Evaluation of Two Different Preparation Protocols for MALDI-TOF MS Nontuberculous Mycobacteria Identification from Liquid and Solid Media.

Nontuberculous mycobacteria (NTM) identification is essential for establishing the relevance of the isolate and for appropriate antimicrobial therapy. Traditionally, NTM identification is performed by using Line Probe Assays (LPA), a costly and time-consuming technique requiring trained personnel. MALDI-TOF MS is a promising tool for NTM identification, and its use is rapidly growing. We evaluated the newly introduced MBT Mycobacteria kit (MBT) and the MycoEx preparation protocol (Bruker Daltonics, Germany) for NTM MALDI-TOF MS identification using LPA results as a reference. Fifty NTM grown on 7H11 agar and MGIT broth were analyzed with both protocols using the Bruker Microflex® LT MALDI-TOF MS (Bruker Daltonics) instrument. MBT and MycoEx provided identification results in 97.0% and 95.0% of the cases, respectively. With both protocols, 100% of the provided results agreed with LPA with no registered mismatch. MBT achieved an elevated number of highly probable identifications (88.0% vs. 83.0%) and a higher reproducibility rate of correct results (86.6% vs. 75.8%) in comparison to MycoEx. This study provides results about MBT performance for liquid and solid media, underlining the strengths and weakness under different conditions. Our results suggest that MALDI-TOF MS could provide a great advantage for timely and cost-saving NTM identification with potential implications for patient outcome.

Epidemiological analysis of Trueperella abortisuis isolated from cases of pig abortion of a single farm.

The present study was designed to characterize six Trueperella (T.) abortisuis strains, cultured over a period of 5 months from fetus and abortion material of six pigs of a single farm in Mecklenburg-West Pomerania federal state, Germany. It was of interest to investigate the epidemiological relationships of the six strains among each other and whether a single bacterial clone was responsible for the abortion situation of the single farm. All six strains were identified phenotypically, by MALDI-TOF MS analysis and by phylogenetic analysis based on 16S rRNA gene and gap (encoding the glyceraldehyde 3-phosphate dehydrogenase) and tuf (encoding elongation factor tu) gene sequencing. Further genotypic comparison was performed using different genomic DNA fingerprint methods including BOX-PCR, (GTG)5-PCR, and three RAPD-PCRs. The sequence analysis of the genes gap and tuf and the genomic DNA fingerprinting results revealed, as noval findings, that the six T. abortisuis strains cultured from a single farm represent six different bacterial clones showing a genetic variability of this bacterial species in the pig population. All six T. abortisuis strains were isolated in mixed culture with several other bacterial species. However, the T. abortisuis strain, generally found in high numbers, seemed to be responsible for the abortion situation in the farm.

Confirmation and Identification of Salmonella spp., Cronobacter spp., and Other Gram-Negative Organisms by the Bruker MALDI Biotyper Method: Collaborative Study Method Extension to Include Campylobacter Species, Revised First Action 2017.09.

Background: The Bruker MALDI Biotyper® method utilizes matrix-assisted laser desorption/ionization time-of-flight MS for the rapid and accurate identification and confirmation of Gram-negative bacteria from select media types. The alternative method was evaluated in a method extension study of AOAC INTERNATIONAL First Action Official MethodSM 2017.09 using nonselective and selective agars to identify Cronobacter spp., Salmonella spp., Campylobacter spp., and select Gram-negative bacteria. Results obtained by the Bruker MALDI Biotyper were compared to the traditional biochemical methods as prescribed in the appropriate reference methods. Methods: Two collaborative studies were organized, one in the United States focusing on Cronobacter spp. and other Gram-negative bacteria and one in Europe focusing on Salmonella spp. and other Gram-negative bacteria. Fourteen collaborators from seven laboratories located within the United States participated in the first collaborative study for Cronobacter spp. Fifteen collaborators from 15 service laboratories located within Europe participated in the second collaborative study for Salmonella spp. For each target organism (either Salmonella spp. or Cronobacter spp.), a total of 24 blind-coded isolates were evaluated. In each set of 24 organisms, there were 16 inclusivity organisms (Cronobacter spp. or Salmonella spp.) and 8 exclusivity organisms (non-Cronobacter spp. and non-Salmonella spp. closely related Gram-negative organisms). For the Campylobacter spp. method extension, 17 collaborators from eight laboratories located within the United States (seven laboratories) and Canada (one laboratory) participated in the collaborative study. A total of 24 blind-coded isolates were evaluated. In each set of 24 organisms, there were 16 inclusivity organisms (Campylobacter spp.) and 8 exclusivity organisms (non-Campylobacter spp. closely related Gram-negative organisms). Results: After testing was completed, the total percentage of correct identifications from each agar type for each strain was determined at a percentage of 100.0% to the genus level for the Cronobacter study and a percentage of 100.0% to the genus level for the Salmonella study. For the Campylobacter method extension, a correct identification and confirmation rate of 100.0% was obtained for the Campylobacter organisms at the species level. For non-Cronobacter, non-Salmonella, and non-Campylobacter organisms, 100.0% were correctly identified. Conclusions: The results indicated that the alternative method produced equivalent results when compared to the confirmatory procedures specified by each reference method. Highlights: The method extension can be modified to include the identification and confirmation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari.

