Carola Fleige

Tigecycline-resistant Enterococcus faecalis strain isolated from a German intensive care unit patient

Institute for LaboratoryMedicine, Vinzenz von Paul Clinics, MarienhospitalStuttgart, Bo¨heimstr. 37, D-70199 Stuttgart, GermanyKeywords: reserpine, omeprazole, glycylcyclines, tetX*Corresponding author. Tel: þ49-3943-679210; Fax: þ49-3943-679207; E-mail: wernerg@rki.de†Present address. Centre for Diagnostic Medicine, Robert BoschHospital Stuttgart, Auerbachstr. 110, D-70376 Stuttgart,Germany.Sir,Tigecycline is a member of the new group of glycylcyclines anda promising new antibiotic of last resort, active against manybacteria including Enterococcus spp.

Insufficient discriminatory power of MALDI-TOF mass spectrometry for typing of Enterococcus faecium and Staphylococcus aureus isolates.

MALDI-TOF mass spectrometry (MALDI-TOF MS) is increasingly used as a reliable technique for species identification of bacterial pathogens. In this study we investigated the question of whether MALDI-TOF MS can be used for accurate sub-differentiation of strains and isolates of two important nosocomial pathogens Enterococcus faecium and Staphylococcus aureus. For this purpose, a selection of 112 pre-characterized E. faecium isolates (clonal complexes CC2, CC5, CC9, CC17, CC22, CC25, CC26, CC92 altogether 52 multilocus sequence types) and 59 diverse S. aureus isolates (mostly methicillin resistant; CC5, CC8, CC22, CC30, CC45, CC398) were studied using a combination of MALDI-TOF MS and advanced methods of spectral data analysis. The strategy of MS data evaluation included manual peak inspection on the basis of pseudo gel views, unsupervised hierarchical cluster analysis and supervised artificial neural network (ANN) analysis. We were capable of differentiating patterns of hospital-associated E. faecium isolates (CC17) from other strains of E. faecium with 87% accuracy, but failed to identify lineage-specific biomarker peaks. For S. aureus pattern analyses we were able to confirm a number of signals described in previous studies, but often failed to identify biomarkers that would allow a consistent and reliable identification of phylogenetic lineages, clonal complexes or sequence types. Hence, the discriminatory power of MALDI-TOF MS was found to be insufficient for reliably sub-differentiating E. faecium and S. aureus isolates to the level of distinct clones or clonal complexes, such as assessed by MLST. Further, a comparison between peak patterns of susceptible and resistant isolates did not identify statistically relevant marker peaks linked to glycopeptide resistance determinants (vanA, vanB) in E. faecium, or the methicillin resistance determinant (mecA) in S. aureus.

High-level ciprofloxacin resistance among hospital-adapted Enterococcus faecium (CC17)

Hospital-adapted Enterococcus faecium differ from their colonising variants in humans and animals by additional genomic content. Molecular typing based on multilocus sequence typing (MLST) allows allocation of isolates to specific clonal complexes (CCs), such as CC17 for hospital-adapted strains. Acquired ampicillin resistance is a specific feature of these hospital isolates, especially in Europe. A few recent reports have described acquired high-level ciprofloxacin resistance as a supposed feature of hospital-adapted E. faecium strains. In the present retrospective analysis, ciprofloxacin minimum inhibitory concentrations (MICs) of 609 clinical isolates from German hospital patients (1997-2007) were determined and a breakpoint for high-level resistance was deduced (>16mg/L). Acquired high-level ciprofloxacin resistance was distributed among isolates of 26 different MLST types (all CC17), indicating a wide prevalence of this acquired resistance trait among the hospital-adapted E. faecium population. High-level ciprofloxacin resistance was linked to gyrA and parC mutations in 98 investigated isolates. Eleven different allele types or combinations thereof were identified. Their allocation to specific MLST and pulsed-field gel electrophoresis (PFGE) types revealed differences in the emergence and spread of corresponding mutations and strains.

Improved identification including MALDI-TOF mass spectrometry analysis of group D streptococci from bovine mastitis and subsequent molecular characterization of corresponding Enterococcus faecalis and Enterococcus faecium isolates

We examined 199 group D streptococci isolated from clinically defined and epidemiologically unrelated cases of bovine mastitis. Samples were collected during a 5-month period from 2010 to 2011 from diseased animals in 199 herds (1 isolate per herd) raised in different counties and federal states in Germany. A classical enterococcal species identification procedure started with PYRase and catalase assays, growth on Enterococcoselagar(®) and GCG(®) agar plates and in 6.5% NaCl followed by a biochemical reaction panel. All 199 isolates were also subjected to MALDI-TOF MS diagnostics in which a simple and an extended direct transfer protocol were compared. The latter revealed a much better performance (higher log (score) values) although the same result was obtained in all but three cases. Classical and MALDI TOF MS analyses identified 64 Enterococcus faecalis and 37 Enterococcus faecium isolates which were confirmed by species-specific PCRs. These 101 enterococcal isolates did not display a specific multi-resistance phenotype and resistances to glycopeptides and antibiotics of last resort (linezolid, daptomycin, tigecycline) were absent, resistance to tetracycline was the most frequent resistance feature. Molecular typing of the 64 E. faecalis isolates revealed 3 main PFGE clusters of related strains represented by three MLST types (ST40, ST211, ST268). PFGE and MLST analysis of E. faecium isolates revealed several smaller clusters of only a few related strains and identified a number of previously unknown allele and MLST types (n=6; ST624-ST629) besides known variants (ST22, ST32). One of the 37 E. faecium strains showed properties of hospital-associated E. faecium strains (ampicillin resistance, IS16-positive; MLST CC17).

