An update on the taxonomy and functional properties of the probiotic Enterococcus faecium SF68.

Enterococcus faecium SF68 (SF68) is a well-known probiotic with a long history of safe use. Recent changes in the taxonomy of enterococci have shown that a novel species, Enterococcus lactis, is closely related with E. faecium and occurs together with other enterococci in a phylogenetically well-defined E. faecium species group. The close phylogenetic relationship between the species E. faecium and E. lactis prompted a closer investigation into the taxonomic status of E. faecium SF68. Using phylogenomics and ANI, the taxonomic analysis in this study showed that probiotic E. faecium SF68, when compared to other E. faecium and E. lactis type and reference strains, could be re-classified as belonging to the species E. lactis. Further investigations into the functional properties of SF68 showed that it is potentially capable of bacteriocin production, as a bacteriocin gene cluster encoding the leaderless bacteriocin EntK1 together with putative Lactococcus lactis bacteriocins LsbA, and LsbB-like putative immunity peptide (LmrB) were found located in an operon on plasmid pF9. However, bacteriocin expression was not studied. Competitive exclusion experiments in co-culture over 7 days at 37 °C showed that the probiotic SF68 could inhibit the growth of specific E. faecium and Listeria monocytogenes strains, while showing little or no inhibitory activity towards an entero-invasive Escherichia coli and a Salmonella Typhimurium strain, respectively. In cell culture experiments with colon carcinoma HT29 cells, the probiotic SF68 was also able to strain-specifically inhibit adhesion and/or invasion of enterococcal and L. monocytogenes strains, while such adhesion and invasion inhibition effects were less pronounced for E. coli and Salmonella strains. This study therefore provides novel data on the taxonomy and functional properties of SF68, which can be reclassified as Enterococcus lactis SF68, thereby enhancing the understanding of its probiotic nature.

Comprehensive integrated NGS-based surveillance and contact-network modeling unravels transmission dynamics of vancomycin-resistant enterococci in a high-risk population within a tertiary care hospital.

Vancomycin-resistant E. faecium (VRE) are an important cause of nosocomial infections, which are rapidly transmitted in hospitals. To identify possible transmission routes, we applied combined genomics and contact-network modeling to retrospectively evaluate routine VRE screening data generated by the infection control program of a hemato-oncology unit. Over 1 year, a total of 111 VRE isolates from 111 patients were collected by anal swabs in a tertiary care hospital in Southern Germany. All isolated VRE were whole-genome sequenced, followed by different in-depth bioinformatics analyses including genotyping and determination of phylogenetic relations, aiming to evaluate a standardized workflow. Patient movement data were used to overlay sequencing data to infer transmission events and strain dynamics over time. A predominant clone harboring vanB and exhibiting genotype ST117/CT469 (n = 67) was identified. Our comprehensive combined analyses suggested intra-hospital spread, especially of clone ST117/CT469, despite of extensive screening, single room placement, and contact isolation. A new interactive tool to visualize these complex data was designed. Furthermore, a patient-contact network-modeling approach was developed, which indicates both the periodic import of the clone into the hospital and its spread within the hospital due to patient movements. The analyzed spread of VRE was most likely due to placement of patients in the same room prior to positivity of screening. We successfully demonstrated the added value for this combined strategy to extract well-founded knowledge from interdisciplinary data sources. The combination of patient-contact modeling and high-resolution typing unraveled the transmission dynamics within the hospital department and, additionally, a constant VRE influx over time.

High Phenotypic and Genotypic Diversity of Enterococcus faecium from Clinical and Commensal Isolates in Third Level Hospital.

Background: The use of antimicrobials and myeloablative chemotherapy regimens has promoted multiresistant microorganisms to emerge as nosocomial pathogens, such as vancomycin-resistant Enterococcus faecium (VREfm). We described a polyclonal outbreak of bloodstream infection caused by Efm in a hemato-oncological ward in Mexico. Our aim was to describe the clonal complex (CC) of the Efm strains isolated in the outbreak in comparison with commensal and environmental isolates. Methodology: Sixty Efm clinical, environmental, and commensal strains were included. We constructed a cladogram and a phylogenetic tree using Vitek and Multilocus sequence typing data, respectively. Results: We reported 20 new sequence types (ST), among which 17/43 clinical isolates belonged to CC17. The predominant ST in the clinical strains were ST757, ST1304, ST412, and ST770. Neither environmental nor commensal isolates belonged to CC17. The phylogeny of our collection shows that the majority of the clinical isolates were different from the environmental and commensal isolates, and only a small group of clinical isolates was closely related with environmental and commensal isolates. The cladogram revealed a similar segregation to that of the phylogeny. Conclusions: We found a high diversity among clinical, environmental, and commensal strains in a group of samples in a single hospital. Highest diversity was found between commensal and environmental isolates.

