N-Terminal guanidine derivatives of teicoplanin antibiotics strongly active against glycopeptide resistant Enterococcus faecium.

Antibiotic resistance is one of the major challenges in healthcare of our time. To meet this challenge, we designed and prepared guanidine and lipophilic guanidine derivatives of the glycopeptide antibiotic teicoplanin to armed them with activity against the most threatening nosocomial bacteria, multiresistant enterococci. From teicoplanin and its pseudoaglycone, a series of N-terminal guanidine derivatives have been prepared with free and amide C-terminal parts. Six aliphatic and aromatic lipophilic carbodiimides were prepared and used for the synthesis of lipophilic guanidine teicoplanin conjugates. All new N-terminal guanidine antibiotics showed high activity against a standard panel of Gram-positive bacteria. Four selected derivatives displayed excellent antibacterial activity against a series of nosocomial VanA Enterococcus faecium strains.

Assessing the intestinal carriage rates of vancomycin-resistant enterococci (VRE) at a tertiary care hospital in Hungary.

Excessive use of antibiotics contributes to the selection of resistant bacteria and intestinal colonization with multiresistant pathogens poses a risk factor for subsequent infections. The present study assessed vancomycin-resistant enterococci (VRE) carriage rates in patients admitted to our tertiary care hospital. Stool samples sent for routine culturing were screened with vancomycin containing solid or broth enrichment media. VRE isolates were identified with matrix-assisted laser desorption/ionization-time of flight mass spectrometry and antibiotic susceptibilities were tested by E-test. Vancomycin resistance genes were detected by polymerase chain reaction. Medical records of carriers were examined for suspected risk factors for colonization. Altogether 3025 stool specimens were analyzed. Solid media identified a VRE carriage rate of 2.2% while broth enrichment detected 5.8%. Seventy percent of the isolates were Enterococcus faecium. VanB genotype was detected in 38.2%, VanA in 37.3%, VanC1 in 22.6%, and VanC2 in 1.9%. All VRE were sensitive to linezolid, daptomycin, and tigecycline. Collective risk factors for carriage were diabetes, normal flora absence, Clostridioides difficile positivity, longer hospital stay, and advanced age. 78.5% of the carriers received antibiotic therapy which was metronidazole in most cases (47.3%). We recommend regular screening of risk groups such as patients with diabetes, history of recent hospitalization, or former C. difficile infection as an imperative step for preventing VRE dissemination.

Investigation of silver nanoparticles on titanium surface created by ion implantation technology.

Objectives: Using dental Ti implants has become a well-accepted and used method for replacing missing dentition. It has become evident that in many cases peri-implant inflammation develops. The objective was to create and evaluate the antibacterial effect of silver nanoparticle (Ag-NP) coated Ti surfaces that can help to prevent such processes if applied on the surface of dental implants. Methods: Annealing I, Ag ion implantation by the beam of an Electron Cyclotron Resonance Ion Source (ECRIS), Ag Physical Vapor Deposition (PVD), Annealing II procedures were used, respectively, to create a safely anchored Ag-NP layer on 1x1 cm2 Grade 2 titanium samples. The antibacterial effect was evaluated by culturing Staphylococcus aureus (ATCC 29213) on the surfaces of the samples for 8 hours, and comparing the results to that of glass as control and of pure titanium samples. Alamar Blue assay was carried out to check cytotoxicity. Results: It was proved that silver nanoparticles were present on the treated surfaces. The average diameter of the particles was 58 nm, with a 25 nm deviation and Gaussian distribution, the the filling factor was 25%. Antibacterial evaluation revealed that the nanoparticle covered samples had an antibacterial effect of 64.6% that was statistically significant. Tests also proved that the nanoparticles are safely anchored to the titanium surface and are not cytotoxic. Conclusion: Creating a silver nanoparticle layer can be an option to add antibacterial features to the implant surface and to help in the prevention of peri-implant inflammatory processes. Recent studies demonstrated that silver nanoparticles can induce pathology in mammal cells, thus safe fixation of the particles is essential to prevent them from getting into the circulation.

Characterization of Clinical Vancomycin-Resistant Enterococcus faecium Isolated in Eastern Hungary.

