Validating a screening agar for linezolidresistant 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 accuracyPeer Reviewe

Determination of a Tentative Epidemiological Cut-Off Value (ECOFF) for Dalbavancin and Enterococcus faecium

Dalbavancin is a lipoglycopeptide antibiotic that shows potent activity against Gram-positive bacteria. It circumvents vanB-type glycopeptide resistance mechanisms; however, data on the in vitro activity of dalbavancin for Enterococcus faecium (E. faecium) are scarce, and thus, no breakpoints are provided. In recent years, there has been a continuing shift from vanA-type to vanB-type vancomycin-resistance in enterococci in Central Europe. Therefore, we aimed to investigate the in vitro activity of dalbavancin against different van-genotypes, with particular focus on vanB-type E. faecium. Dalbavancin susceptibility was determined for 25 van-negative, 50 vanA-positive, and 101 vanB-positive clinical E. faecium isolates (typed by cgMLST). Epidemiological Cut-Off Values (ECOFFs) were determined using ECOFFinder. For vanB-type E. faecium isolates, dalbavancin MICs were similar to those of vancomycin-susceptible isolates reaching values no higher than 0.125 mg/L. ECOFFs for van-negative and vanB-positive isolates were 0.5 mg/l and 0.25 mg/L respectively. In contrast, E. faecium possessing vanA predominantly showed dalbavancin MICs >8 mg/L, therefore preventing the determination of an ECOFF. We demonstrated the potent in vitro activity of dalbavancin against vancomycin-susceptible and vanB-type E. faecium. On the basis of the observed wildtype distribution, a dalbavancin MIC of 0.25 mg/L can be suggested as a tentative ECOFF for E. faecium.Dalbavancin is a lipoglycopeptide antibiotic that shows potent activity against Gram-positive bacteria. It circumvents vanB-type glycopeptide resistance mechanisms; however, data on the in vitro activity of dalbavancin for Enterococcus faecium (E. faecium) are scarce, and thus, no breakpoints are provided. In recent years, there has been a continuing shift from vanA-type to vanB-type vancomycin-resistance in enterococci in Central Europe. Therefore, we aimed to investigate the in vitro activity of dalbavancin against different van-genotypes, with particular focus on vanB-type E. faecium. Dalbavancin susceptibility was determined for 25 van-negative, 50 vanA-positive, and 101 vanB-positive clinical E. faecium isolates (typed by cgMLST). Epidemiological Cut-Off Values (ECOFFs) were determined using ECOFFinder. For vanB-type E. faecium isolates, dalbavancin MICs were similar to those of vancomycin-susceptible isolates reaching values no higher than 0.125 mg/L. ECOFFs for van-negative and vanB-positive isolates were 0.5 mg/l and 0.25 mg/L respectively. In contrast, E. faecium possessing vanA predominantly showed dalbavancin MICs >8 mg/L, therefore preventing the determination of an ECOFF. We demonstrated the potent in vitro activity of dalbavancin against vancomycin-susceptible and vanB-type E. faecium. On the basis of the observed wildtype distribution, a dalbavancin MIC of 0.25 mg/L can be suggested as a tentative ECOFF for E. faecium.Peer Reviewe

Linezolid Resistance Genes and Mutations among Linezolid-Susceptible Enterococcus spp.—A Loose Cannon?

The National Reference Centre for Enterococci receives an increasing number of linezolid-resistant Enterococcus isolates. Linezolid (LIN) resistance is mediated by G2576T 23S rDNA gene mutations and/or acquisition of resistance genes (cfr, optrA, poxtA). There are anecdotal reports that those resistance traits may be present in phenotypically linezolid-susceptible isolates. We aimed to determine the prevalence of LIN resistance genes and mutations in enterococci with a LIN MIC of 4 mg/L in broth microdilution (EUCAST = susceptible) isolated from German hospital patients 2019-2021. LIN MICs were additionally determined by ETEST (R) and VITEK2. Selected strains were subjected to LIN selective pressure and growth was monitored with increasing antibiotic concentrations. We received 195 isolates (LIN MIC = 4 mg/L). In total, 78/195 (40%) isolates contained either a putative resistance gene, the G2576T mutation, or a combination thereof. Very major error was high for broth microdilution. The ability to predict phenotypic resistance from genotypic profile was highest for G2576T-mediated resistance. Selection experiments revealed that, in particular, E. faecium isolates with resistance gene mutations or poxtA rapidly adapt to MICs above the clinical breakpoint. In conclusion, LIN resistance genes and mutations can be observed in phenotypically linezolid-susceptible enterococci. Those isolates may rapidly develop resistance under LIN selective pressure potentially leading to treatment failure