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

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 Sma I- and Ceu I-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 three chromogenic VRE screening agars, two Etest® vancomycin protocols, and different microdilution methods in detecting vanB genotype Enterococcus faecium with varying vancomycin MICs

Frequencies of vanB-type Enterococcus faecium increased in Europe during the last years. VanB enterococci show various levels of vancomycin MICs even below the susceptible breakpoint challenging a reliable diagnostics. The performance of 3 chromogenic vancomycin-resistant enterococci (VRE) screening agars, 2 Etest® vancomycin protocols, and different microdilution methods to detect 129 clinical vanB E. faecium strains was investigated. Altogether, 112 (87%) were correctly identified as VanB-type Enterococcus by microdilution MICs. An Etest® macromethod protocol was more sensitive than the standard protocol while keeping sufficient specificity in identifying 15 vanA/vanB-negative strains. Three chromogenic VRE agars performed similarly with 121 (94%), 123 (95%), and 124 (96%) vanB isolates that grew on Brilliance™ VRE Agar, CHROMagar™ VRE, and chromID™ VRE agar, respectively. Using identical media and conditions, we did not identify different growth behaviour on agar and in broth. A few vanB strains showed growth of microcolonies inside the Etest® vancomycin inhibition zones, suggesting a VanB heteroresistance phenotype.

Population structure and acquisition of the vanB resistance determinant in German clinical isolates of Enterococcus faecium ST192.

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.

Detection of a cfr(B) Variant in German Enterococcus faecium Clinical Isolates and the Impact on Linezolid Resistance in Enterococcus spp.

The National Reference Centre for Staphylococci and Enterococci in Germany has received an increasing number of clinical linezolid-resistant E. faecium isolates in recent years. Five isolates harbored a cfr(B) variant gene locus the product of which is capable of conferring linezolid resistance. The cfr(B)-like methyltransferase gene was also detected in Clostridium difficile. Antimicrobial susceptibility was determined for cfr(B)-positive and linezolid-resistant E. faecium isolates and two isogenic C. difficile strains. All strains were subjected to whole genome sequencing and analyzed with respect to mutations in the 23S rDNA, rplC, rplD and rplV genes and integration sites of the cfr(B) variant locus. To evaluate methyltransferase function, the cfr(B) variant of Enterococcus and Clostridium was expressed in both E. coli and Enterococcus spp. Ribosomal target site mutations were detected in E. faecium strains but absent in clostridia. Sequencing revealed 99.9% identity between cfr(B) of Enterococcus and cfr of Clostridium. The methyltransferase gene is encoded by transposon Tn6218 which was present in C. difficile Ox3196, truncated in some E. faecium and absent in C. difficile Ox3206. The latter finding explains the lack of linezolid and chloramphenicol resistance in C. difficile Ox3206 and demonstrates for the first time a direct correlation of elevated linezolid MICs in C. difficile upon cfr acquisition. Tn6218 insertion sites revealed novel target loci for integration, both within the bacterial chromosome and as an integral part of plasmids. Importantly, the very first plasmid-association of a cfr(B) variant was observed. Although we failed to measure cfr(B)-mediated resistance in transformed laboratory strains the occurrence of the multidrug resistance gene cfr on putatively highly mobile and/or extrachromosomal DNA in clinical isolates is worrisome with respect to dissemination of antibiotic resistances.

Increased frequency of linezolid resistance among clinical Enterococcus faecium isolates from German hospital patients

Linezolid is an antibiotic of last resort for the treatment of infections with vancomycin-resistant enterococci (VRE). Here we report the increasing prevalence of linezolid resistance among clinical Enterococcus faecium strains from German hospital patients. Linezolid minimum inhibitory concentrations (MICs) were determined for 4461 clinical E. faecium strains isolated between 2008 and 2014. Isolates originated from the network of diagnostic laboratories collaborating with the National Reference Centre (NRC) for Staphylococci and Enterococci covering all German federal states. All linezolid-resistant isolates were determined by broth microdilution and confirmed by Etest as well as by analysing the 23S rDNA for putative mutations. Marker genes were determined by PCR. Genotyping was performed by SmaI macrorestriction analysis in pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) for selected isolates. An increase in linezolid resistance was observed, from <1% in 2008 to >9% in 2014. Occasionally, outbreaks with linezolid-resistant VRE (ST117) were observed. In total, 232 (92.4%) of 251 linezolid-resistant E. faecium isolates (including 61 vanA and 29 vanB) contained the G2576T 23S rDNA mutation and showed a varying mixture of wild-type and mutated alleles per genome sufficient to confer linezolid resistance. In vitro growth experiments revealed a stable linezolid MIC. Of the 251 linezolid-resistant isolates, 5 were cfr-positive. In conclusion, these NRC data identified a country-wide ongoing trend of increasing linezolid resistance among clinical E. faecium isolates within the last 5 years.

Evaluation of DiversiLab®, MLST and PFGE typing for discriminating clinical Enterococcus faecium isolates

We evaluated and critically assessed the performance and discriminatory power of a rep-PCR based commercial test DiversiLab® Enterococcus kit (bioMerieux) for typing a set of 65 representative isolates of Enterococcus faecium/VRE and compared it to state-of-the-art typing techniques such as PFGE and MLST.