Glycopeptide resistance in Enterococcus spp. and coagulase-negative staphylococci from hospitalised patients in Germany: occurrence, characteristics and dalbavancin susceptibility.

OBJECTIVES: The aim of this study was to evaluate the occurrence of glycopeptide resistance in enterococci and coagulase-negative staphylococci (CoNS) and to determine the susceptibilities of the identified glycopeptide-resistant isolates to dalbavancin. METHODS: Twenty-two medical laboratories participated in the study conducted in 2016/17 by the Paul-Ehrlich-Society for Chemotherapy. Each laboratory was asked to collect 30 Enterococcus spp. (limited to Enterococcus faecalis and Enterococcus faecium) and 30 CoNS isolates consecutively from hospitalised patients with a proven or suspected infection. RESULTS: A total of 1285 isolates were collected, comprising 364 E. faecalis, 291 E. faecium and 630 CoNS. No E. faecalis isolates (0%) but 76 E. faecium isolates (26.1%) were vancomycin-resistant, of which 21 showed the VanA type and 55 the VanB type. The proportion of vancomycin-resistant strains among E. faecium isolates from patients in intensive care units (21.6%) was significantly lower than that from patients on regular wards (30.5%). Among the CoNS, 67 isolates (10.6%) were teicoplanin-resistant but none were vancomycin-resistant, with resistance only detected in Staphylococcus epidermidis (12.2%), Staphylococcus haemolyticus (17.9%) and Staphylococcus hominis (13.2%). Dalbavancin at ≤0.25 mg/L inhibited all VanB-type enterococci and 95.5% of teicoplanin-resistant CoNS. CONCLUSION: The level of glycopeptide resistance in E. faecalis remains very low in Germany but achieved 26% in E. faecium and was >10% in CoNS. Dalbavancin appears to be a feasible option for treating infections caused by VanB-type vancomycin-resistant E. faecium and teicoplanin-resistant CoNS.

Long-Lasting Decrease of the Acquisition of Enterococcus faecium and Gram-Negative Bacteria Producing Extended Spectrum Beta-Lactamase (ESBL) by Transient Application of Probiotics.

Previously it was shown that application of probiotics stopped the acquisition of vancomycin-resistant Enterococcus faecium (VRE) by patients in an early rehabilitation ward. Once the application of probiotics ended, we examined whether acquisition of VRE reoccurred. Furthermore, we examined whether probiotics altered prevalence of vancomycin-susceptible E. faecium (VSE) and Gram-negative bacteria, which produce extended spectrum beta-lactamase (ESBL). Although probiotic application ceased in April 2018, VRE-colonized patients rarely presented on that ward until 2019. Probiotic treatment also resulted in a decreased number of patients with VSE and ESBL. While decreased incidence of VRE occurred immediately, decreased VSE and ESBL numbers occurred months later. A probiotic-mediated decrease of VSE and ESBL incidence cannot be explained when assuming bacterial transmission exclusively as a linear cause and effect event. The decrease is better understood by considering bacterial transmissions to be stochastic events, which depend on various driving forces similar to an electric current. We hypothesize that VRE, VSE and ESBL uptake by patients and by staff members mutually reinforced each other, leading staff members to form a bacterial reservoir, similar to a condenser that stores electrical energy. Probiotic treatment then inhibited regeneration of that store, resulting in a breakdown of the driving force.

A Nosocomial Cluster of Tigecycline- and Vancomycin-Resistant Enterococcus faecium Isolates and the Impact of rpsJ and tet(M) Mutations on Tigecycline Resistance.

Tigecycline-resistant enterococci are only rarely detected worldwide. In 2017, the National Reference Centre for Staphylococci and Enterococci noticed a nosocomial cluster of tigecycline- and vancomycin-resistant Enterococcus faecium (TVRE) in a hospital of tertiary care in Northern Germany. Nineteen E. faecium isolates were analyzed by means of antimicrobial susceptibility testing and pulsed-field gel electrophoresis. A subset of isolates was subjected to whole-genome sequencing. The genetic basis of tigecycline resistance was assessed by ResFinder and by comparative analyses to known tetracycline and tigecycline resistance genes. Phylogenetic investigations revealed the clustering of 11 TVRE that exhibited genotype ST117/CT1489. Two tigecycline-susceptible isolates were unrelated. Characterization of the genetic determinant putatively responsible for tigecycline resistance revealed two chromosomal changes in the TVRE population: (1) a deletion within the ribosomal protein gene rpsJ and (2) a serine insertion in and removal of transcriptional regulation of the ribosomal protection protein Tet(M). We here report the first nosocomial cluster of TVRE in a German hospital and disclosed the resistance mechanism that was most likely causative for tigecycline insusceptibility. Clonal spread of TVRE isolates can be assumed because all isolates were highly related and harbored identical chromosomal alterations associated with tigecycline resistance.