Confirmation and Identification of Salmonella spp., Cronobacter spp., and Other Gram-Negative Organisms by the Bruker MALDI Biotyper Method: Collaborative Study, First Action 2017.09.

The Bruker MALDI Biotyper® method utilizes matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for the rapid and accurate identification and confirmation of Gram-negative bacteria from select media types. The alternative method was evaluated using nonselective and selective agars to identify Cronobacter spp., Salmonella spp., and select Gram-negative bacteria. Results obtained by the Bruker MALDI Biotyper were compared to the traditional biochemical methods as prescribed in the appropriate reference methods. Two collaborative studies were organized, one in the United States focusing on Cronobacter spp. and other Gram-negative bacteria, and one in Europe focusing on Salmonella spp. and other Gram-negative bacteria. Fourteen collaborators from seven laboratories located within the United States participated in the first collaborative study for Cronobacter spp. Fifteen collaborators from 15 service laboratories located within Europe participated in the second collaborative study for Salmonella spp. For each target organism (either Salmonella spp. or Cronobacter spp.), a total of 24 blind-coded isolates were evaluated. In each set of 24 organisms, there were 16 inclusivity organisms (Cronobacter spp. or Salmonella spp.) and 8 exclusivity organisms (closely related non-Cronobacter spp. and non-Salmonella spp. Gram-negative organisms). After testing was completed, the total percentage of correct identifications from each agar type for each strain was determined at a percentage of 100.0% to the genus level for the Cronobacter study and a percentage of 100.0% to the genus level for the Salmonella study. For both non-Cronobacter and non-Salmonella organisms, a percentage of 100.0% was correctly identified. The results indicated that the alternative method produced equivalent results when compared to the confirmatory procedures specified by each reference method.

Confirmation and Identification of Listeria monocytogenes, Listeria spp. and Other Gram-Positive Organisms by the Bruker MALDI Biotyper Method: Collaborative Study, First Action 2017.10.

The Bruker MALDI Biotyper® method utilizes matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for the rapid and accurate confirmation and identification of Gram-positive bacteria from select media types. This alternative method was evaluated using nonselective and selective agar plates to identify and confirm Listeria monocytogenes, Listeria species, and select Gram-positive bacteria. Results obtained by the Bruker MALDI Biotyper were compared with the traditional biochemical methods as prescribed in the appropriate reference method standards. Sixteen collaborators from 16 different laboratories located within the European Union participated in the collaborative study. A total of 36 blind-coded isolates were evaluated by each collaborator. In each set of 36 organisms, there were 16 L. monocytogenes strains, 12 non-monocytogenes Listeria species strains, and 8 additional Gram-positive exclusivity strains. After testing was completed, the total percentage of correct identifications (to both genus and species level) and confirmation from each agar type for each strain was determined at a percentage of 99.9% to the genus level and 98.8% to the species level. The results indicated that the alternative method produced equivalent results when compared with the confirmatory procedures specified by each reference method.

Identification of Arcanobacterium phocae isolated from fur animals by phenotypic properties, by MALDI-TOF MS analysis and by detection of phocaelysin encoding gene phl as probable novel target.

In the present study 12 Arcanobacterium phocae strains isolated from fur animals in Finland, including foxes, minks and Finnraccoons, could successfully be identified phenotypically, by matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) and genotypically by sequencing 16S rDNA and phocaelysin (PHL) encoding gene phl. The PHL of all 12 A. phocae strains in the present study and reference strains A. phocae DSM 10002T and A. phocae DSM 10003 displayed, as typical members of the cholesterol dependent cytolysin-group of toxins, the variant undecapeptide sequence EATGLAWDPWW which appeared to be most closely related to arcanolysin of Arcanobacterium haemolyticum and pyolysin of Trueperella pyogenes. In addition, gene phl could be determined with a newly designed loop-mediated isothermal amplification (LAMP) assay. The detection of mass spectra by MALDI-TOF MS and the LAMP assay based on gene phl might help to reliably identify A. phocae in future and also elucidate the role this species plays in infections of fur animals.

