Vancomycin-resistant vanB-type Enterococcus faecium isolates expressing varying levels of vancomycin resistance and being highly prevalent among neonatal patients in a single ICU

Abstract

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.

Performance of the EUCAST Disk Diffusion Method, the CLSI Agar Screen Method, and the Vitek 2 Automated Antimicrobial Susceptibility Testing System for Detection of Clinical Isolates of Enterococci with Low- and Medium-Level VanB-Type Vancomycin Resistance: a Multicenter Study

“…Moreover, the CLSI agar screen method re- quires experienced personnel who can prepare and store the agar plates correctly, so that the vancomycin concentration in the plates is accurate. It is well known that VanB-type VRE can be difficult to detect, due to the inducible mechanism of resistance and the variable levels of vancomycin resistance expressed (12,37). The strain collection was designed to be genetically and epidemiologically diverse and to include troublesome isolates with low vancomycin MICs in addition to ATCC reference strains ( Table 1).…”

“…The wide range of vancomycin MICs in VanB-type enterococci is well known and has been observed within the same clone during outbreaks (12,21) Sundsfjord, and K. Hegstad, submitted for publication). The MIC clinical breakpoints defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for Enterococcus spp.…”

“…The vanA genotype is the most prevalent genotype in vancomycin-resistant enterococci (VRE) worldwide, but infections with VanB-type VRE (mainly E. faecium) have shown dramatic increases in several European countries and are predominant in Australia (10)(11)(12)(13)(14)(15)(16). The vanB ligase gene has been divided into three subtypes, vanB1 to vanB3, based on phylogenetic diversity (17)(18)(19).…”

Evaluation of the Xpert vanA / vanB Assay Using Enriched Inoculated Broths for Direct Detection of vanB Vancomycin-Resistant Enterococci

“…Moreover, amoxicillin limits the growth of amoxicillin-sensitive anaerobic bacteria, like Clostridium species, which are some of the most relevant species that may contain vanB genes (8,17,17). Vancomycin was not added to the broth, as this would hamper the detection of vanB VRE exhibiting low vancomycin MICs (12,14). From the broths, 100 l was used in the Xpert vanA/vanB assay, and 10 l of broth was subcultured on VRE Brilliance agars (Oxoid).…”

“…Therefore, positive vanB VRE results still need to be confirmed by culture. An additional problem is that VanB-type resistance is sometimes difficult to detect, since the vancomycin MIC can be below the antimicrobial susceptibility breakpoint of Յ4 mg/liter defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (12)(13)(14).…”