Validating a screening agar for linezolid-resistant enterococci.

BACKGROUND: Linezolid is an alternative treatment option for infections with multidrug-resistant Gram-positive bacteria including vancomycin-resistant enterococci. Some countries report an increasing number of isolates with resistance to linezolid. The recent publication of the Commission for Hospital Hygiene in Germany on enterococci/VRE recommends screening for linezolid-resistant enterococci (LRE). However, a suitable selective medium or a genetic test is not available. Our aim was to establish a selective screening agar for LRE detection and validate its application with a comprehensive collection of clinical LRE and linezolid-susceptible enterococci. METHODS: We decided to combine the selective power of an enterococcal screening agar with a supplementation of linezolid. Several rounds of analyses with reference, control and test strains and under varying linezolid concentrations of a wider and a smaller range were investigated and assessed. The collection of linezolid-resistant enterococcal control strains included isolates with different resistance mechanisms (23S rDNA mutations, cfr(B), optrA, poxtA). Finally, we validated our LRE screening agar with 400 samples sent to our National Reference Centre in 2019. RESULTS: Several rounds of pre-tests and confirmatory analyses favored Enterococcosel® Agar supplemented with a concentration of 2 mg/L linezolid. A 48 h incubation period was essential for accurate identification of LRE strains. Performance of the LRE screening agar revealed a sensitivity of 96.6% and a specificity of 94.4%. CONCLUSIONS: Here we describe preparation of a suitable screening agar and a procedure to identify LRE isolates with high accuracy.

Comparison of VITEK® 2, three different gradient strip tests and broth microdilution for detecting vanB-positive Enterococcus faecium isolates with low vancomycin MICs.

OBJECTIVES: In 2018, EUCAST issued a warning regarding unreliable results of gradient strip tests for confirming vancomycin resistance in enterococci. We compared the performance of various diagnostic standard and confirmatory tests to identify and determine vanB-type vancomycin resistance. METHODS: We analysed a collection of vanB-positive Enterococcus faecium isolates (n = 68) with low vancomycin MICs and compared the performance of VITEK® 2 (bioMérieux), broth microdilution and three gradient strip tests from different providers (Oxoid, Liofilchem and bioMérieux). For the latter we compared the standard procedure with a protocol with increased inoculum, a rich agar medium and a longer incubation time ('macromethod'). RESULTS: The sensitivity of VITEK® 2 was 81% compared with 72% for broth microdilution and 61%-63% for the three gradient strip tests using standard conditions. The macromethod substantially improved the performance of all strip tests resulting in a sensitivity of 89%-96% after 48 h of incubation. CONCLUSIONS: We recommend that EUCAST changes the present warning against the general use of MIC strips. When MIC strips are used to either exclude or confirm suspected vancomycin resistance in E. faecium, and a PCR is not available, the macromethod should be employed. For clinically relevant enterococci, where a rapid therapeutic decision is needed, a molecular test (e.g. PCR) should be favoured in order to save time and to further increase sensitivity.

Development of a multiplex-PCR to simultaneously detect acquired linezolid resistance genes cfr, optrA and poxtA in enterococci of clinical origin.

Linezolid-resistant enterococcus spp. are increasingly recognized by diagnostic laboratories. Resistance can be mediated by the expression of cfr, optrA or poxtA. We developed a multiplex-PCR to simultaneously detect all three genes. The PCR is suitable for microbiological diagnostics in order to restrict further spread of resistances in enterococci.

Treatment with Saccharomyces boulardii and Escherichia coli Nissle is safe and associated with reduced nosocomial transmission of vanB vancomycin-resistant Enterococcus faecium on an early rehabilitation ward in Germany: a retrospective analysis.