OBJECTIVES: The aim of our study was to characterize and elicit the genetic relatedness of emerging vancomycin-resistant enterococci (VRE) isolated between 2012 and 2015 at a teaching hospital in Debrecen, Hungary. RESULTS: Altogether 43 nonduplicate vancomycin-resistant Enterococcus faecium (VREfm) clinical isolates were obtained. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used for species identification. Isolates showed 100% resistance to ampicillin and ciprofloxacin while 81.4% were resistant to gentamicin. PCR analysis revealed the presence of VanB in 40 and VanA in 3 isolates. Among ace, agg, and esp virulence genes only esp was found in seven cases. Modified microtiter-plate test showed 13 weak and 4 moderate biofilm producer isolates. Pulsed-field gel electrophoresis revealed nine pulsotypes. According to multilocus sequence typing all of the tested isolates belonged to clonal complex 17 (CC17). CONCLUSIONS: We report on the alarming emergence of multidrug-resistant VREfm belonging to CC17 at a tertiary hospital in Eastern Hungary. This is the first report of sequence types 412 and 364 from this region. Although outbreak did not occur the increasing prevalence of VREfm is of concern and dissemination must be prevented with proper infection control measures and regular VRE screening.

Dissemination of VanA-Type Enterococcus faecium Isolates in Hungary.

Although vanA carrying Enterococcus faecium human clinical isolates have been rarely found in Hungary before 2012, they have been detected in continuously increasing numbers since then. To identify factors associated with their dissemination, we investigated the clonal relatedness and plasmids of 30 vanA carrying E. faecium isolates originating from different Hungarian healthcare institutions from 2012 to 2014. Molecular typing of the isolates (n = 30) was performed with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, Tn1546 polymerase chain reaction mapping, plasmid restriction fragment length polymorphism analysis, and sequencing. A single Tn1546 variant was detected in all of the isolates. It harbored IS1251 in the vanS-vanH intergenic region, had an entire deletion of the transposase gene and a partial deletion of the resolvase gene, and was located on a pRUM-like plasmid. Based on PFGE, the isolates could be grouped into 13 pulsotypes. Representative strains of these pulsotypes belonged to ST17, ST18, ST80, ST117, and ST203, which are known to be part of the hospital-adapted clades. The increase in the number of vanA carrying E. faecium clinical isolates in Hungary could be explained by the dissemination of pRUM-like vancomycin resistance plasmids in hospital-adapted clonal lineages.

Foodborne Listeria monocytogenes: A Real Challenge in Quality Control.

Listeria monocytogenes is a foodborne pathogen, and the detection and differentiation of this bacterium from the nonpathogenic Listeria species are of great importance to the food industry. Differentiation of Listeria species is very difficult, even with the sophisticated MALDI-TOF MS technique because of the close genetic relationship of the species and the usual gene transfer. The present paper emphasizes the difficulties of the differentiation through the standardized detection and confirmation according to ISO 11290-1:1996 and basic available L. monocytogenes detection methods and tests (such as API Listeria test, MALDI-TOF MS analysis, and hly gene PCR). With the increase of reports on the pathogenesis of atypical Listeria strains in humans, the significance of species level determination has become questionable, especially in food quality control, and the detection of pathogenic characteristics seems to be more relevant.

Re-evaluation of in vitro activity of primycin against prevalent multiresistant bacteria.

With the increasing emergence of antibiotic resistances old antibiotics became a valuable source to find agents suitable to address this problem. More than 20 years after the last report, our purpose was to re-evaluate the in vitro antibacterial activity of the topical agent primycin against current important bacterial pathogens. Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of primycin were tested in comparison with agents widely applied topically, and with those of mupirocin and vancomycin, the topical and the non-topical gold-standard anti-MRSA agents. Primycin was ineffective (MIC>64 µg/ml) against all the Gram-negative isolates tested. On the other hand, all the tested Gram-positive isolates were susceptible with MIC90 values of 0.06 µg/ml for staphylococci and 0.5-1 µg/ml for enterococci, streptococci, and P. acnes isolates, including all the multiresistant strains. Against MRSA isolates primycin showed slightly higher activity than mupirocin, and inhibited the mupirocin-resistant strains also. MBC90 values ranged from 0.25 to 2 µg/ml for the investigated Gram-positive species. The bactericidal effect proved to be concentration-dependent in time-kill experiments. Spontaneous resistant mutants did not emerge in single-step mutation experiments and the resistance development was very slow by serial passaging. Passaged S. aureus strains showing increased primycin MIC values exhibited elevated vancomycin and daptomycin MIC values also. Though elucidation of the mechanisms behind warrants further investigations, these correlations can be related to development of vancomycin-intermediate phenotype. From the point of view of medical practice it is noteworthy that the increased primycin MIC values remained far below the concentration accessible by local application of the agent. These data make primycin a remarkable object of further investigations as well as a promising candidate for topical application against multiresistant Gram-positive pathogens.

