The current MLVA typing scheme for Enterococcus faecium is less discriminatory than MLST and PFGE for epidemic-virulent, hospital-adapted clonal types

BACKGROUND: MLVA (multiple-locus variable-number tandem repeat analysis) is a reliable typing technique introduced recently to differentiate also isolates of Enterococcus faecium. We used the established VNTR (variable number of tandem repeats) scheme to test its suitability to differentiate 58 E. faecium isolates representing mainly outbreaks and clusters of infections and colonizations among patients from 31 German hospitals. All isolates were vancomycin-resistant (vanA type). Typing results for MLVA are compared with results of macrorestriction analysis in PFGE (pulsed-field gel electrophoresis) and MLST (multi-locus sequence typing). RESULTS: All 51 but one hospital isolates from 1996–2006 were assigned to the clonal complex (CC) of epidemic-virulent, hospital-adapted lineages (MLST CC-17; MLVA CC-1) and differed from isolates of sporadic infections and colonizations (n = 7; 1991–1995) and other non-hospital origins (n = 27). Typing of all 58 hospital VRE revealed MLVA as the least discriminatory method (Simpson's diversity index 0.847) when compared to MLST (0.911) and PFGE (0.976). The two most common MLVA types MT-1 (n = 16) and MT-159 (n = 14) combined isolates of several MLST types including also major epidemic, hospital-adapted, clonal types (MT-1: ST-17, ST-18, ST-280, ST-282; MT-159: ST-78, ST-192, ST-203). These data clearly indicate that non-related E. faecium could possess an identical MLVA type being especially critical when MLVA is used to elucidate supposed outbreaks with E. faecium within a single or among different hospitals. Stability of a given MLVA profile MT-12 (ST-117) during an outbreak over a period of five years was also shown. CONCLUSION: MLVA is a suitable method to assign isolates of E. faecium into distinct clonal complexes. To investigate outbreaks the current MLVA typing scheme for E. faecium does not discriminate enough and cannot be recommended as a standard superior to PFGE

Tolerance and safety of the potentially probiotic strain Lactobacillus rhamnosus PRSF-L477 : a randomised, double-blind placebo-controlled trial in healthy volunteers

In Europe, the species Lactobacillus rhamnosus is currently on the Qualified Presumption of Safety list used by the European Food Safety Authority (EFSA) for internal safety assessment, but according to the EFSA the species should remain a topic of surveillance. In the present study, the safety and tolerance of the potentially probiotic strain L. rhamnosus PRSF-L477 was investigated in a placebo-controlled double-blind volunteer trial following FAO/WHO guidelines. A total of thirty-four subjects received daily doses of 1 x 10(11) colony-forming units (cfu) of L. rhamnosus PRSF-L477 (n 17) or placebo (n 17) for a period of 3 weeks, followed by a wash-out period of another 3 weeks. A questionnaire on gastrointestinal tolerance and a diary was kept daily to record compliance throughout these 6 weeks. Faecal and blood samples were collected for microbiological and haematological analysis. The recorded gastrointestinal symptoms, defecation frequency and stool consistency were not influenced indicating that L. rhamnosus PRSF-L477 was well tolerated. The species L. rhamnosus was detected in the faeces of sixteen out of seventeen subjects of the probiotic group during the intervention period. Using pulsed-field gel electrophoresis, re-isolates of L. rhamnosus PRSF-L477 were confirmed in nine of these subjects. Antibiotic susceptibility profiles of these re-isolates were unchanged compared with PRSF-L477. No clinically relevant changes in blood parameters such as liver and kidney function and no serious adverse events appeared during and after administration. Therefore, we conclude that L. rhamnosus PRSF-L477 can safely be administrated to healthy subjects at a daily dose of 1 x 10(11) cfu

Cross-transmission rates of enterococcal isolates among newborns in a neonatal intensive care unit

Enterococci are important pathogens causing nosocomial infections and patients at risk include also premature babies requiring intensive care treatment. Our aim was to assess occurrence and cross transmission rates of enterococci among neonatal patients of a hospital ward during a two months period. Rectal and skin samples were taken between day one and 60 of infants' age. Colonization with various potentially pathogenic bacteria was correlated with developing a subsequent infection. Enterococcal isolates were identified by colony morphology. The bacterial species was assessed and antibiotic susceptibilities were determined. A molecular analysis of 20 investigated enterococcal isolates revealed prevalence of commensal strain types; hospital-associated strain types or multi-resistant variants were absent. Cross transmission of E. faecium and E. faecalis isolates among neonatal patients attending the intensive crare unit at the same time was demonstrable. Introduction of hospital-associated, multi-resistant variants into this special setting has to be avoided to reduce the risk of subsequent infections

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

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

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.Peer Reviewe

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

Antibiotikaresistenz bei bakteriellen Infektionserregern

In den vergangenen zehn Jahren traten bei bakteriellen Infektionserregern weltweit neue Resistenzeigenschaften auf (z.B. gegen Glykopeptide, Chinolone, Carbapeneme). Es kam zu einer Zunahme mehrfachresistenter Bakterienstämme. Dies betrifft zum einen Erreger von Krankenhausinfektionen (S. aureus, Enterokokken, Enterobacteriaceae, Nonfermenter) sowie auch von ambulant erworbenen Infektionen (z.B. S. pneumoniae, M. tuberculosis, S. typhimurium). Die Möglichkeit, daß neuartige Resistenzeigenschaften bei weiteren Erregergruppen auftreten und diese gegen zusätzliche antibakterielle Chemotherapeutika resistent werden (z.B. Glykopeptidresistenz bei Staphylokokken, Carbapenemase bei gram-negativen Bakterien, Resistenz gegen neue Chinolone bei S. pneumoniae), erfordert besondere Aufmerksamkeit. Die Auswirkungen der Resistenzentwicklung auf die Veränderung von Chemotherapieregimen, auf Morbidität und Behandlungskosten sind gut dokumentiert. Für begründete Aussagen hinsichtlich der Mortalität fehlen jedoch oft weiterführende Studien an vergleichbaren Patientengruppen. Für die Prävention haben ein zurückhaltender und rationaler Antibiotikaeinsatz sowie Hygienemaßnahmen, die an die jeweilige Situation angepaßt sind, eine vorrangige Bedeutung