Evaluation of a Novel MALDI Biotyper Algorithm to Distinguish Mycobacterium intracellulare From Mycobacterium chimaera.

Accurate and timely mycobacterial species identification is imperative for successful diagnosis, treatment, and management of disease caused by nontuberculous mycobacteria (NTM). The current most widely utilized method for NTM species identification is Sanger sequencing of one or more genomic loci, followed by BLAST sequence analysis. MALDI-TOF MS offers a less expensive and increasingly accurate alternative to sequencing, but the commercially available assays used in clinical mycobacteriology cannot differentiate between Mycobacterium intracellulare and Mycobacterium chimaera, two closely related potentially pathogenic species of NTM that are members of the Mycobacterium avium complex (MAC). Because this differentiation of MAC species is challenging in a diagnostic setting, Bruker has developed an improved spectral interpretation algorithm to differentiate M. chimaera and M. intracellulare based on differential spectral peak signatures. Here, we utilize a set of 185 MAC isolates that have been characterized using rpoB locus sequencing followed by whole genome sequencing in some cases, to test the accuracy of the Bruker subtyper software to identify M. chimaera (n = 49) and M. intracellulare (n = 55). 100% of the M. intracellulare and 82% of the M. chimaera isolates were accurately identified using the MALDI Biotyper algorithm. This subtyper module is available with the MALDI Biotyper Compass software and offers a promising mechanism for rapid and inexpensive species determination for M. chimaera and M. intracellulare.

Trueperella pyogenes isolated from a brain abscess of an adult roebuck (Capreolus capreolus).

The present study was designed to characterize phenotypically and genotypically a Trueperella pyogenes strain isolated from a brain abscess of an adult roebuck (Capreolus capreolus). The species identity could be confirmed by phenotypical investigations, by MALDI-TOF MS analysis, and by sequencing the 16S ribosomal RNA (rRNA) gene, the 16S-23S rRNA intergenic spacer region (ISR); by sequencing the target genes rpoB, gap, and tuf; and by detection of T. pyogenes chaperonin-encoding gene cpn60 with a previously developed loop-mediated isothermal amplification (LAMP) assay. The T. pyogenes strain could additionally be characterized by PCR-mediated amplification of several known and putative virulence factor-encoding genes which revealed the presence of the genes plo encoding pyolysin and nanH and nanP encoding neuraminidases; the genes fimA, fimC, and fimE encoding the fimbrial subunits FimA, FimC, and FimE; and the gene cbpA encoding collagen-binding protein CbpA. The present data give a detailed characterization of a T. pyogenes strain isolated from a brain abscess of a roebuck. However, the route of infection of the roebuck remains unclear.

Accurate differentiation of Mycobacterium chimaera from Mycobacterium intracellulare by MALDI-TOF MS analysis.

PURPOSE: The increasing number of infections caused by nontuberculous mycobacteria (NTM) has prompted the need for rapid and precise identification methods of these pathogens. Several studies report the applicability of MALDI-TOF mass spectrometry (MS) for identification of NTM. However, some closely related species have very similar spectral mass fingerprints, and until recently, Mycobacterium chimaera and M. intracellulare could not be separated from each other by MALDI-TOF MS. METHODOLOGY: The conventional identification methods used in routine diagnostics have similar limitations. Recently, the differentiation of these two species within the Mycobacterium avium complex has become increasingly important due to reports of M. chimaera infections related to open heart surgery in Europe and in the USA. In this report, a method for the distinct differentiation of M. chimaera and M. intracellulare using a more detailed analysis of MALDI-TOF mass spectra is presented. KEY FINDINGS: Species-specific peaks could be identified and it was possible to assign all isolates (100 %) from reference strain collections as well as clinical isolates to the correct species. CONCLUSIONS: We have developed a model for the accurate identification of M. chimaera and M. intracellulare by MALDI-TOF MS. This approach has the potential for routine use in microbiology laboratories, as the model itself can be easily implemented into the software of the currently available systems by MALDI-TOF MS manufacturers.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Combined Species Identification and Drug Sensitivity Testing in Mycobacteria.

