Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates

This study compared virulence and antibiotic resistance traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E, esp and acm were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.8% of E. faecalis and 70.4% of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linezolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.2% of E. faecalis and 70.3% of E. faecium) compared to water isolates (only 5.7% E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.     

Bacteriocin-producing strain of Enterococcus faecium EK 13 with probiotic character and its application in the digestive tract of rabbits

Enterococcus faecium EK 13 is a bacteriocin-enterocin A producing strain with probiotic properties. In this study its colonization, stability and effect on microflora in rabbits was studied as well as its influence on zootechnical parameters. Fifty rabbits of both sexes (HYPLUS, 30-day old; after weaning) were divided into control (CG) and experimental (EG) groups. They were fed a standard diet. Moreover, 25 rabbits in EG were fed daily (for 4 weeks) 15 g (separate doses ∼1.6 g) of lyophilized EK13 strain (rifampicin resistant variant — rif^R; 10⁹ cfu/g) dissolved in drinking water. After cessation of EK13 (rif^R) strain application, the rabbits in both groups were fed a standard diet for the next 2 weeks. Sampling was performed in double on day 0 (at the beginning of experiment), weekly during EK13 (rif^R) strain application as well as on week 1 and 2 after cessation of EK13 (rif^R) strain application. The counts of EK13 (rif^R) strain reached 7.1 ± 2.6 log₁₀ cfu/g after 4 weeks and even on week 2 after its cessation the counts 5.6 ± 2.3 log₁₀ cfu/g were determined. The total counts of enterococci in the rabbits were already increased in EG comparing with CG (p < 0.05); even 2 weeks after EK13 (rif^R) strain cessation, their counts in EG were 7.2 ± 2.6 log₁₀ cfu/g (p < 0.001). Enterococci in CG reached at the same time the value 3.7 ± 2.6 log₁₀ cfu/g. The counts of E. coli were significantly reduced in EG during 4 weeks (p < 0.05, p < 0.001). Even 2 weeks after EK13 (rif^R) strain cessation significant difference in E. coli counts between CG and EG was detected (p < 0.001). Enterobacteria in EG were significantly reduced (p < 0.001). Average daily gain in EG was 41.0 ± 3.83 in comparison to CG (40.6 ± 3.72); it means almost the same; although rabbits in EG showed higher feed intake per kg of gain than rabbits in CG. Preliminary results demonstrated that EK13 is a perspective probiotic candidate for rabbits. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Selenium enhances the antimutagenic activity of probiotic bacterium Enterococcus faecium M-74

The antibacterial activity of the probiotic bacterium Enterococcus faecium M-74 was assessed on De Man–Rogosa–Sharpe (MRS), Todd–Hewitt (T–H), M17 (M-17) and brain heart infusion (BHI) media with sodium selenite pentahydrate (+Se) and without sodium selenite pentahydrate (–Se) under aerobic or anaerobic conditions against nine bacterial pathogens. The highest antibacterial activity was found to be in the MRS medium under anaerobic conditions. There were no differences in the antibacterial activity between MRS(+Se) and MRS(–Se) media. The antimutagenic activity of MRS(+Se) and MRS(–Se) extracts after culture with E. faecium M-74 as well as of live and killed cells of E. faecium M-74 grown in the presence or absence of Se against the genotoxicity of ofloxacin (OFL) and acridine orange (AO) was determined in the Euglena gracilis assay. The MRS(+Se) extracts showed a significantly higher activity in reducing the genotoxicity of OFL and AO than MRS(–Se) extracts. The live cells of the probiotic strain M-74 exhibited higher antimutagenic activity than the killed bacterial cells, but differed depending on the mutagen used. However, the live bacterial cells grown in the presence of Se showed significantly higher antimutagenic activity. These results suggest a potential benefit for the future development of new Se-enriched probiotics exhibiting higher antimutagenic properties.     