Characterization of a new transferable MDR plasmid carrying the pbp5 gene from a clade B commensal Enterococcus faecium.

OBJECTIVES: To evaluate the transferability of antibiotic resistance from an MDR clade B Enterococcus faecium and to characterize the genetic elements involved. METHODS: The erm(B)-positive strain E. faecium 37BA (donor) and strains E. faecium 64/3 and Listeria welshimeri 11857RF (recipients) were used in mating experiments. Donors and transconjugants were characterized using MIC assays, PFGE, Southern blotting and hybridization, quantitative RT-PCR (RT-qPCR), next-generation sequencing and PCR mapping. RESULTS: One E. faecium and one L. welshimeri transconjugant were selected for in-depth investigation. Both acquired an ∼40 kb plasmid carrying erm(B). An additional plasmid of ∼200 kb, encoding the full conjugation machinery, was detected in the donor and in the E. faecium transconjugant. Next-generation sequencing revealed a new 40 396 bp plasmid that was designated pEf37BA; it contained 10 antibiotic resistance genes, tet(M), tet(L), erm(B), aadE, sat4, aphA, spw, lsa(E), lnu(B) and pbp5, resulting from the recombination of pM7M2 of E. faecium with an MDR chromosomal region of Erysipelothrix rhusiopathiae. A pbp5-carrying circular form was also detected. The PBP5 amino acid sequence differed from the C46 variant by two mutations (S39T and D644N). Its expression was documented in both transconjugants. pEf37BA persisted in the absence of selective pressure. CONCLUSIONS: The MDR clade B E. faecium plasmid, deriving from the recombination of two different resistance regions, carried a pbp5 element and was transferable to different bacterial species. This finding further documents the dissemination of ampicillin resistance among community-associated E. faecium and the key role of commensal strains in the spread of antibiotic resistance.

The commensal microbiome is associated with anti-PD-1 efficacy in metastatic melanoma patients.

Anti-PD-1-based immunotherapy has had a major impact on cancer treatment but has only benefited a subset of patients. Among the variables that could contribute to interpatient heterogeneity is differential composition of the patients' microbiome, which has been shown to affect antitumor immunity and immunotherapy efficacy in preclinical mouse models. We analyzed baseline stool samples from metastatic melanoma patients before immunotherapy treatment, through an integration of 16S ribosomal RNA gene sequencing, metagenomic shotgun sequencing, and quantitative polymerase chain reaction for selected bacteria. A significant association was observed between commensal microbial composition and clinical response. Bacterial species more abundant in responders included Bifidobacterium longum, Collinsella aerofaciens, and Enterococcus faecium. Reconstitution of germ-free mice with fecal material from responding patients could lead to improved tumor control, augmented T cell responses, and greater efficacy of anti-PD-L1 therapy. Our results suggest that the commensal microbiome may have a mechanistic impact on antitumor immunity in human cancer patients.

Effects of Enterococcus faecium SLB 120 on growth performance, blood parameters, relative organ weight, breast muscle meat quality, excreta microbiota shedding, and noxious gas emission in broilers.

