The MLST scheme currently used for Enterococcus faecium typing was designed in 2002 and is based on putative gene functions and Enterococcus faecalis gene sequences available at that time. As a result, the original MLST scheme does not correspond to the real genetic relatedness of E. faecium strains and often clusters genetically distant strains to the same sequence types (ST). Nevertheless, typing has a significant impact on the subsequent epidemiological conclusions and introduction of appropriate epidemiological measures, thus it is crucial to use a more accurate MLST scheme. Based on the genome analysis of 1,843 E. faecium isolates, a new scheme, consisting of 8 highly discriminative loci, was created in this study. These strains were divided into 421 STs using the new MLST scheme, as opposed to 223 STs assigned by the original MLST scheme. The proposed MLST has a discriminatory power of D = 0.983 (CI95% 0.981 to 0.984), compared to the original scheme's D = 0.919 (CI95% 0.911 to 0.927). Moreover, we identified new clonal complexes with our newly designed MLST scheme. The scheme proposed here is available within the PubMLST database. Although whole genome sequencing availability has increased rapidly, MLST remains an integral part of clinical epidemiology, mainly due to its high standardization and excellent robustness. In this study, we proposed and validated a new MLST scheme for E. faecium, which is based on genome-wide data and thus reflects the tested isolates' more accurate genetic similarity. IMPORTANCE Enterococcus faecium is one of the most important pathogens causing health care associated infections. One of the main reasons for its clinical importance is a rapidly spreading resistance to vancomycin and linezolid, which significantly complicates antibiotic treatment of infections caused by such resistant strains. Monitoring the spread and relationships between resistant strains causing severe conditions represents an important tool for implementing appropriate preventive measures. Therefore, there is an urgent need to establish a robust method enabling strain monitoring and comparison at the local, national, and global level. Unfortunately, the current, extensively used MLST scheme does not reflect the real genetic relatedness between individual strains and thus does not provide sufficient discriminatory power. This can lead directly to incorrect epidemiological measures due to insufficient accuracy and biased results.

• MeSH
• Anti-Bacterial Agents MeSH
• Enterococcus faecium * genetics   MeSH
• Gram-Positive Bacterial Infections * epidemiology   MeSH
• Humans MeSH
• Multilocus Sequence Typing methods   MeSH
• Whole Genome Sequencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The beneficial influence of bacteriocin-producing, probiotic, mostly non-autochthonous bacteria has already been reported in various animals. However, their use in horses provides limited information, and results with autochthonous bacteria have not been reported. Therefore, the main objective of this model study was to test the effect of autochthonous, bacteriocin-producing faecal strain Enterococcus faecium EF 412 application in horses. One gram of freeze-dried EF 412 strain (109 CFU/mL for 21 days) was applied to horses in a small feed ball. Clinically healthy horses (12), Slovak warm-blood breed of various ages (5-13 years), were involved in a 35-day-long experiment, also functioning as control for themselves. They were stabled in separate boxes (university property), fed twice a day (hay, whole oats or grazed) with water access ad libitum. Sampling was performed at the start of the experiment, i.e. at days 0/1, 21 (3 weeks of EF 412 application) and at day 35 (2 weeks of EF 412 cessation). EF 412 colonized GIT of horses was 3.54 ± 0.75 CFU/g (log 10) at day 21. The eggs of the nematode Strongylus spp. were not found in horses after EF 412 application, and Eimeria spp. oocysts were similarly not found. The other microbiota were not reduced as evaluated by the use of standard method. Using next-generation sequencing, at phylum level, phyla Bacteroidetes and Firmicutes dominated and at family level, they were Bacteroidales BS11 and S24-7 gut goups and Lentisphaerae. In horses, the increasing tendency in phagocytic activity was noted after EF 412 application. Biochemical parameters were in the physiological range. Total protein value was significantly decreased at day 21 compared with day 0/1 as well as with day 35 (P < 0.05). Cholesterol and triglycerides were influenced (decreased) at day 21 compared with day 0/1 and day 35. Neither nematode eggs Strongylus spp. nor Eimeria spp. oocysts were found in faeces after EF 412 application. Autochthonous, faecal strain E. faecium EF 412 showed promising application potential.