Species identification and drug susceptibility testing (DST) of mycobacteria are important yet complex processes traditionally reserved for reference laboratories. Recent technical improvements in matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has started to facilitate routine mycobacterial identifications in clinical laboratories. In this paper, we investigate the possibility of performing phenotypic MALDI-based DST in mycobacteriology using the recently described MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA). We randomly selected 72 clinical Mycobacterium tuberculosis and nontuberculous mycobacterial (NTM) strains, subjected them to MBT-ASTRA methodology, and compared its results to current gold-standard methods. Drug susceptibility was tested for rifampin, isoniazid, linezolid, and ethambutol (M. tuberculosis, n = 39), and clarithromycin and rifabutin (NTM, n = 33). Combined species identification was performed using the Biotyper Mycobacteria Library 4.0. Mycobacterium-specific MBT-ASTRA parameters were derived (calculation window, m/z 5,000 to 13,000, area under the curve [AUC] of >0.015, relative growth [RG] of <0.5; see the text for details). Using these settings, MBT-ASTRA analyses returned 175/177 M. tuberculosis and 65/66 NTM drug resistance profiles which corresponded to standard testing results. Turnaround times were not significantly different in M. tuberculosis testing, but the MBT-ASTRA method delivered on average a week faster than routine DST in NTM. Databases searches returned 90.4% correct species-level identifications, which increased to 98.6% when score thresholds were lowered to 1.65. In conclusion, the MBT-ASTRA technology holds promise to facilitate and fasten mycobacterial DST and to combine it directly with high-confidence species-level identifications. Given the ease of interpretation, its application in NTM typing might be the first in finding its way to current diagnostic workflows. However, further validations and automation are required before routine implementation can be envisioned.

Evaluation of MALDI Biotyper Mycobacteria Library v3.0 for Identification of Nontuberculous Mycobacteria.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has demonstrated its ability to promptly identify nontuberculous mycobacteria using the Mycobacteria Library v2.0. However, some species are particularly difficult to identify reliably using this database, providing a low log(score). In this study, the identification power of an updated Mycobacteria Library (v3.0) has been evaluated. Overall, 109 NTM isolates were analyzed with both databases. The v3.0 database allowed a high-level confidence in the identification [log(score) value, ≥1.8] of 91.7% of the isolates versus 83.5% with the v2.0 version (P< 0.01).

Phenotypic and genotypic approach to characterize Arcanobacterium pluranimalium isolated from bovine milk samples.

In the present study, three Arcanobacterium pluranimalium strains isolated from bovine milk samples of three cows of three farms (two cows with subclinical mastitis) could successfully be identified by phenotypical investigations, by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis and genotypically by sequencing the molecular targets 16S rDNA, 16S-23S rDNA intergenic spacer region (ISR), the ß subunit of bacterial RNA polymerase encoding gene rpoB, the glyceraldehyde 3-phosphate dehydrogenase encoding gene gap, the elongation factor tu encoding gene tuf, and the pluranimaliumlysin encoding gene pla. The latter could also be identified by a loop-mediated isothermal amplification (LAMP) assay. The presented phenotypic and genotypic approaches might support the identification of A. pluranimalium in future and might help to understand the role this species plays in bovine mastitis.

Phenotypic and genotypic characteristics of Arcanobacterium haemolyticum isolated from clinical samples in a Danish hospital.

Six Arcanobacterium haemolyticum strains isolated from six patients of two hospitals in Denmark were identified phenotypically, also including matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, and by genotypic methods. The latter were performed by sequencing 16S rDNA and glyceraldehyde 3-phosphate dehydrogenase encoding gene gap and by amplification of an A. haemolyticum specific region of 16S-23S rDNA intergenic spacer region and 23S rDNA. The six A. haemolyticum strains were further investigated for the presence of seven potential virulence genes encoding arcanolysin, phospholipase D, hemolysin A, CAMP factor family protein, collagen binding protein, neuraminidase A and neuraminidase H which appeared to be present in two (seven virulence genes), two (six virulence genes) and two strains (four virulence genes), respectively. The phenotypic and genotypic properties described in the present study might help to reliably identify and further characterize A. haemolyticum isolated from human patients, a species which seems to be of increasing importance.

Properties of an Arcanobacterium haemolyticum strain isolated from a donkey.

The present study was designed to characterize phenotypically and genotypically an Arcanobacterium haemolyticum strain (A. haemolyticum P646) isolated from a purulent nasal discharge of a donkey. A. haemolyticum P646 showed, compared to sheep blood, an enhanced hemolytic reaction on rabbit blood agar, a synergistic CAMP-like reaction with Streptococcus agalactiae and Rhodococcus equi as indicator strains, a reverse CAMP reaction in the zone of Staphylococcus aureus beta-hemolysin and the typical biochemical properties of this species. The species identity could be confirmed by MALDI-TOF MS analysis, by sequencing the 16S rDNA and glyceraldehyde-3-phosphate dehydrogenase encoding gene gap and by amplification of A. haemolyticum specific parts of 16S-23S rDNA intergenic spacer region and 23S rDNA. A. haemolyticum P646 and the reference strain A. haemolyticum DSM 20595 were further characterized by amplification of the putative virulence genes encoding arcanolysin, phospholipase D, hemolysin A, CAMP factor family protein, a collagen binding protein and two neuraminidases which were present for A. haemolyticum DSM 20595. A. haemolyticum P646 showed a comparable gene spectrum but was negative for the genes encoding collagen binding protein and neuraminidase H. To our knowledge, the present study is the first phenotypic and genotypic characterization of an A. haemolyticum strain isolated from a donkey.