This 5-week study was conducted to determine the effects of Enterococcus faecium (SLB 120) on growth performance, blood parameters, relative organ weight, breast muscle meat quality, excreta microbiota shedding, and noxious gas emission in broilers. A total of 816 one-day-old male broilers were allocated to 4 groups with 12 replications (17 broilers/pen) according to body weight (43.2 ± 0.32 g). Dietary treatment groups were: (1) CON, basal diet, (2) T1, CON + 0.05% E. faecium, (3) T2, CON + 0.10% E. faecium, (4) T3, CON + 0.20% E. faecium. From day 1 to 21, dietary E. faecium supplementation showed linear increase (P < 0.05) in gain:feed ratio. From day 21 to 35 and the overall period, dietary E. faecium supplementation showed a linear increase (P < 0.05) in body weight gain and gain:feed ratio. On day 35, dietary E. faecium supplementation showed a linear increase (P < 0.05) in the apparent total tract digestibility of dry matter and nitrogen, and the relative weight of bursa of Fabricius; no differences were observed in white blood cells, red blood cells or lymphocyte counts. Dietary E. faecium supplementation showed a linear decrease (P < 0.05) in excreta E.coli counts on day 7 and 35, while excreta Lactobacillus counts were linearly increased (P < 0.05) on day 35. On day 35, dietary E. faecium supplementation linearly decreased (P < 0.05) excreta NH3, H2S, and total mercaptans emission, but only H2S emission was linearly decreased (P < 0.05) on day 7. In conclusion, the supplementation of E. faecium improved growth performance, the digestibility of dry matter and nitrogen, the relative weight of bursa of Fabricius, and shifted excreta microbiota by increasing Lactobacillus and decreasing E.coli counts, as well as decreased excreta NH3, H2S, and total mercaptans gas emission.

Long-term clonal dynamics of Enterococcus faecium strains causing bloodstream infections (1995-2015) in Spain.

OBJECTIVES: To investigate the population structure of Enterococcus faecium causing bloodstream infections (BSIs) in a tertiary Spanish hospital with low glycopeptide resistance, and to enhance our knowledge of the dynamics of emergence and spread of high-risk clonal complexes. METHODS: All available E. faecium causing BSIs (n = 413) in our hospital (January 1995-May 2015) were analysed for antibiotic susceptibility (CLSI), putative virulence traits (PCR, esp, hylEfm) and clonal relationship (SmaI-PFGE, MLST evaluated by goeBURST and BAPS). RESULTS: The increased incidence of BSIs caused by enterococci [2.3‰ of attended patients (inpatients and outpatients) in 1996 to 3.0‰ in 2014] significantly correlated with the increase in BSIs caused by E. faecium (0.33‰ of attended patients in 1996 to 1.3‰ in 2014). The BSIs Enterococcus faecalis:E. faecium ratio changed from 5:1 in 1996 to 1:1 in 2014. During the last decade an increase in E. faecium BSIs episodes in cancer patients (10.9% in 1995-2005 and 37.1% in 2006-15) was detected. Ampicillin-susceptible E. faecium (ASEfm; different STs/BAPS) and ampicillin-resistant E. faecium (AREfm; ST18/ST17-BAPS 3.3a) isolates were recovered throughout the study. Successive waves of BAPS 2.1a-AREfm (ST117, ST203 and ST80) partially replaced ASEfm and ST18-AREfm since 2006. CONCLUSIONS: Different AREfm clones (belonging to BAPS 2.1a and BAPS 3.3a) consistently isolated during the last decade from BSIs might be explained by a continuous and dense colonization (favouring both invasion and cross-transmission) of hospitalized patients. High-density colonization by these clones is probably enhanced in elderly patients by heavy and prolonged antibiotic exposure, particularly in oncological patients.

A polyclonal outbreak of bloodstream infections by Enterococcus faecium in patients with hematologic malignancies.

BACKGROUND: Enterococcus faecium causes bloodstream infection (BSI) in patients with hematologic malignancies (HMs). We studied the clinical features and outcomes of patients with HM with vancomycin-sensitive E faecium (VSE) and vancomycin-resistant E faecium (VRE) BSI and determined the genetic relatedness of isolates and circumstances associated with the upsurge of E faecium BSI. METHODS: Case-control study of patients with HM and E faecium-positive blood culture from January 2008-December 2012; cases were patients with VRE and controls were VSE isolates. The strains were tested for Van genes by polymerase chain reaction amplification and we performed pulsed-field gel electrophoresis to determine genetic relatedness. RESULTS: Fifty-eight episodes of E faecium BSI occurred: 35 sensitive and 23 resistant to vancomycin. Mortality was 46% and 57%, attributable 17% and 40%, respectively. Early stage HM was associated with VSE (P = .044), whereas an episode of BSI within the 3 months before the event (P = .039), prophylactic antibiotics (P = .013), and vancomycin therapy during the previous 3 months (P = .001) was associated with VRE. The VanA gene was identified in 97% of isolates studied. E faecium isolates were not clonal. CONCLUSIONS: E faecium BSI was associated with high mortality. This outbreak of VRE was not clonal; it was associated with antibiotic-use pressure and highly myelosuppressive chemotherapy.