PURPOSE: According to the WHO vancomycin-resistant Enterococcus faecium (VRE) belongs to the microorganisms for which new antibiotics are urgently needed. Depending on the type of vancomycin resistance vanA gene VRE is differentiated from vanB VRE and other types. In this retrospective analysis the results of VRE surveillance performed at a German tertiary hospital with approximately 1,200 beds between 2013 and 2017 are shown. PATIENTS AND METHODS: Rectal screening swabs were taken at admission and once per week on the early rehabilitation ward of Ingolstadt Hospital (ERWIN) but not at other wards. The number of VRE colonized patients was evaluated by using appropriate computer software (LabCentre, Hybase). The Hybase program was also used to find out the number of Saccharomyces boulardii and multi-susceptible Escherichia coli Nissle in blood cultures of patients at ERWIN. The mechanism of vancomycin resistance was examined by PCR and clonality of VRE strains was analyzed by pulsed-field gel electrophoresis. RESULTS: Between 2013 and 2015 the number of VRE increased from 30 to 78 per year whereas in 2016 and 2017 the number declined to 51. Systematic analysis of the laboratory data revealed that this increase was driven by oligoclonal transmission of vanB VRE on ERWIN until August 2016 despite performing intensified infection control measures. However, afterward the number of VRE decreased at ERWIN and subsequently at the other wards. While searching for the reason behind this beneficial development we noticed that at ERWIN, patients treated with antibiotics received two probiotic medications simultaneously (S. boulardii, E. coli Nissle) for the duration of the antibiotic therapy plus an additional 2 days. There was no indication of side effects caused by these microorganisms, particularly no infections. CONCLUSION: Application of S. boulardii and E. coli Nissle was safe and associated with reduced transmission of VRE from patient to patient at ERWIN. Therefore, in our setting, probiotic treatment of patients receiving antibiotics contributed to the increase of patients' safety.

A Core Genome Multilocus Sequence Typing Scheme for Enterococcus faecalis.

Among enterococci, Enterococcus faecalis occurs ubiquitously, with the highest incidence of human and animal infections. The high genetic plasticity of E. faecalis complicates both molecular investigations and phylogenetic analyses. Whole-genome sequencing (WGS) enables unraveling of epidemiological linkages and putative transmission events between humans, animals, and food. Core genome multilocus sequence typing (cgMLST) aims to combine the discriminatory power of classical multilocus sequence typing (MLST) with the extensive genetic data obtained by WGS. By sequencing a representative collection of 146 E. faecalis strains isolated from hospital outbreaks, food, animals, and colonization of healthy human individuals, we established a novel cgMLST scheme with 1,972 gene targets within the Ridom SeqSphere+ software. To test the E. faecalis cgMLST scheme and assess the typing performance, different collections comprising environmental and bacteremia isolates, as well as all publicly available genome sequences from the NCBI and SRA databases, were analyzed. In more than 98.6% of the tested genomes, >95% good cgMLST target genes were detected (mean, 99.2% target genes). Our genotyping results not only corroborate the known epidemiological background of the isolates but exceed previous typing resolution. In conclusion, we have created a powerful typing scheme, hence providing an international standardized nomenclature that is suitable for surveillance approaches in various sectors, linking public health, veterinary public health, and food safety in a true One Health fashion.

Rapid emergence of highly variable and transferable oxazolidinone and phenicol resistance gene optrA in German Enterococcus spp. clinical isolates.

The number of linezolid-resistant Enterococcus spp. isolates received by the National Reference Centre for Staphylococci and Enterococci in Germany has been increasing since 2011. Although the majority are E. faecium, clinical linezolid-resistant E. faecalis have also been isolated. With respect to the newly discovered linezolid resistance protein OptrA, the authors conducted a retrospective polymerase chain reaction screening of 698 linezolid-resistant enterococcus clinical isolates. That yielded 43 optrA-positive strains, of which a subset was analysed by whole-genome sequencing in order to infer linezolid resistance-associated mechanisms and phylogenetic relatedness, and to disclose optrA genetic environments. Multiple optrA variants were detected. The originally described variant from China (optrAWT) was the only variant shared between the two Enterococcus spp.; however, distinct optrAWT loci were detected for E. faecium and E. faecalis. Generally, optrA localized to a plethora of genetic backgrounds that differed even for identical optrA variants. This suggests transmission of a mobile genetic element harbouring the resistance locus. Additionally, identical optrA variants detected on presumably identical plasmids, that were present in unrelated strains, indicates dissemination of the entire optrA-containing plasmid. In accordance, in vitro conjugation experiments verified transfer of optrA plasmids between enterococci of the same and of different species. In conclusion, multiple optrA variants located on distinct plasmids and mobile genetic elements with the potential for conjugative transfer are supposedly causative for the emergence of optrA-positive enterococci. Hence, rapid dissemination of the resistance determinant under selective pressure imposed by extensive use of last-resort antibiotics in clinical settings could be expected.