Phagocytosis and intracellular killing of heterogeneous vancomycin-intermediate Staphylococcus aureus strains.

Risk factors for invasive infections by heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) may involve resistance to opsonophagocytosis and bacterial killing. hVISA strains typically have a thickened cell wall with altered peptidoglycan cross-linking. To determine whether hVISA may be endowed with an increased resistance to phagocytosis, this study assessed the characteristics of uptake and killing by granulocytes of three hVISA strains. All isolates were analysed by multilocus sequence typing and staphylococcal chromosome cassette mec typing. One of the strains belonged to the Hungarian meticillin-resistant S. aureus (MRSA) clone ST239-MRSA-III and the other two to the New York/Japan MRSA clone ST5-MRSA-II. In the presence of 10 % normal serum, the extent of phagocytosis and killing by blood granulocytes was equivalent for hVISA, MRSA and meticillin-sensitive S. aureus (MSSA) strains. Using granulocytes and serum from one patient who survived hVISA infection, the rate of phagocytosis and killing was also found to be comparable to that by control cells in the presence of 10 % serum. However, phagocytosis and killing of hVISA and MRSA (ATCC 25923) strains by normal granulocytes was markedly decreased in the presence of low concentrations (1 and 2.5 %) of serum from the patient who survived hVISA infection compared with that found with normal human serum. These data suggest that hVISA and MRSA isolates may be more resistant to opsonophagocytosis and bacterial killing than MSSA isolates, at least in some cases.

Prevalence of vanC vancomycin-resistant enterococci in the teaching hospitals of the University of Debrecen, Hungary.

Vancomycin-resistant enterococci (VRE) are common nosocomial pathogens; however, until now they have been rarely encountered in Hungary. In the present study, we investigated the prevalence of VRE in the teaching hospitals of the University of Debrecen. Of 7,271 Enterococcus-containing clinical samples collected between 2004 and 2009, we identified 16 VRE. Species-specific polymerase chain reaction was used to detect Enterococcus faecalis, Enterococcus faecium, Enterococcus casseliflavus, and Enterococcus gallinarum. Multiplex polymerase chain reaction was performed to identify the vancomycin resistance genes: vanA, vanB, vanC1/C2, vanD, vanE, and vanG. Restriction digestion with SalI and HindIII was introduced to differentiate the vanC1 and vanC2 genes from each other. Genetic relationships between the strains were investigated by pulsed-field gel electrophoresis. Overall, we identified the vanC1 resistance gene in 14 E. gallinarum and the vanC2 resistance gene in two E. casseliflavus strains. Except for two samples, the isolates had different pulsed-field gel electrophoresis types, suggesting sporadic emergence of the resistant bacteria. In addition, antibiotic resistance profile was determined by E-test. Three E. gallinarum strains proved to be resistant to gentamicin because of the presence of the aacA-aphD gene. Although the prevalence of VRE in Debrecen is rather low, the appearance of multiple resistances is of concern.

Comparison of the VITEK 2 system with the E-test for the determination of glycopeptide susceptibility of vanA and vanC positive enterococci.

The performance of the VITEK 2 System (bioMérieux) version 3.01 software was compared to that of the E-test (AB Biodisk, Solna, Sweden) for antibiotic susceptibility testing of 17 clinical isolates of vancomycin resistant enterococcus (VRE). Antibiotic Susceptibility Testing (AST) by VITEK 2 produced 10 minor (59%) errors, resulting in false phenotypes. Reporting of vancomycin resistance in enterococcal strains has enormous therapeutic and epidemiological consequences. Therefore, at laboratories using automated systems (e.g. VITEK 2) for routine microbiological susceptibility testing data must be confirmed by independent validated methods, e.g. E-test, or microdilution.