MALDI-TOF MS meets WGS in a VRE outbreak investigation.

The use of MALDI-TOF MS (matrix-assisted laser desorption/ ionization-time of flight mass spectrometry) and WGS (whole genome sequencing) has been described for identification and strain relatedness determination. We describe the complementary use of MALDI-TOF MS and WGS in a VRE (vancomycin-resistant enterococci) outbreak investigation, and discuss some of the challenges with defining strain similarity across these two platforms. Although both assays indicated multiple clusters involved in the outbreak of vancomycin resistant Enterococcus faecium isolates from positive blood cultures of four haematology-oncology patients, the small cohort and discrepancies between findings indicate the limitations of MALDI-TOF MS and the cautious interpretation of MALDI-TOF MS dendrograms during outbreaks. For definitive determination of the evolutionary distance between isolates, WGS can be used.

The rise of ampicillin-resistant Enterococcus faecium high-risk clones as a frequent intestinal colonizer in oncohaematological neutropenic patients on levofloxacin prophylaxis: a risk for bacteraemia?

Levofloxacin extended prophylaxis (LEP), recommended in oncohaematological neutropenic patients to reduce infections, might select resistant bacteria in the intestine acting as a source of endogenous infection. In a prospective observational study we evaluated intestinal emergence and persistence of ampicillin-resistant Enterococcus faecium (AREfm), a marker of hospital adapted high-risk clones. AREfm was recovered from the faeces of 52 patients with prolonged neutropenia after chemotherapy, at admission (Basal), during LEP, and twice weekly until discharge (Pos-LEP). Antibiotic susceptibility, virulence traits and population structure (pulsed-field gel electrophoresis and multilocus sequence typing) were determined and compared with bacteraemic isolates. Gut enterococcal population was monitored using a quantitative PCR quantification approach. AREfm colonized 61.4% of patients (194/482 faecal samples). Sequential AREfm acquisition (25% Basal, 36.5% LEP, 50% Pos-LEP) and high persistent colonization rates (76.9-89.5%) associated with a decrease in clonal diversity were demonstrated. Isolates were clustered into 24 PFGE-patterns within 13 sequence types, 95.8% of them belonging to hospital-associated Bayesian analysis of population structure subgroups 2.1a and 3.3a. Levofloxacin resistance and high-level streptomycin resistance were a common trait of these high-risk clones. AREfm-ST117, the most persistent clone, was dominant (60.0% isolates, 32.6% patients). It presented esp gene and caused 18.2% of all bacteraemia episodes in 21% of patients previously colonized by this clone. In AREfm-colonized patients, intestinal enrichment in the E. faecium population with a decline in total bacterial load was observed. AREfm intestinal colonization increases during hospital stay and coincides with enterococci population enrichment in the gut. Dominance and intestinal persistence of the ST117 clone might increase the risk of bacteraemia.

Multiple hospital outbreaks of vanA Enterococcus faecium in Denmark, 2012-13, investigated by WGS, MLST and PFGE.

OBJECTIVES: In Denmark, the incidence of vancomycin-resistant Enterococcus faecium (VREfm) has increased since 2012. The aim of this study was to investigate the epidemiology and clonal relatedness of VREfm isolates in Danish hospitals in 2012-13 using WGS. The second aim was to evaluate if WGS-based typing could replace PFGE for typing of VREfm. METHODS: A population-based study was conducted including all VREfm isolates submitted for national surveillance from January 2012 to April 2013. All isolates were investigated by WGS, MLST and PFGE. RESULTS: One-hundred and thirty-two isolates were included. The majority of the isolates were from clinical samples (77%). Gastroenterology/abdominal surgery (29%) and ICUs (29%) were the predominant departments with VREfm. Genomics revealed a polyclonal structure of the VREfm outbreak. Seven subgroups of 3-44 genetically closely related isolates (separated by <17 SNPs) were identified using WGS. Direct or indirect transmission of VREfm between patients and intra- and inter-regional spreading clones was observed. We identified 10 STs. PFGE identified four major clusters (13-43 isolates) and seven minor clusters (two to three isolates). The results from the typing methods were highly concordant. However, WGS-based typing had the highest discriminatory power. CONCLUSIONS: This study emphasizes the importance of infection control measures to limit transmission of VREfm between patients. However, the diversity of the VREfm isolates points to the fact that other important factors may also affect the VREfm increase in Denmark. Finally, WGS is suitable for typing of VREfm and has replaced PFGE for typing of VREfm in Denmark.