Chemical structure of wall teichoic acid isolated from Enterococcus faecium strain U0317

Wall teichoic acid (WTA) was isolated from Enterococcus faecium strain U0317 and structurally characterized using ¹H, ¹³C, and ³¹P NMR spectroscopy, including two-dimensional COSY, TOCSY, ROESY, HMQC, and HMBC experiments. Further compositional determination was undertaken using classical chemical methods and HF treatment followed by GLC and GLC–MS analyses. The repeating unit of WTA consisted of two residues of 2-acetamido-2-deoxy-d-galactose, glycerol (Gro), and phosphate, and has the structure shown below:→6)-α-d-GalpNAc-(1→3)-β-d-GalpNAc-(1→2)-Gro-(3→P→     

利用胞内代谢物变化预测大肠埃希菌耐药性

目的利用胞内代谢物量的差异达到预测大肠埃希氏菌的药物敏感性结果。方法收集临床分离大肠埃希菌120株,在头孢他啶存在条件下培养4 h。收集菌体,测定细胞内化合物然后进行多变量分析。结果 120株大肠埃希菌的聚类分析结果很好地体现了药物敏感性特征,57株耐药菌中,有5株被误判,准确率为91%;47株中敏菌株中,有8株被误判,准确率为83%;16株敏感菌中有2株被误判;准确率为88%。结论该方法能够有效预测菌株的药敏结果,而且快速可靠。     

Virulence-related DNA sequences and adherence patterns in strains of enteropathogenic Escherichia coli

The presence of the genes for Escherichia coli adherence factor (EAF), attaching and effacing lesion (eae) and bundle-forming pili (bfp) in 72 strains identified as enteropathogenic E. coli (EPEC) by slide agglutination was evaluated using hybridization and PCR. The adherence property of these strains was assayed using 3h HeLa cells adherence assay. The results obtained indicated that virulence-associated genes were present in 65% of the strains but only ten (13.9%) isolates were positive for all the three markers (typical EPEC), 37 (51.4%) isolates carried either one or two of these determinants (atypical EPEC) and the remaining 25 (34.7%) were negative for all these genes. In vitro adherence assay showed that 44 (61.1%) strains adhered to HeLa cells with a defined pattern, 13 (18.1%) isolates adhered loosely with no definite pattern and the remaining 15 (20.8%) were non-adherent. Analysis of the results showed a statistically significant association between the presence of the virulence-related genes with adherence of the strains with a defined pattern (P相似文献   

Use of a 'Miniprep' for rapid extraction of plasmids from vancomycin- and gentamicin-resistant Enterococcus faecium

Conventional methods of plasmid extraction are largely unsuited to diagnostic laboratories. The 'Miniprep' is a rapid method that utilises a centrifugable column to separate plasmid DNA from chromosomal DNA. We have modified this technique to extract plasmid DNA from seven strains of vancomycin- and gentamicin-resistant Enterococcus faecium (VGREF): 1% mannitol was added to the growth medium and cell lysis was achieved by incubation in 10 mg of lysozyme/ml in 10 mM Tris, 1 mM EDTA and 25% sucrose at pH 8.0. RNase A was added to plasmid eluate rather than at the lytic step. In comparison to a standard phenol/chloroform method, Miniprep completely eliminated chromosomal interference in gel electrophoresis but otherwise produced identical plasmid profiles. Plasmids obtained from the VGREF ranged from 42 to 1.3 Md. Band densities on a single elution from the Miniprep varied from 8.3 to 106.3 relative units. Double elution increased band densities from the same preparation from 30.4 to 196 relative units; mean percentage increases per track between 7.0 and 34.6%. This method is suitable to achieve plasmid DNA extraction from VGREF within 1 h, making the process more suitable for diagnostic laboratories.     

Impact of Enterococcus faecium on specific activity of disaccharidases in small intestine of gnotobiotic pigs

The aim of the present work was to study the changes in the activity of disaccharidase enzymes (lactase. maltase, saccharase) in the small intestine of gnotobiotic pigs aged 0–35 days and inoculated with Enterococcus faecium. The continual decrease of lactase activity was observed from the 14^th day of age up to the end of the experiment. The most significant decrease of specific lactase activity in the duodenum (2.1 μmol/mg protein/hour) was noted from the 21^st to the 28^th day of age. On the other hand, the specific saccharase activity increased moderately during the post weaning period and maltase activity maintained a constant level. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

In vitro activity of three different antimicrobial agents against ESBL producing Escherichia coli and Klebsiella pneumoniae blood isolates