Emergence and control of linezolid-resistant Staphylococcus epidermidis in an ICU of a German hospital.

Objectives: To investigate an outbreak of linezolid-resistant Staphylococcus epidermidis (LRSE) in an interdisciplinary ICU, linezolid consumption and infection control measures taken. Methods: Routine surveillance of nosocomial infections revealed colonization and infection with LRSE affecting 14 patients during a 15 month period. LRSE isolates were analysed with respect to their clonal relatedness, antimicrobial susceptibility, the presence of cfr and/or mutations in the 23S rRNA, rplC, rplD and rplV genes. cfr plasmids were characterized by Illumina sequencing. Medical records were reviewed and antibiotic consumption was determined. Results: Molecular typing identified the presence of three different LRSE clusters: PFGE type I/ST168 (n = 5), PFGE type II/ST5 (n = 10) and PFGE type III/ST2 (n = 1). Ten strains harboured the cfr gene; we also detected mutations in the respective ribosomal protein genes. WGS revealed an almost identical 39 kb cfr plasmid obtained from strains of different genetic background (ST2, ST5, ST168) that shows high similarity to the recently published LRSE plasmid p12-02300. Due to an increase in the number of patients treated for infections with MRSA, a significant increase in linezolid usage was noted from January to July 2014 (from 5.55 to 20.41 DDDs/100 patient-days). Conclusions: Here, we report the molecular epidemiology of LRSE in an ICU. Our results suggest the selection of resistant mutants under linezolid treatment as well as the spread of cfr-carrying plasmids. The reduction of linezolid usage and the strengthening of contact precautions proved to be effective infection control measures.

Comparative evaluation of VITEK® 2 and three commercial gradient strip assays for daptomycin susceptibility testing of Staphylococcus aureus.

OBJECTIVES: MRSA remains a major cause of severe nosocomial infections and the increased use of vancomycin and daptomycin for MRSA treatment over the last decade has led to the isolation of MRSA strains with decreased daptomycin susceptibility. In addition, a growing number of MSSA isolates with reduced susceptibility to daptomycin have been described lately. Surveillance of the emergence of such a daptomycin-non-susceptible MSSA population requires prompt and reliable daptomycin susceptibility testing. Therefore, this work aimed to evaluate the ability of commonly used methods to detect daptomycin resistance in clinical microbiological laboratories. METHODS: We used commercially available manual and automated test systems, including VITEK® 2 and three gradient strip assays, in comparison with broth microdilution, to detect daptomycin resistance in a representative Staphylococcus aureus strain collection. RESULTS: We found high inter-assay concordance as well as congruence with the reference method. This is demonstrated by essential agreement between commercial test systems and reference broth microdilution ranging from 98.1% to 100% and by categorical agreement from 98.2% to 99.1%. Thus, all systems used were able to detect daptomycin non-susceptibility in MRSA and MSSA isolates. CONCLUSIONS: Our data indicate that routine laboratories are at limited risk of overlooking further daptomycin resistance development, as long as commercially available test systems are used according to the manufacturer's recommendations. However, laboratories must be aware of an increasing number of daptomycin-non-susceptible MSSA isolates, including those exhibiting elevated MICs of glycopeptides.

Nursing staff fluctuation and pathogenic burden in the NICU - effective outbreak management and the underestimated relevance of non-resistant strains.

