1 Robert Koch Institute, Wernigerode Branch, Burgstr. 37, 38855 Wernigerode; 2 Institute of Medical Microbiology and Epidemiology of Infectious Diseases, University Leipzig, Liebigstraße 24, 04103 Leipzig, Germany
Received 20 February 2003; returned 2 April 2003; revised 28 April 2003; accepted 28 April 2003
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Abstract |
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Materials and methods: Multiply-resistant E. faecium were isolated from three female patients aged 9 months, 2 and 15 years during a 10 day time span. Antibiotic susceptibilities were determined by microbroth dilution. Clonal relatedness among the isolates was investigated via SmaI-macrorestriction analysis by PFGE, multilocus sequence typing (MLST), and plasmid profiling. Presence of virulence and resistance determinants was tested by polymerase chain reaction (PCR). Selected resistance genes were localized by Southern hybridizations.
Results: A single E. faecium isolate per patient was investigated. All exhibited resistances to quinupristin/dalfopristin, vancomycin/teicoplanin, streptomycin (high-level), penicillin/ampicillin, erythromycin, oxytetracycline, chloramphenicol, rifampicin and fusidic acid. The isolates were susceptible to linezolid only and intermediately resistant to fluoroquinolones including moxifloxacin. PFGE revealed identical patterns for all three isolates. PCRs for virulence determinants hyaluronidase and enterococcal surface protein, esp, were negative, whereas PCR for the enterocin A gene was positive. MLST identified clonal type [8-5-1-1-1-1-1] belonging to a clonal subgroup C1 of hospital- and outbreak-related E. faecium. Southern hybridizations located several resistance genes (erm(B), vat(D), vanA) on a large plasmid, which was transferable in mating experiments with an E. faecium recipient.
Conclusions: These data show routes of dissemination of resistance to multiple antibiotics including streptogramins and glycopeptides in E. faecium via vertical and/or horizontal gene transfer. The isolates spread in the absence of a direct selective pressure, as none of the patients had received earlier streptogramin or glycopeptide therapy.
Keywords: quinupristin/dalfopristin, vancomycin, vanA, vat(D), VRE
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Introduction |
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Materials and methods |
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The index case was a 9-month-old patient who was under medical support since birth. As a result of multiple disorders, intensive care treatment including mechanical ventilation was necessary several times. The patient received ceftazidime/cefuroxime for treatment of a urinary tract infection (UTI) of unknown bacterial origin. Bacteriological investigation revealed E. faecium counts of 10 000 cfu/mL urine 3 days after the onset of therapy. Case two was a 15-year-old patient who was hospitalized for 1 month due to a progressing Guillain-Barré syndrome. She received mechanical ventilation via a tracheostomy. The patient was treated with cefuroxime due to a supposed UTI. Enterococcus spp. of 10 000 cfu/mL was isolated from the catheter urine (later identified as a mixture of E. faecalis and E. faecium). Case three was a 2-year-old patient with a progressing peritonitis. The patient received several courses of antibiotic treatment including meropenem, ampicillin/sulbactam, and teicoplanin. E. faecium was obtained from wound swabs in low numbers. These three isolates were collected during 10 days, and all patients involved were female. In none of the cases were the enterococci considered to be the cause of infection and no appropriate anti-enterococcal therapy was initiated. The E. faecium isolates from the three cases were already identified as multiply resistant in the hospital microbiology laboratory and subjected to further tests for clonal relatedness and other molecular characterizations.
Antibiotic susceptibilities were identified using microbroth dilution according to German standard DIN58940 as already described.3 According to the resistance phenotype, PCRs for resistance determinants erm(B), vat(D/E), vanA/B, tetM/L and aadEaphA-3 were carried out.4,5 PCRs for virulence markers hyaluronidase (primers hyl-1: 5'-GAGTAGAGGAATATCTTAGC, hyl-2: 5'-AGGCTCCAATTCTGT), enterocin A (entA-F: 5'-TATGGGGGTACCACTCATAG, entA-R: 5'-ACCTAAAAAACCACCTAT), and enterococcal surface protein, esp (esp5: 5'-ACGTGGATGTAGAGTTTGC, esp6: 5'-GAATATGTCACTACAACCGTAC) were carried out as described elsewhere.6,7 Plasmid analysis and Southern hybridizations were done using digoxigenin-labelled probes and the fluorescence detection system Attophos (Roche Biochemicals, Mannheim, Germany).3,5 Resistance determinants were transferred into a rifampicin and fusidic acid high-level resistant recipient (MICs for both >128 mg/L) by filter-mating and transconjugants were selected on agar plates supplemented with oxytetracycline/rifampicin (5/30 mg/L). Fifty transconjugants were randomly chosen and tested on agar plates supplemented with either 30 mg rifampicin or 20 mg fusidic acid excluding high-level rifampicin resistant donor mutants. Phylogenetic analyses including PFGE as well as the corresponding data evaluation were carried out according to recommended schemes.3 Multilocus sequence typing (MLST) was done for isolate three as described elsewhere.8
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Results |
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Analysis of phylogenetic relationships of isolate three within the species E. faecium by MLST revealed the profile [8-5-1-1-1-1-1]. It included a new allele type for the adk gene which was deposited in GenBank (AY205312) and designated allele type adk-8 (in reference to R. J. L. Willems and D. Tribe; www.mlst.net). The corresponding MLST profile suggested that this strain belongs to clonal group C1 (definition of clonal group: 5/7 allele types identical).8 Subgroup C1 defines a cluster of epidemic E. faecium strains disseminated world-wide and isolated mainly from outbreaks or single infections. Most of these isolates also possessed the esp gene serving as an epidemic marker.6 However, when tested by PCR and dot blot hybridizations of genomic DNA, none of the three isolates was esp positive. PCR tests for two other virulence determinants frequently found in E. faecium hospital isolates,7 hyaluronidase and enterocin A, were negative and positive, respectively.
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Discussion |
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Footnotes |
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References |
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2 . Werner, G., Klare, I. & Witte, W. (2002). Molecular analysis of streptogramin resistance in enterococci. International Journal of Medical Microbiology 292, 8194.[ISI][Medline]
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