Cluster of linezolid-resistant Enterococcus faecium ST117 in Norwegian hospitals.

A linezolid-resistant, vancomycin-susceptible Enterococcus faecium strain was isolated from 3 patients who had not received linezolid. The first patient was hospitalized in the same hospitals and wards as the 2 following patients. The E. faecium isolates were resistant to linezolid (minimum inhibitory concentration 8-32 mg/l), ampicillin, and high levels of gentamicin. Resistance to linezolid was associated with a G2576T mutation in 23S rDNA. The cfr linezolid resistance gene was not detected. The 3 isolates showed identical DNA fingerprints by pulsed-field gel electrophoresis, belonged to ST117, and harboured virulence genes esp, hyl, acm, efaAfm, srgA, ecbA, scm, pilA, pilB, and pstD typically associated with high-risk E. faecium genotypes. The linezolid-resistant E. faecium high-risk clone caused bacteraemia in the first 2 cancer patients and survived in the hospital environment for more than a year before appearing in the urethral catheter of the third patient.

Antioxidant activity of exopolysaccharide from probiotic strain Enterococcus faecium (BDU7) from Ngari.

"Ngari" is a traditional fermented fish of Manipur and considered for its therapeutic value in healing stomach ulcers. In the present study, an attempt was made to isolate and identify an efficient antioxidant probiotic isolate from Ngari. BDU7 with potent antioxidant property was isolated and characterized. The isolate was identified by 16S rRNA genotyping as Enterococcus faecium. E. faecium showed auto aggregation and hydrophobicity of 72.7 and 54.8% respectively. The extrapolysaccharide (EPS) was extracted from the culture free supernatant and assayed for its radical scavenging activity. The EPS showed significant 2, 2-diphenyl-1-picrylhydrazyl (DPPH) (63.5%), superoxide (77.3%) and hydroxyl (38.4%) radical scavenging ability. The structural analysis of the extracted and purified EPS was performed by FTIR and NMR analysis. From the present study E. faecium BDU7 can be claimed as a promising and an efficient probiotic candidate. The present study evidenced that EPS from E. faecium BDU7 showed strong DPPH and superoxide radical scavenging ability in vitro. Considering its potency as a potential antioxidant the extracted EPS can find wide application in functional food and pharmaceutical formulations.

Increase in bloodstream infection due to vancomycin-susceptible Enterococcus faecium in cancer patients: risk factors, molecular epidemiology and outcomes.

We conducted a prospective study to assess the risk factors, molecular epidemiology and outcome of bloodstream infection (BSI) due to Enterococcus faecium in hospitalized cancer patients. Between 2006 and 2012, a significant increase in vancomycin-susceptible E. faecium BSI was observed among cancer patients. Comparison of 54 episodes of BSI due to E. faecium with 38 episodes of BSI due to E. faecalis showed that previous use of carbapenems was the only independent risk factor for E. faecium acquisition (OR 10.24; 95% CI, 1.35-77.66). All E. faecium isolates were susceptible to glycopeptides, whereas 97% showed high-level resistance to ampicillin and ciprofloxacin. All 30 isolates available for genotyping belonged to the hospital-associated E. faecium lineages 17, 18 and 78. After 2009, most of the isolates belonged to ST117 (lineage 78). Patients with E. faecium BSI were more likely to receive inadequate initial empirical antibiotic therapy than patients with E. faecalis BSI, and time to adequate empirical antibiotic therapy was also longer in the former group. No significant differences were found between the two groups regarding early and overall case-fatality rates. Independent risk factors for overall case-fatality were current corticosteroids (OR 4.18; 95% CI, 1.34-13.01) and intensive care unit admission (OR 9.97; 95% CI, 1.96-50.63). The emergence of E. faecium among cancer patients is a concern since there are limited treatment options and it may presage the emergence of vancomycin-resistant enterococci. A rationale approach that combines infection control with antimicrobial stewardship.

Effects of probiotic supplementation in different energy and nutrient density diets on performance, egg quality, excreta microflora, excreta noxious gas emission, and serum cholesterol concentrations in laying hens.