Further Studies on Arcanobacterium phocisimile: a Novel Species of Genus Arcanobacterium.

Arcanobacterium phocisimile, a newly described species with the type strain A. phocisimile 2698(T) isolated from a vaginal swab of a harbour seal and four additional A. phocisimile strains also isolated from four harbour seals could reliably be identified by phenotypic properties, by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), and by sequencing the genomic targets 16S rDNA and 16S-23S rDNA intergenic spacer region and the genes rpoB and gap. The A. phocisimile strains investigated in the present study were isolated together with several other bacterial species indicating that the pathogenic importance of A. phocisimile remains unclear. However, the detection of peptidic spectra by MALDI-TOF MS and the presented phenotypic and genotypic approach might help to identify A. phocisimile in future.

Comparison of MALDI-TOF MS with HPLC and nucleic acid sequencing for the identification of Mycobacterium species in cultures using solid medium and broth.

OBJECTIVES: The genus Mycobacterium contains over 150 species including pathogenic and nonpathogenic strains. Accurate species level identification can aid in differentiating environmental contamination from true infection, and also can aid in selection of antimicrobial therapy. METHODS: We evaluated the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the routine identification of clinical isolates of mycobacteria using 2 commercially available spectral reference libraries, and also assessed the impact of mycobacterial culture using solid medium and broth on MALDI-TOF MS-based identification. RESULTS: All results were compared with those obtained on high-pressure liquid chromatography and nucleic acid sequencing. Optimal results were obtained with a mycobacterium-specific reference library (Mycobacterium Library v1.0). The identification rate was 89.8% (79/88) for isolates cultured on solid medium and 98.8% (85/86) for isolates analyzed directly from broth. Among these, the proportion identified with a high confidence level was 50.0% (44/88) for isolates cultured on solid medium and 80.2% (69/86) for isolates cultured in broth. CONCLUSIONS: Agreement with nucleic acid sequencing for species present in Mycobacterium Library v1.0 was 97.6% (81/83) for isolates cultured on solid medium and 97.5% (79/81) for those cultured in broth.

Interlaboratory comparison of intact-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry results for identification and differentiation of Brucella spp.

Classical microbiological diagnosis of human brucellosis is time-consuming, hazardous, and subject to variable interpretation. Intact-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated for the routine identification of Brucella spp. Analysis of mass peak patterns allowed accurate identification to the genus level. However, statistical models based on peak intensities were needed for definite species differentiation. Interlaboratory comparison confirmed the reproducibility of the results.

Trueperella pyogenes as cause of a facial abscess in a grey slender loris (Loris lydekkerianus nordicus)--a case report.

In the present study a Trueperella (T.) pyogenes strain isolated from an abscess on the left side of the face of a six year old grey slender loris (Loris lydekkerianus nordicus) could successfully be identified phenotypically, by MALDI-TOF MS analysis and genotypically using T. pyogenes superoxide dismutase A encoding gene sodA and T. pyogenes 16S-23S rDNA intergenic spacer region specific oligonucleotide primers. The T. pyogenes strain could additionally be characterized by PCR-mediated amplification of several known and putative virulence factor encoding genes which revealed the presence of the genes plo encoding pyolysin, nanH encoding neuraminidase NanH and the genes fimA, fimC, fimE encoding the fimbrial subunits FimA, FimC and FimE but not the genes cbpA and nanP encoding collagen-binding protein CbpA and neuraminidase NanP, respectively. The present data give the first information about properties of T. pyogenes isolated from a monkey.

Further characteristics of Actinomyces weissii, a novel species isolated from the oral cavity of dogs.

Comparable to previously conducted phenotypical and genotypical investigations (Hijazin et al., 2011c), three strains of the newly described species Actinomyces weissii, isolated from infections of the oral cavity of three dogs could be classified by matrix-assisted laser desorption ionization-time of flight mass spectrometry and by sequencing the target genes 23S rDNA and cpn60 as novel species of genus Actinomyces. The detection of peptidic spectra and both genotypic approaches might help to identify A. weissii in future and elucidate the role this species plays in infections of dogs.

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).