In the course of a hospital management takeover, a microbial outbreak took place in a tertiary neonatal intensive care unit (NICU). Here, we characterize the outbreak and its management. About 4 months prior to takeover, there was a sharp increase in positive isolates for MSSA and multidrug-resistant organisms (MDROs). Simultaneously, the nursing staff sick leave rate increased dramatically which directly correlated with the number of infection/colonization per week (r2 = 0.95, p = 0.02). During the following months we observed several peaks in positive isolates of methicillin-sensitive staphylococcus aureus (MSSA), MDROs and subsequently a vancomycin-resistant enterococcus (VRE) outbreak. Interventional outbreak management measures were only successful after substantial recruitment of additional nursing staff. None of the VRE, but 44% (n = 4) of MDRO and 32% (n = 23) of MSSA colonized infants developed symptomatic infections (p = 0.02). Among the latter, 35% suffered from serious consequences such as osteomyelitis. The most important risk factors for colonization-to-infection progression were low gestational age and birth weight. Nursing staff fluctuation poses a substantial risk for both bacterial colonization and infection in neonates. Comprehensive outbreak management measures are only successful if adequate nursing staff is available. Non resistant strains account for most neonatal infections - possibly due to their limited perception as being harmful.

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.

Does antibiotic resistance impair plasma susceptibility of multi-drug resistant clinical isolates of enterococci in vitro?

BACKGROUND: Cold atmospheric plasma could constitute an alternative against multi-drug resistant pathogens. Susceptibility of enterococci to cold atmospheric plasma was investigated in vitro. METHODS: 39 clinical isolates of enterococci were grouped dependent on the most important resistance patterns and treated on agar using dielectric barrier discharge plasma. These included enterococci with combined vancomycin- and high-level gentamicin resistance, high-level resistance to gentamicin (HLGR) only, vancomycin resistance alone (VRE), and enterococci susceptible to both. Susceptibility to cold atmospheric plasma was evaluated based on the zones of inhibition and examined in terms of the enterococcal group and the "degree" of drug resistance. RESULTS: Cold atmospheric plasma treatment killed all groups. Comparison of VRE and HLGR strains with non-VRE and non-HLGR isolates concerning zones of inhibition revealed that enterococci with special resistance patterns (VRE and HLGR) showed significantly smaller zones of inhibition than the sensitive ones. The mean of all isolates, irrespective of belonging to groups, showed smaller zones of inhibition with increasing "degree" of drug resistance. CONCLUSIONS: Cold atmospheric plasma treatment killed all isolates of enterococci, but its efficacy depended on the "degree" of drug resistance and on membership in special resistance groups with particular clinical-outbreak importance. However, a possible role of the different genetic lineages, which might be prone to acquiring more or less resistance phenotypes, may also play a role in this context.

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.

Complete Genome Sequence of the Gut Commensal and Laboratory Strain Enterococcus faecium 64/3.

The genome sequence of the commensal and widely used laboratory strain Enterococcus faecium 64/3 was resolved by means of PacificBioscience and Illumina whole-genome sequencing. The genome comprises 2,575,333 bp with 2,382 coding sequences as assigned by NCBI.

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.

Pre-Use Susceptibility to Ceftaroline in Clinical Staphylococcus aureus Isolates from Germany: Is There a Non-Susceptible Pool to be Selected?

Ceftaroline is a new cephalosporin active against Methicillin-resistant Staphylococcus aureus (MRSA). Based on a representative collection of clinical S. aureus isolates from Germany, supplemented with isolates of clonal lineages ST228 and ST239, we demonstrate the in-vitro susceptibility towards ceftaroline prior to its introduction into clinical use for a total of 219 isolates. Susceptibility testing was performed by broth microdilution, disc diffusion and Etest, respectively. Results were interpreted according to EUCAST guidelines and showed considerable variance in dependence on clonal affiliation of the isolates tested. Among isolates of widespread hospital-associated lineages we found a high proportion of clinical isolates with MICs close to the EUCAST breakpoint (MIC50/90 1.0/1.5 mg/L); currently, interpretation of these "borderline" MICs is complicated by a lack of concordant susceptibility testing methods and reasonable breakpoint determination. Isolates of clonal lineages ST228 and ST239 demonstrated increased MIC50/90 values of 2.5/3.33 mg/L. Sequencing of mecA revealed no association of resistance to a specific mecA polymorphism, but rather reveals two regions in the non-penicillin-binding domain of PbP2a which displayed different combinations of mutations putatively involved in resistance development. This study provides national baseline data to (i) adjust susceptibility testing methods and current breakpoints to clinical and epidemiological requirements, (ii) evaluate current breakpoints with respect to therapeutic outcome and (iii) monitor further resistance evolution.

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.

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

BACKGROUND: Vancomycin-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. METHODS: During 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. RESULTS: Majority 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. CONCLUSIONS: Low 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.