This 6-wk study was conducted to determine the effects of probiotic (Enterococcus faecium DSM 7134) supplementation of different energy and nutrient density diets on performance, egg quality, excreta microflora, excreta noxious gas emission, and serum cholesterol concentrations in laying hens. A total of 432 Hy-Line brown layers (40 wk old) were allotted into 4 dietary treatments with 2 levels of probiotic supplementation (0 or 0.01%) and 2 levels of energy (2,700 or 2,800 kcal ME/kg) and nutrient density. Weekly feed intake, egg quality, and daily egg production were determined. Eighteen layers per treatment (2 layers/replication) were bled to determine serum cholesterol concentrations at wk 3 and 6. Excreta microbial shedding of Lactobacillus, Escherichia coli, and Salmonella and noxious gas emission were determined at the end of the experiment. Hens fed the high-energy and high-nutrient-density diets had less (P < 0.01) ADFI than those fed the low-energy and low-nutrient-density diets throughout the experimental period. During wk 4 to 6 and overall, hens fed the diets supplemented with the probiotic had greater (P < 0.01) egg production, egg weight, and eggshell thickness than hens fed the diets without the probiotic. Dietary supplementation of the probiotic increased (P = 0.01) excreta Lactobacillus counts and decreased (P = 0.02) Escherichia coli counts compared with hens fed the diets without the probiotic. The excreta ammonia emission was decreased (P = 0.02) in hens fed the probiotic diets compared with hens fed the diets without the probiotic. Serum total cholesterol concentration was decreased (P < 0.01) by feeding hens with the probiotic at wk 3 and 6. Layers fed the probiotic-incorporated diets had greater (P < 0.01) high-density lipoprotein (HDL) cholesterol and lower (P = 0.03) low-density lipoprotein (LDL) cholesterol concentrations than hens fed the nonsupplemented diets at wk 6. Interactive effects (P < 0.05) of energy and nutrient density and the probiotic on excreta Lactobacillus counts and serum HDL cholesterol concentration were observed at wk 6. In conclusion, dietary supplementation of 0.01% probiotic improved egg production and egg quality and decreased excreta ammonia emission. The use of a probiotic in the high-energy and high-nutrient-density diets may be more favorable than the low-energy and low-nutrient-density diets in laying hens.

Clonal outbreak of ST17 multidrug-resistant Enterococcus faecium harbouring an Inc18-like::Tn1546 plasmid in a haemo-oncology ward of a Spanish hospital.

OBJECTIVES: To report a clonal outbreak of ST17 vancomycin-resistant Enterococcus faecium (VREfm) carrying Tn1546 (vanA) in a haemo-oncology ward of a tertiary teaching hospital in the south of Spain (January-September 2009). PATIENTS AND METHODS: Twenty-two VREfm strains from 13 patients were characterized by PFGE, multiple-locus variable-number tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST). Genes encoding antibiotic resistance and putative virulence traits and the Tn1546 backbone were investigated by PCR. Plasmid characterization included determination of size (S1-PFGE) and replication modules (PCR, hybridization and sequencing). Patient clinical records were analysed retrospectively. RESULTS: A single ST17 E. faecium clone (MT-7 MLVA type) carrying esp and hyl plus a 30 kb Inc18-like::Tn1546 (IS1216) plasmid was identified. Ampicillin resistance was linked to PBP5 showing mutations at positions 24, 27, 34, 66, 68, 85, 100, 144, 172, 177, 204, 216, 324, 462, 466', 470, 485, 496, 499, 525, 546, 558, 582, 586, 629, 632, 642 and 667. Other resistance genes identified were erm(B), ant(6')-Ia and aph(3')-IIIa. Fluoroquinolone resistance was attributable to ParC (Arg-61 → Gly and Ser-80 → Arg) and GyrA (Ser-83 → Arg) mutations. CONCLUSIONS: A nosocomial outbreak caused by an ST17 (CC17) E. faecium clone harbouring Esp and Hyl and a 30 kb Inc18-like::Tn1546 plasmid among haemo-oncology patients is reported. The failure of early infection control practices indicates an undetected reservoir and the ability of this strain to persist over long periods. The potential spread of epidemic clones and broad host plasmids carrying vancomycin resistance in Spain is of concern since it might contribute towards a higher rate of VREfm infection.

FTIR-based polyphasic identification of lactic acid bacteria isolated from traditional Greek Graviera cheese.

