Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center Rotterdam, Dr Molewaterplein 40, 3015 GD Rotterdam, The Netherlands
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Abstract |
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Introduction |
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Materials and methods |
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Enterococcal strains (n = 243) were isolated from blood cultures taken from 243 patients hospitalized at the Erasmus University Medical Center in Rotterdam between June 1991 and June 1997 [19911992 (n = 34), 19921993 (n = 28), 19931994 (n = 42), 19941995 (n = 39), 19951996 (n = 48), 19961997 (n = 52)]. Five strains from the 19911992 collection were lost and were not available for further study.
Identification
A presumptive identification of all HLGRE was made on the basis of colony morphology, Gram's stain, catalase and pyrase (Dryslide Pyrkit, Difco Laboratories, Detroit, MI, USA). Definitive identification was done by RAPID ID32 STREP (bioMérieux, Hertogenbosch, The Netherlands). The identification strips were read after 5 and 24 h of incubation at 37°C.
Antimicrobial susceptibility tests
All enterococcal strains isolated were tested for high-level gentamicin resistance on MuellerHinton agar (Difco Laboratories) with Etest strips (AB Biodisk, Solna, Sweden) following the manufacturer's instructions. All plates were incubated at 37°C and read after 18 h.
Pulsed field gel electrophoresis
PFGE was performed as described in our previous studies.9 The gel was stained with ethidium bromide and de-stained in distilled water for 1 h before photography. The gel pictures were inspected visually by two different investigators. The PFGE patterns were interpreted according to Tenover et al.10 Isolates that differed by one to three bands, consistent with a single differentiating genetic event, were assigned a numbered subtype. Four or more band differences between two strains defined different genotypes.
DNA isolation and PCR detection of aminoglycoside resistance genes
DNA was isolated according to Boom et al.11 Diagnostic PCR assays targeting the various resistance genes (aphA3; aacA/aphD; aadC) were performed as described by Van Asselt et al.7 Three different primer pairs were used: aphA3 5'-GCCGATGTGGATTGCGAAAA and aphA3 3'-GCTTGATCCCCAGTAAGTCA (292 bp); aacA/aphD 5'-CCAAGAGCAATAAGGGCATA and aacA/aphD 3'-CCAAGAGCAATAAGGGCATA (220 bp); aadC 59-GCAAGGACCGACAACATTTC and aadC39-TGGACAGATGGTCATAACC (165 bp). Per reaction 50 pmol of each primer was included. A PCR aiming for the 16S rRNA gene was used in each reaction as an internal positive control [rrs5'-GGATTAGATACCCTGGTAGTCC ; rrs39-TCGTTGCGGGACTTAACCCAAC (340 bp)]. Amplification of DNA was performed in a Biomed thermocycler (Model 60, Theres, Germany), using predenaturation at 94°C for 5 min. This was followed by 32 cycles of 30 s at 94°C, 45 s at 55°C and 2 min at 72°C. Amplicons were analysed by electrophoresis on 2% agarose gels (Hispanagar; Sphaero Q, Leiden, The Netherlands) in the presence of a 100 bp DNA ladder (Gibco/BRL Life Technologies, Breda, The Netherlands).
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Results |
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During the study period the following HLGRE incidences were found; 19911992: 4/29 (14%), 19921993: 5/28 (18%), 19931994: 12/42 (29%), 19941995: 9/39 (23%), 19951996: 11/48 (23%) and 19961997: 16/52 (31%). All enterococcal strains isolated had gentamicin MICs of >1024 mg/L. Two out of 57 HLGRE were identified as Enterococcus faecium, the rest appeared to be Enterococcus faecalis.
Pulsed field gel electrophoresis
A total of 57 HLGRE isolates were analysed by PFGE. Most of the PFGE banding patterns consisted of 1520 bands. Nineteen different genotypes were identified. However, two genotypes [respectively 12/57 (Type A) and 23/57 (Type B)] predominated (see Table and Figure 1). The incidence of these two epidemic PFGE types increased during the study period from 10% to 15%. The prevalence of strains with a unique PFGE pattern increased from 4% to 16%.
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Genes that encode for aminoglycoside-modifying enzymes were found in all 57 HLGR enterococci. All 57 strains carried the gene aacA/aphD. Sixteen E. faecalis (28%) also had the aphA3 gene and one E. faecalis (2%) contained the aadCgene (see Table). Nine of 12 clonal A-types carried both the aacA/aphD and aphA3 genes. All clonal B-types identified during this study contained only the aacA/aphD gene.
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Discussion |
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In this study, we report a significant increase in the prevalence of HLGR in enterococci isolated from hospitalized patients with bloodstream infections, from 14% in 1991 to 31% in 1997. A similar rise in HLGRE detection was reported by others. Lavery et al.2 documented an increase of HLGRE isolates from 17% to 60% in two Dublin hospitals during the period 19914. In South London a prevalence of 44% HLGRE was reported in the first quarter of 1991.13 A study in Oklahoma reported 16/27 (59%) enterococci isolated from blood that were highly resistant to gentamicin.14 Watanakunakorn15 reported an absence of HLGRE in blood cultures before 1985 in a teaching hospital in Ohio. However, in the periods 19851988 and 19891991 the prevalence of HLGRE increased from 9% to 36%. In contrast, a recent study by Barteloni et al.5 reported a significant decrease in the prevalence of E. faecalis highly resistant to gentamicin during the period 19931995, compared with the rate of HLGR in enterococci in 1990, 1991 and 1993. PFGE of the HLGRE found in our study revealed two possibly epidemic genotypes isolated in diverse years and different wards. These two epidemic enterococcal subtypes represent the majority of HLGR strains. However, this endemicity obscured another serious trend: unique types also increased in numbers during the years. Straut et al.16 reported a clonal spread of one HLGRE genotype. However, these strains were isolated from sources other than blood cultures. The emergence of new genotypes is a potential risk since these types may eventually colonize the hospital environment. Moreover, when selective pressure is applied (for example by antibiotics) these resistant strains may be selected.
With the use of the polymerase chain reaction we identified the genes that encode aminoglycoside-modifying enzymes in enterococci. All strains carried the gene aacA/aphD, 28% of all enterococci also carried the aphA3 gene and 2% harboured the aadC gene. It remains to be assessed whether other genes involved in gentamicin resistance can be detected in the present collection of strains. Since all strains discussed are sensitive to vancomycin, the detection of the recently described, associated gene aphA-Id is not likely; also because this gene has only been documented in the absence of aacA/aphD.17
Nosocomial occurrence of HLGRE can follow either of two scenarios. First, HLGRE can be selected from the patients' gut flora, which presumes community-based carriage of HLGRE in otherwise healthy people. Since the prevalence of HLGRE in the community is unknown, surveillance studies involving non-hospitalized patients and healthy adults are warranted. Secondly, some strains of HLGRE may become endemic in the hospital and spread from one patient to another. Nosocomial spread of either resistant strains or resistance determinants may be reduced by prudent use of antimicrobial agents and by implementation of strict infection control measures.
The increasing and alarming problem of glycopeptide resistance in Gram-positive microorganisms worldwide has, somewhat diverted our attention from aminoglycoside resistance. However, against a background of increasing glycopeptide resistance, the increase in HLGR in enterococci reported in this study is a cause for additional alarm. More studies that clarify the epidemiology of aminoglycoside resistance in Gram-positive organisms in hospitals as well as in the community are required.
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Notes |
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References |
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Received 5 March 1999; returned 14 May 1999; revised 10 June 1999; accepted 8 July 1999