• MeSH
• Bacteriocins * metabolism   MeSH
• Enterococcus faecium * metabolism   MeSH
• Feces microbiology   MeSH
• Horses MeSH
• Communicable Disease Control MeSH
• Microbiota * MeSH
• Probiotics * metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

A small receptor molecule composed of a porphyrin core with tetrakis-ammonium glycine pickets (liptin 3e) appears to target anionic phosphatidylglycerol (PG) lipid head groups through multifunctional binding-pocket complementarity. Although a major component of bacterial cell membranes, PG is not widely found in animal cells, making PG potentially selective for bacterial targeting. Growth of microbial isolates was monitored in liquid cultures treated with liptin 3e by dilution plate counts and turbidity. Inhibition of growth by liptin 3e was observed for the ESKAPE human pathogens (Enterobacter aerogenes, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus faecium), Escherichia coli, Mycobacterium smegmatis, Streptococcus sobrinus, and methicillin-resistant S. aureus (MRSA), with certain species suppressed at <1 μg/mL (sub-μM) concentrations. Prolonged lag phases were observed, although cell viability was mainly unaffected, suggesting that liptin treatment caused bacteriostasis. Cultures treated with liptin 3e eventually recovered, resumed growth, and reached the same final densities as untreated cultures. Growth of the fungus Candida albicans was not appreciably inhibited by liptin 3e. If liptins exhibit bacteriostasis through broad extracellular binding to PG head groups, thereby disrupting cellular processes, liptins may be considered for development into preclinical drug candidates or be useful as a targeting system for molecular beacons or antibacterial drugs.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Enterococcus faecium * MeSH
• Escherichia coli MeSH
• Phosphatidylglycerols MeSH
• Humans MeSH
• Methicillin-Resistant Staphylococcus aureus * MeSH
• Microbial Sensitivity Tests MeSH
• Receptors, Artificial * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

The study aimed to identify colonized patients as a possible source of eventual VRE (vancomycin-resistant enterococci) infection from stool samples positive for glutamate dehydrogenase antigen, as well as for Clostridioides difficile toxins A and B. The study was carried out from 7/2020 to 9/2021. Stool samples were grown in a brain heart infusion medium with a gram-positive non-spore-forming bacteria supplement under aerobic conditions. The samples for VRE identification were grown on CHROMID® VRE agar, and the MICs for vancomycin and teicoplanin were also estimated. The presence of the vanA/vanB genes was tested using the PCR method. The total number of 113 stool samples positive for Clostridioides difficile toxins was analyzed. Of these samples, 44 isolates with VRE characters were identified. The most prevalent isolates in our set of isolates were Enterococcus faecium (27 isolates, 62%), Enterococcus faecalis (9 isolates, 21%), Enterococcus solitarius (4 isolates, 9%), Enterococcus durans (2 isolates, 4%), 1 isolate Enterococcus sulfurous (2%), and Enterococcus raffinosus (2%). In total, 26 isolates were detected in the study in the presence of vanA genes (24 isolates E. faecium, 2 isolates E. faecalis) and 18 isolates detected in the presence of vanB genes (7 isolates E. faecalis, 4 isolates E. solitarius, 3 isolates E. faecium, 2 isolates E. durans, 1 isolate E. sulfurous, and E. raffinosus). The results of this study showed the local dominance character of the vanA gene of hospital VRE isolates that were carriers of genes associated with high resistance to vancomycin, teicoplanin, and occasionally linezolid.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Bacterial Proteins genetics   MeSH
• Clostridioides difficile * genetics   MeSH
• Enterococcus faecium * genetics   MeSH
• Vancomycin-Resistant Enterococci * genetics   MeSH
• Gram-Positive Bacterial Infections * microbiology   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Teicoplanin pharmacology   MeSH
• Vancomycin pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Slovakia MeSH

In the microbiological diagnosis of bloodstream infections (BSI), blood culture (BC) is considered the gold standard test despite its limitations such as low sensitivity and slow turnaround time. A new FDA-cleared and CE-marked platform utilizing magnetic resonance to detect amplified DNA of the six most common and/or problematic BSI pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli; referred to as ESKAPEc) is available and may shorten the time to diagnosis and potentially improve antimicrobial utilization. Whole blood samples from hospitalized patients with clinical signs of sepsis were analyzed using the T2Bacteria Panel (T2Biosystems) and compared to simultaneously collected BC. Discrepant results were evaluated based on clinical infection criteria, combining supporting culture results and the opinion of treating physicians. A total of 55 samples from 53 patients were evaluated. The sensitivity and specificity of the T2Bacteria panel was 94% (16 out of 17 detections of T2Bacteria-targeted organisms) and 100%, respectively, with 36.4% (8 of 22) causes of BSI detected only by this method. The T2Bacteria Panel detected pathogens on average 55 hours faster than standard BC. In our study, 9 of 15 patients with positive T2Bacteria Panel results received early-targeted antibiotic therapy and/or modification of antimicrobial treatment based on T2Bacteria Panel findings. Given the high reliability, faster time to detection, and easy workflow, the technique qualifies as a point-of-care testing approach.

