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® 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.

CHROMAgar™ LIN-R as an efficient screening tool to assess the prevalence of linezolid-resistant enterococci in German hospital patients-a multicentre study approach, 2021-2022.

BACKGROUND: In recent years, an increasing number of linezolid-resistant enterococci (LRE) was recognized at the German National Reference Centre (NRC) for Enterococci. National guidelines on infection prevention recommend screening for LRE in epidemiologically linked hospital settings without referring to a reliable and rapid diagnostic method. Since 2020, CHROMAgar™ provide a chromogenic linezolid screening agar, LIN-R, suitable to simultaneously screen for linezolid-resistant staphylococci and enterococci. OBJECTIVES: To assess the applicability of CHROMAgar™ LIN-R in clinical settings for detecting LRE directly from patient material and to infer prevalence rates of LRE amongst German hospital patients. METHODS: During the 3-month trial period, clinical samples were plated on CHROMAgar™ LIN-R. Antimicrobial susceptibility testing was performed using VITEK2 or disc diffusion. At the NRC, linezolid resistance was determined by broth microdilution, multiplex-PCR for cfr/optrA/poxtA and by a restriction-based assay for 23S rDNA mutations. RESULTS: The 12 participating study sites used 13 963 CHROMAgar™ LIN-R plates during the study period. Of 442 presumptive LRE, 192 were confirmed by phenotypic methods. Of these, 161 were received by the NRC and 121 (75%) were verified as LRE. Most of LR-E. faecium 53/81 (65%) exhibited a 23S rRNA gene mutation as the sole resistance-mediating mechanism, whereas optrA constituted the dominant resistance trait in LR-E. faecalis [39/40 (98%)]. Prevalence of LRE across sites was estimated as 1% (ranging 0.18%-3.7% between sites). CONCLUSIONS: CHROMAgar™ LIN-R represents a simple and efficient LRE screening tool in hospital settings. A high proportion of false-positive results demands validation of linezolid resistance by a reference method.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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

BACKGROUND: Hospital strains of Enterococcus faecium 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 E. faecium 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, IS16, to identify hospital E. faecium isolates within a collection of 260 strains of hospital, animal and human commensal origins. METHODS: Specific primers were selected amplifying a 547-bp fragment of IS16. Presence of IS16 was determined by PCR screenings among the 260 E. faecium isolates. Distribution of IS16 was compared with a prevalence of commonly used markers for hospital strains, esp and hylEfm. All isolates were typed by MLST and partly by PFGE. Location of IS16 was analysed by Southern hybridization of plasmid and chromosomal DNA. RESULTS: IS16 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 (vanA/B/negative). The five invasive IS16-negative strains did not belong to the clonal complex of hospital-associated strains (CC17). IS16 was absent in all but three isolates from 100 livestock, food-associated and human commensal strains ("non-CC17"; 64 MLST types). The three IS16-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 IS16 was 100% and thus superior to screening for the presence of esp (66%) and/or hylEfm (46%). Southern hybridizations revealed chromosomal as well as plasmid localization of IS16. CONCLUSIONS: This simple screening assay for insertion element IS16 is capable of differentiating hospital-associated from human commensal, livestock- and food-associated E. faecium strains and thus allows predicting the epidemic strengths or supposed pathogenic potential of a given E. faecium isolate identified within the nosocomial setting.

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.