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

Rezensionen

Sammelrezension: 1) David N. Aspin/ Judith Chapman/ Karen Evans/ Richard Bagnall (Hg.): Second International Handbook of Lifelong Learning. Springer International Handbook of Education, Vol. 26. Dordrecht: Springer 2012. 978-94-007-2360-3. 2) Karin Dollhausen/ Timm C. Feld/ Wolfgang Seitter (Hg.): Erwachsenenpädagogische Kooperations- und Netzwerkforschung. Schriftenreihe Theorie und Empirie Lebenslangen Lernens. Wiesbaden: Springer VS 2013. 978-3-658-03218-0. 3) Peter Faulstich: Menschliches Lernen: eine kritisch-pragmatistische Lerntheorie. Bielefeld: transcript Verlag 2013. 978-3-8376-2425-0. 4) Martin Fromm: Einführung in didaktisches Denken. Münster: Waxmann 2012. 978-3-8309-2753-2. 5) Birgit Hilliger: Paradigmenwechsel als Feld strukturellen Lernens: Konsequenzen für die Herausbildung von Lernkulturen in der Transformationsgesellschaft. Opladen: Budrich UniPress 2012. 978-3-86388-005-7. 6) Andrea Hoffmeier/ Dolores Smith (Hg.): Interkulturelle Kompetenz und Kulturelle Erwachsenenbildung: Erfahrungsfelder, Möglichkeitsräume, Entwicklungsperspektiven. Bielefeld: W. Bertelsmann Verlag 2013. 978-3-7639-5233-5. 7) Ralf Lottmann: Bildung im Alter - für alle? Altersbilder, Ziele und Strukturen in der nachberuflichen Bildung in Deutschland und den USA. Bielefeld: W. Bertelsmann Verlag 2013. 978-3-7639-5111-6. 8) Matthias Pilz (Hg.): The Future of Vocational Education and Training in a Changing World. Wiesbaden: VS Verlag 2012. 978-3-531-18527-9. 9) Burkhard Schäffer/Olaf Dörner (Hg.): Weiterbildungsbeteiligung als Teilhabe- und Gerechtigkeitsproblem. München: Herbert Utz Verlag 2012. 978-3-8316-4076-8

IS element IS16 as a molecular screening tool to identify hospital-associated strains of Enterococcus faecium

<p>Abstract</p> <p>Background</p> <p>Hospital strains of <it>Enterococcus faecium </it>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 <it>E. faecium </it>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, IS<it>16</it>, to identify hospital <it>E. faecium </it>isolates within a collection of 260 strains of hospital, animal and human commensal origins.</p> <p>Methods</p> <p>Specific primers were selected amplifying a 547-bp fragment of IS<it>16</it>. Presence of IS<it>16 </it>was determined by PCR screenings among the 260 <it>E. faecium </it>isolates. Distribution of IS<it>16 </it>was compared with a prevalence of commonly used markers for hospital strains, <it>esp </it>and <it>hyl</it>_<it>Efm</it>. All isolates were typed by MLST and partly by PFGE. Location of IS<it>16 </it>was analysed by Southern hybridization of plasmid and chromosomal DNA.</p> <p>Results</p> <p>IS<it>16 </it>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 (<it>van</it>A/B/negative). The five invasive IS<it>16</it>-negative strains did not belong to the clonal complex of hospital-associated strains (CC17). IS<it>16 </it>was absent in all but three isolates from 100 livestock, food-associated and human commensal strains ("non-CC17"; 64 MLST types). The three IS<it>16</it>-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 IS<it>16 </it>was 100% and thus superior to screening for the presence of <it>esp </it>(66%) and/or <it>hyl</it>_{<it>Efm </it>}(46%). Southern hybridizations revealed chromosomal as well as plasmid localization of IS<it>16</it>.</p> <p>Conclusions</p> <p>This simple screening assay for insertion element IS<it>16 </it>is capable of differentiating hospital-associated from human commensal, livestock- and food-associated <it>E. faecium </it>strains and thus allows predicting the epidemic strengths or supposed pathogenic potential of a given <it>E. faecium </it>isolate identified within the nosocomial setting.</p

IS element IS16 as a molecular screening tool to identify hospital-associated strains of Enterococcus faecium

<p>Abstract</p> <p>Background</p> <p>Hospital strains of <it>Enterococcus faecium </it>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 <it>E. faecium </it>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, IS<it>16</it>, to identify hospital <it>E. faecium </it>isolates within a collection of 260 strains of hospital, animal and human commensal origins.</p> <p>Methods</p> <p>Specific primers were selected amplifying a 547-bp fragment of IS<it>16</it>. Presence of IS<it>16 </it>was determined by PCR screenings among the 260 <it>E. faecium </it>isolates. Distribution of IS<it>16 </it>was compared with a prevalence of commonly used markers for hospital strains, <it>esp </it>and <it>hyl</it>_<it>Efm</it>. All isolates were typed by MLST and partly by PFGE. Location of IS<it>16 </it>was analysed by Southern hybridization of plasmid and chromosomal DNA.</p> <p>Results</p> <p>IS<it>16 </it>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 (<it>van</it>A/B/negative). The five invasive IS<it>16</it>-negative strains did not belong to the clonal complex of hospital-associated strains (CC17). IS<it>16 </it>was absent in all but three isolates from 100 livestock, food-associated and human commensal strains ("non-CC17"; 64 MLST types). The three IS<it>16</it>-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 IS<it>16 </it>was 100% and thus superior to screening for the presence of <it>esp </it>(66%) and/or <it>hyl</it>_{<it>Efm </it>}(46%). Southern hybridizations revealed chromosomal as well as plasmid localization of IS<it>16</it>.</p> <p>Conclusions</p> <p>This simple screening assay for insertion element IS<it>16 </it>is capable of differentiating hospital-associated from human commensal, livestock- and food-associated <it>E. faecium </it>strains and thus allows predicting the epidemic strengths or supposed pathogenic potential of a given <it>E. faecium </it>isolate identified within the nosocomial setting.</p

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

Excellent performance of CHROMagarTM LIN-R to selectively screen for linezolid-resistant enterococci and staphylococci

The increasing number of nosocomial pathogens with resistances against last resort antibiotics like linezolid leads to a pressing need for the reliable detection of these drug-resistant bacteria. National guidelines on infection prevention, e.g., in Germany, have already recommend screening for linezolid-resistant bacteria, although a corresponding screening agar medium has not been provided. In this study we analyzed the performance and reliability of a commercial, chromogenic linezolid screening agar. The medium was capable to predict more than a hundred linezolid-resistant isolates of E. faecium, E. faecalis, S. aureus, S. epidermidis, and S. hominis with excellent sensitivity and specificity. All isolates were collected at the National Reference Centre between 2010 and 2020.Peer Reviewe