• MeSH
• Acinetobacter baumannii drug effects   genetics   isolation & purification   MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Antimicrobial Stewardship methods   MeSH
• Bacteremia blood   drug therapy   microbiology   MeSH
• Enterococcus faecium drug effects   genetics   isolation & purification   MeSH
• Escherichia coli drug effects   genetics   isolation & purification   MeSH
• Klebsiella pneumoniae drug effects   genetics   isolation & purification   MeSH
• Blood microbiology   MeSH
• Blood Culture MeSH
• Humans MeSH
• Prospective Studies MeSH
• Pseudomonas aeruginosa drug effects   genetics   isolation & purification   MeSH
• Staphylococcus aureus drug effects   genetics   isolation & purification   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In Slovakia, dairy products made from ewes' milk have a long tradition. These products include the lactic acid product called "žinčica" which is a by-product occurring during the preparation of ewes' lump cheese. There is no information in the literature regarding the special properties of the microbiota, especially lactic acid Firmicutes, which can survive in "žinčica." From the safety aspect, enterococci are a controversial group of bacteria, and those from "žinčica" have never been tested for their properties. The "žinčica" used in our study was supplied by several different agrofarms producing ewes' lump cheese in central Slovakia. The species Enterococcus faecium (strains EF30E1, EF32E1, EF34E1, EF34E5) and Enterococcus faecalis (strains EE30E4, EE35E1, E31E2, altogether 7) were detected in samples from "žinčica" identified using MALDI-TOF spectrometry with secure genus identification/probable species identification and then confirmed by means of PCR. Enterococci were hemolysis-negative and the genes of the typical enterococcal virulence factors were mostly absent; the gelE gene was found in two E. faecium strains (EF30E1 and EF32E1), the agg gene was detected in E. faecalis EE35E1, and the esp gene was found in two E. faecalis strains (EE30E4 and EE31E2). No strains harbored the cytolysin A gene. Biofilm formation was detected in four strains (EF30E1, EF32E1, EF34E1, and EF34E5), indicating highly positive and low-grade positive biofilm formation. Enterococci were mostly susceptible to antibiotics tested for their phenotype. This is the first study to analyze enterococci in "žinčica."

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Bacterial Proteins genetics   MeSH
• Food Safety * MeSH
• Biofilms growth & development   MeSH
• Enterococcus faecalis drug effects   genetics   MeSH
• Enterococcus faecium drug effects   genetics   MeSH
• Enterococcus classification   drug effects   pathogenicity   MeSH
• Virulence Factors genetics   MeSH
• Microbial Sensitivity Tests MeSH
• Microbiota MeSH
• Sheep MeSH
• Food Microbiology * MeSH
• Cheese microbiology   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Slovakia MeSH

Here, we describe a fluorescent assay developed to study competitive binding of the glycopeptide antibiotics to live bacteria cells. This assay demonstrated that the mechanism of action of the lipoglycopeptide antibiotics strongly depends on the hydrophobicity of the substitutes, with the best antibacterial activity of the glycopeptide antibiotics equally sharing properties of binding to D-Ala-D-Ala residues of the nascent peptidoglycan and to the membrane.

• MeSH
• Anti-Bacterial Agents metabolism   MeSH
• Staining and Labeling MeSH
• Cell Wall microbiology   MeSH
• Enterococcus faecium metabolism   MeSH
• Vancomycin-Resistant Enterococci metabolism   MeSH
• Fluorescence MeSH
• Glycopeptides metabolism   MeSH
• Lipoglycopeptides chemistry   metabolism   MeSH
• Microbial Sensitivity Tests MeSH
• Peptidoglycan metabolism   MeSH
• Rhodamines chemistry   MeSH
• Staphylococcus aureus metabolism   MeSH
• Teicoplanin analogs & derivatives   chemistry   metabolism   MeSH
• Vancomycin chemistry   metabolism   MeSH
• Protein Binding physiology   MeSH
• Publication type
• Journal Article MeSH

