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BackgroundHospital strains of Enterococcus faecium could be characterized and typed by various molecular methods (MLST, AFLP, MLVA) and allocated to a distinct clonal complex known as MLST CC17. However, these techniques are laborious, time-consuming and cost-intensive. Our aim was to identify hospital E. faecium strains and differentiate them from colonizing and animal variants by a simple, inexpensive and reliable PCR-based screening assay. We describe here performance and predictive value of a single PCR detecting the insertion element, IS16, to identify hospital E. faecium isolates within a collection of 260 strains of hospital, animal and human commensal origins.MethodsSpecific primers were selected amplifying a 547-bp fragment of IS16. Presence of IS16 was determined by PCR screenings among the 260 E. faecium isolates. Distribution of IS16 was compared with a prevalence of commonly used markers for hospital strains, esp and hylEfm. All isolates were typed by MLST and partly by PFGE. Location of IS16 was analysed by Southern hybridization of plasmid and chromosomal DNA.ResultsIS16 was exclusively distributed only among 155 invasive strains belonging to the clonal complex of hospital-associated strains ("CC17"; 28 MLST types) and various vancomycin resistance genotypes (vanA/B/negative). The five invasive IS16-negative strains did not belong to the clonal complex of hospital-associated strains (CC17). IS16 was absent in all but three isolates from 100 livestock, food-associated and human commensal strains ("non-CC17"; 64 MLST types). The three IS16-positive human commensal isolates revealed MLST types belonging to the clonal complex of hospital-associated strains (CC17). The values predicting a hospital-associated strain ("CC17") deduced from presence and absence of IS16 was 100% and thus superior to screening for the presence of esp (66%) and/or hylEfm (46%). Southern hybridizations revealed chromosomal as well as plasmid localization of IS16.ConclusionsThis simple screening assay for insertion element IS16 is capable of differentiating hospital-associated from human commensal, livestock- and food-associated E. faecium strains and thus allows predicting the epidemic strengths or supposed pathogenic potential of a given E. faecium isolate identified within the nosocomial setting.

In the context of the global action plan to reduce the dissemination of antibiotic resistances it is of utmost importance to understand the population structure of resistant endemic bacterial lineages and to elucidate how bacteria acquire certain resistance determinants. Vancomycin resistant enterococci represent one such example of a prominent nosocomial pathogen on which nation-wide population analyses on prevalent lineages are scarce and data on how the bacteria acquire resistance, especially of the vanB genotype, are still under debate. With respect to Germany, an increased prevalence of VRE was noted in recent years. Here, invasive infections caused by sequence type ST192 VRE are often associated with the vanB-type resistance determinant. Hence, we analyzed 49 vanB-positive and vanB-negative E. faecium isolates by means of whole genome sequencing. Our studies revealed a distinct population structure and that spread of the Tn1549-vanB-type resistance involves exchange of large chromosomal fragments between vanB-positive and vanB-negative enterococci rather than independent acquisition events. In vitro filter-mating experiments support the hypothesis and suggest the presence of certain target sequences as a limiting factor for dissemination of the vanB element. Thus, the present study provides a better understanding of how enterococci emerge into successful multidrug-resistant nosocomial pathogens.

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BackgroundVancomycin-resistant isolates of E. faecalis and E. faecium are of special concern and patients at risk of acquiring a VRE colonization/infection include also intensively-cared neonates. We describe here an ongoing high prevalence of VanB type E. faecium in a neonatal ICU hardly to identify by routine diagnostics.MethodsDuring a 10 months’ key period 71 E. faecium isolates including 67 vanB-type isolates from 61 patients were collected non-selectively. Vancomycin resistance was determined by different MIC methods (broth microdilution, Vitek® 2) including two Etest® protocols (McFarland 0.5/2.0. on Mueller-Hinton/Brain Heart Infusion agars). Performance of three chromogenic VRE agars to identify the vanB type outbreak VRE was evaluated (BrillianceTM VRE agar, chromIDTM VRE agar, CHROMagarTM VRE). Isolates were genotyped by SmaI- and CeuI-macrorestriction analysis in PFGE, plasmid profiling, vanB Southern hybridisations as well as MLST typing.ResultsMajority of vanB isolates (n = 56, 79%) belonged to a single ST192 outbreak strain type showing an identical PFGE pattern and analyzed representative isolates revealed a chromosomal localization of a vanB2-Tn5382 cluster type. Vancomycin MICs in cation-adjusted MH broth revealed a susceptible value of ≤4 mg/L for 31 (55%) of the 56 outbreak VRE isolates. Etest® vancomycin on MH and BHI agars revealed only two vanB VRE isolates with a susceptible result; in general Etest® MIC results were about 1 to 2 doubling dilutions higher than MICs assessed in broth and values after the 48 h readout were 0.5 to 1 doubling dilutions higher for vanB VRE. Of all vanB type VRE only three, three and two isolates did not grow on BrillianceTM VRE agar, chromIDTM VRE agar and CHROMagarTM VRE, respectively. Permanent cross contamination via the patients’ surrounding appeared as a possible risk factor for permanent VRE colonization/infection.ConclusionsLow level expression of vanB resistance may complicate a proper routine diagnostics of vanB VRE and mask an ongoing high VRE prevalence. A high inoculum and growth on rich solid media showed the highest sensitivity in identifying vanB type resistance.