This study used a combination of phenotypic, physical (Fourier Transformed Infra-Red [FTIR] spectroscopy) and molecular (RFLP and SSCP analysis of 16S rRNA genes) methods to identify the lactic acid bacteria (LAB) flora present in traditional Greek Graviera cheese after five weeks of ripening. A total of 300 isolates collected from high dilution plates of TSAYE (incubated at 30 °C), M-17 (22 °C) and M-17 (42 °C) agar media were clustered by FTIR and then representative strains of each cluster were cross-identified blindly by all methods. Based on their FTIR spectra, 282 isolates were LAB grouped in 28 clusters. The LAB species identified and their prevalence in the cheese samples were: Lactobacillus casei/paracasei (68.8%), Lactobacillus plantarum (19.5%), Streptococcus thermophilus (8.9%), Enterococcus faecium (2.1%), and Lactococcus lactis (0.7%). Also, Staphylococcus equorum (11 isolates), Corynebacterium sp. (5 isolates) and Brevibacterium sp. (1 isolate) were recovered from TSAYE. Comparative identification results showed that phenotypic and molecular methods were in mutual agreement as regards the LAB species identified. The present polyphasic identification approach based on rapid FTIR screening of 10-fold more isolates than a previous classical identification approach allowed or improved detection of few sub-dominant species; however the predominant LAB species in the cheese samples were the same with both approaches.

[Is the iron an essential nutrient for enterococci?]. / Zelazo niezbednym substratem pokarmowym enterokoków?

Enterococci were considered as not requiring iron. The aim of study was evaluation of relationship between enterococci and iron. This study examined these relationships in a 71 strains belonging to two species--Enterococcus faecalis and Enterococcus faecium, which are often isolated from human infections. The iron is an essential nutrient for enterococci. Demonstrated that iron--regardless of the concentration in the medium--is collected during growth. Iron deficiency in the nutrient medium resulted in changes in the kinetics of growth of enterococci. Inhibiting the growth of enterococci by iron chelators and lack of inhibition are further proof of this demand for iron bacteria. Enterococci have the ability to acquire this important element of its connections with natural and synthetic chetators with different strength of chemical bonding and structure. Bacteria of the genus Enterococcus have a natural resistance to many antimicrobial agents. In the hospital environment can easily acquire resistance genes to many other classes of antimicrobial compounds. For these reasons, treatment of enterococal infections poses more difficulties. Inhibition of iron uptake in enterococci can be helpful in reducing and combating enterococal infections.

Effect of Enterococcus faecium AL41 and Thymus vulgaris essential oil on small intestine integrity and antioxidative status of laying hens.

We investigated the effect of Enterococcus faecium on phagocytic activity, antioxidative status in vivo and the effect of E. faecium and 0.4% concentration of Thymus vulgaris essential oil (EO) on the duodenal tissue integrity in vitro in laying hens. The birds were fed the same standard diets and were divided into four groups. E. faecium was added to the drinking water for the second and fourth groups. EO was added to special chambers for measuring trans-epithelial electrical resistance (TEER) for the third and fourth groups only. TEER was lower in groups where EO was added, but in the group with E. faecium TEER was not changed significantly. Our results show that EO at 0.4% concentration may negatively affect intestine integrity, and the probiotic strain E. faecium AL41 is able to eliminate this effect and can strengthen non-specific immunity. To confirm our findings further histopathological investigations of intestinal tissue are needed.

Molecular epidemiology of vancomycin-resistant Enterococcus faecium strains isolated from haematological malignancy patients in a research hospital in Turkey.

Infections and outbreaks of vancomycin-resistant enterococci (VRE) still appear to be rare in Turkey. In the present study, VRE strains isolated during an outbreak in a haematology unit of a training and research hospital in Turkey were typed and their antimicrobial-resistance patterns were characterized by molecular methods. Twelve vancomycin-resistant Enterococcus faecium strains isolated from patients with haematological malignancies were investigated by PCR for the presence of genes encoding resistance to vancomycin, tetracycline, chloramphenicol, gentamicin and erythromycin. Their clonal relationship was evaluated by PFGE and multilocus sequence typing. All strains were resistant to vancomycin and erythromycin, and had the vanA and ermB genes, respectively. PFGE was used to determine the presence of two pulsotypes and determine their subtypes. Pulsotype A belonged to sequence type (ST) 17 and pulsotype B belonged to ST 78. All strains with the vanA gene were not the same clone, indicating multiple acquisitions of resistant isolates, even over such a short time period.