Vancomycin-resistant enterococci (VRE) are nosocomial pathogens of increasing medical importance. This study involved 121 VRE selectively obtained from a representative set of 1464 samples collected from various sources in the north-eastern part of the Czech Republic. In total, 119 VRE belonged to Enterococcus faecium and two to Enterococcus faecalis. All isolates of E. faecium were resistant to at least three antibiotic classes. The resistance genes vanA, erm(B), tet(M), tet(L), aac(3)-IIIa and aac(6')-aph(2'') were detected. We assigned the E. faecium to sequence types ST5, ST18, ST38, ST64, ST92, ST273, ST549 and ST640. In E. faecium isolates, we identified the presence of replicases rep20pLG1 , rep2pRE25 , rep17pRUM , rep21pVEF1/2 and rep14pRI1 , as well as relaxases relpEF1 , relpLG1 , relpCIZ2 , relpRE25 and relpRUM . The presence of the toxin-antitoxin system axe-txe was detected mainly among isolates of hospital origin. The A and D types of transposon Tn1546 were those occurring most frequently. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first extensive study of vancomycin-resistant enterococci of diverse origin in a single well-defined area of the Czech Republic. The isolates were investigated for their antibiotic resistance, epidemiological characteristics and plasmid characteristics. Based on the results obtained, we can make assumptions as to the ways that vancomycin resistance is disseminated throughout the environment including humans and animals.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Bacterial Proteins genetics   MeSH
• Enterococcus faecalis drug effects   genetics   isolation & purification   MeSH
• Enterococcus faecium drug effects   genetics   isolation & purification   MeSH
• Vancomycin-Resistant Enterococci classification   genetics   isolation & purification   MeSH
• Gram-Positive Bacterial Infections epidemiology   microbiology   MeSH
• Humans MeSH
• Plasmids genetics   MeSH
• Vancomycin Resistance genetics   MeSH
• Toxin-Antitoxin Systems genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic MeSH

As potential probiotic traits of human milk-isolated bacteria have increasingly been recognized, this study aimed to evaluate the probiotic properties of bacteriocin-producing Enterococcus faecium strains isolated from human milk and colostrum. Among 118 human milk- and colostrum-isolated lactic cocci, only 29 were identified as Enterococcus. Of these, only four Enterococcus faecium isolates exhibited bacteriocigenic activity against several pathogenic Gram-positive bacteria, including Listeria monocytogenes. These isolates exhibited high acid (up to pH 3.0) and bile tolerance (0.5% oxgall) in simulated gastrointestinal conditions, demonstrating their ability to survive through the upper gastrointestinal tract. All of the E. faecium strains were shown to be sensitive to most of the antibiotics including vancomycin, tetracycline, rifampicin, and erythromycin, while they were resistant to kanamycin and chloramphenicol. None of the strains showed any virulence (gelE, agg2, clyA, clyB, clyM) and antibiotic resistance genes (vanA, vanB, ermB, tetM, and aac(6')-le-aph(2″)-la). In addition, all the strains were able to assimilate cholesterol, ranging between 25.2-64.1% and they exhibited variable adherence (19-36%) to Caco-2 cells. Based on the overall results of this in vitro study, four of the E. faecium strains isolated from human milk and colostrum can be considered as promising probiotic candidates; however, further in vivo evaluations are required.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Antibiosis MeSH
• Bacterial Adhesion MeSH
• Drug Resistance, Bacterial MeSH
• Bacteriocins metabolism   MeSH
• Caco-2 Cells MeSH
• Cholesterol metabolism   MeSH
• Enterococcus faecium drug effects   genetics   isolation & purification   metabolism   MeSH
• Colostrum microbiology   MeSH
• Humans MeSH
• Listeria monocytogenes physiology   MeSH
• Milk, Human microbiology   MeSH
• Microbial Sensitivity Tests MeSH
• Probiotics * MeSH
• Gastric Juice MeSH
• Bile Acids and Salts pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

• MeSH
• Corynebacterium diphtheriae isolation & purification   MeSH
• Enterococcus faecium isolation & purification   MeSH
• Humans MeSH
• Microbiological Techniques * MeSH
• Staphylococcus aureus isolation & purification   MeSH
• Laboratory Proficiency Testing * MeSH
• Vibrio cholerae isolation & purification   MeSH
• Check Tag
• Humans MeSH