneAmp2700 (Applied Biosystems) as previously described (1). PCR products were resolved by electrophoresis on agarose-Tris-acetate-EDTA gels containing 0.5 g/ml ethidium bromide and visualized using a Fluor-S MultiImager with Quantity One quantitation software, version 4 (Bio-Rad Laboratories, Hercules, CA). All DNA samples were subjected to four separate independent PCR assays, two assays using the DNA neat and two assays using the DNA at a 1:10 dilution in sterile water. Specimens demonstrating the expected vanB PCR product size of 358 bp were considered positive for vanB. All specimens negative for the expected 466-bp rrs gene product were considered inhibited in PCR. Specimens which were negative for vanB and positive for the rrs gene were considered negative for vanB. Fecal specimens that were positive for vanB in any one of the four PCR tests were completely recultured from the original fecal specimen and retested by PCR (methods described above) with three negative controls per sample tested. In addition, these fecal specimens were also reassessed for VRE by direct plating of the fecal sample and fecal broth culture onto EVA and subsequent identification and characterization of esculin-positive colonies as described above. Only those fecal specimens that were vanB positive in both the initial and confirmatory assessments were recorded as positive for vanB carriage. Specimens were considered VRE positive if VRE were isolated at any time. To confirm the specificity of the vanB PCR, sequencing of the PCR product was undertaken in a randomly selected subset of approximately 20% of positive specimens. PCR products were purified using the MinElute PCR purification kit (Qiagen
Pty. Ltd, Doncaster, Victoria, Australia) and then sequenced using vanBP1 primer and the ABI Prism BigDye Terminator v3.0 Ready Reaction Cycle Sequencing kit (Applied Biosystems) with the ABI Prism 3100 Genetic Analyzer. Chromatograms were read, aligned, and compared for homology with the same region in vanB1 from E. faecalis V583 (GenBank accession no. L06138) and vanB2 from E. faecalis BM4382 (GenBank accession no. AF192329) using Vector NTI Advance version 8.0 (Informax Inc., Bethesda, MD) and the National Center for Biotechnology Information website (www. ncbi.nlm.nih.gov). Statistical comparisons of the proportion of specimens in each cohort positive for NE vanB were undertaken by chisquare (or Fisher’s exact test) and by t test (for continuous variables), where appropriate. Results are shown in Table 1. VRE colonization was infrequent (0%, 1%, and 2%) for the three study populations (children, community adults, and hemodialysis patients, respectively). NE vanB carriage was significantly more common among community adults than among preschool children (63% versus 27%, respectively; P ⬍ 0.001), although the differences between hemodialysis patients and preschool children (45% versus 27%, respectively; P ⫽ 0.083) and between community adults and adult hemodialysis patients (63% versus 45%, respectively; P ⫽ 0.066) were not significant. NE vanB carriage in each study group was not statistically associated with antibiotic use during the previous 2 weeks, hospital admission during either the previous 3 or the previous 12 months, or contact with a health-care worker. Similarly, for dialysis patients there was no statistical association between NE vanB carriage and the presence of diabetes, immunosuppression, or organ transplantation. PCR product was assessed from the positive specimens of 10/57 community adults, 5/15 preschool children, and 6/22 he-
VOL. 52, 2008
modialysis patients. Sequences obtained were 99 to 100% identical to the same region in the sequence of vanB2 and 96% identical to the same region from vanB1 in 20/21 specimens. The sequence of one community adult specimen was 99% identical to vanB1 and only 95% identical to vanB2. In this study, we noted a high rate of NE vanB fecal carriage (27 to 63%) in the three study populations. This finding is consistent with our previous studies examining the specificity/ sensitivity of various vanB PCR detection protocols, where we noticed a significantly high rate of vanB carriage in feces in the absence of cultivable VRE (17 to 70% depending on the protocol and primer pair employed) (1, 21). Others have also recently reported similar findings of vanB detection without identifiable VRE being present, albeit at lower overall rates of vanB positivity (10, 17). Stamper et al. (17) noted that 85% (33/39) of fecal samples/rectal swabs were positive for vanB in the absence of cultivable VRE when tested using the BD GeneOhm VanR assay, while Koh et al. (10) found that 64% (34/53) of specimens cultured in Enterococcosel broth (containing 8 mg/liter vancomycin) were positive for vanB2/B3 in the absence of cultivable VRE when tested using the Roche LightCycler VRE detection kit. In comparison, Domingo et al. (6) noted an overall rate of 4.8% NE vanB fecal carriage (using rectal swabs rather than fecal specimens) among hospitalized patients in North America. Sampling methods and geographical differences between study populations are likely to be important in explaining these findings. Notable in our study is the sequence confirmation of the NE vanB product in a large number of participants and the significant difference in NE vanB fecal carriage between healthy preschool children and community adults (P ⬍ 0.001). This may suggest that cumulative exposure over time to organisms that harbor the vanB gene or organisms capable of acquiring vanB may be important. Our study has some limitations. Firstly, the voluntary nature of recruitment of all our study populations and especially our community adults (approximately 60% of whom were recruited from a bowel cancer prevention program) may have influenced our findings. Secondly, we were surprised that the rate of NE vanB carriage was lower among hemodialysis patients (45%) than community adults (63%; P ⫽ 0.066). However, this may be a reflection of the smaller relative size of the hemodialysis population assessed. Factors that may predict emergence of VRE in vanB carriers warrant investigation (20). We need to assess how people are exposed to NE vanB and what role, if any, diet and/or other environmental exposure may have regarding age-associated NE vanB acquisition given that the food chain, soil, and sewage systems have all previously been implicated in the spread of VRE and that vanB homologues have been identified in soil organisms (8, 9, 12). Monitoring the spread and environmental reservoir of van genes themselves rather than specific bacterial hosts per se may have important implications for future infection control practice. We thank B. Mayall and the members of the Department of Microbiology, Austin Hospital, Australia, for their generous assistance.
NOTES
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