Extended spectrum beta-lactamases (ESBLs) usually associated with multiple drug resistance, including beta-lactam and non-beta-lactam antibiotics. This resistance can cause Limitation in the choice of drugs appropriate for using in clinical practice, especially in life-threatening infections. In this study we aimed to investigate in vitro activity of meropenem, ciprofloxacine and amikacin against ESBL-producing and non-producing blood isolates of Escherichia coli and Klebsiella pneumoniae strains. Fifty-eight E. coli (21 ESBL-producing, 37 non-ESBL producing) and 99 K. pneumoniae (54 ESBL-producing, 45 non-ESBL producing) strains were included in the study. The presence of ESBL was investigated by double disk synergy test and E-test methods. Antibiotic susceptibility test was done by microdilution method according to NCCLS guideline. In vitro susceptibilities of ESBL producing E. coli and K. pneumoniae strains were found as 100% for meropenem, 33.3% and 25.9% for ciprofloxacine, 94.5% and 83.3% for amikacin. It was observed that; meropenem was equally active agent in both ESBL-producing and non-producing strains, and its activity was not affected by ESBL production. Whereas amikacin activity was minimally affected and ciprofloxacine activity was markedly decreased by ESBL production. In conclusion, meropenem seems to be better choice of antibiotic should be used for ESBL positive life-threatening infections, because of remaining highest activity.     

Comparison of genotypic and phenotypic cluster analyses of virulence determinants and possible role of CRISPR elements towards their incidence in Enterococcus faecalis and Enterococcus faecium

Enterococcus faecalis and Enterococcus faecium are human commensals frequently found in fermented foods or used as probiotics, but also recognized as opportunistic pathogens. We investigated 62 Enterococcus strains isolated from clinical, food and environmental origins towards a rationale for safety evaluation of strains in food or probiotic applications. All isolates were characterised with respect to the presence of the virulence determinants fsrB, sprE, gelE, ace, efaAfs/fm, as, esp, cob and the cytolysin operon. In addition RAPD-PCR was used to obtain genomic fingerprints that were clustered and compared to phenotypic profiles generated by MALDI-TOF-MS. The gelatinase phenotype (GelE) and the haemolytic activity (β-haemolysis) were analysed. E. faecium strains contained esp and efaAfm only, and none of them contained any CRISPR elements. The amenability of E. faecalis strains to acquisition of virulence factors was investigated along the occurrence of CRISPR associated (cas) genes. While distribution of most virulence factors, and RAPD versus MALDI-TOF-MS typing patterns were unrelated, 2 out of 5 RAPD clusters almost exclusively contained clinical E. faecalis isolates, and an occurrence of CRISPR elements versus reduced number of virulence factors was observed. The presence of the cytolysin operon, cob and as encoding pheromone and aggregation substance, respectively, significantly corresponded to absence of cas. As their production promote genetic exchange, their absence limits further gene acquisition and distribution. Thus, absence of the cytolysin operon, cob and as in a cas positive environment suggests itself as promising candidate for E. faecalis evaluation towards their occurrence in food fermentation or use as probiotics.     

Characterization of Escherichia coli virulence genes,pathotypes and antibiotic resistance properties in diarrheic calves in Iran

Background

Calf diarrhea is a major economic concern in bovine industry all around the world. This study was carried out in order to investigate distribution of virulence genes, pathotypes, serogroups and antibiotic resistance properties of Escherichia coli isolated from diarrheic calves.

Results

Totally, 76.45% of 824 diarrheic fecal samples collected from Isfahan, Chaharmahal, Fars and Khuzestan provinces, Iran were positive for E. coli and all of them were also positive for cnf2, hlyA, cdtIII, f17c, lt, st, stx1, eae, ehly, stx2 and cnf1 virulence genes. Chaharmahal had the highest prevalence of STEC (84.61%), while Isfahan had the lowest (71.95%). E. coli serogroups had the highest frequency in 1–7 days old calves and winter season. Distribution of ETEC, EHEC, AEEC and NTEC pathotypes among E. coli isolates were 28.41%, 5.07%, 29.52% and 3.49%, respectively. Statistical analyses were significant for presence of bacteria between various seasons and ages. All isolates had the high resistance to penicillin (100%), streptomycin (98.25%) and tetracycline (98.09%) antibiotics. The most commonly detected resistance genes were aadA1, sul1, aac[3]-IV, CITM, and dfrA1. The most prevalent serogroup among STEC was O26.

Conclusions

Our findings should raise awareness about antibiotic resistance in diarrheic calves in Iran. Clinicians should exercise caution when prescribing antibiotics.     

Incidence of antibiotic resistance and virulence determinants among Enterococcus italicus isolates from dairy products

The main objective of this work was to investigate the biosafety of Enterococcus italicus, a recently described enterococcal species widely diffused in dairy products. For this purpose, the antibiotic susceptibility and the incidence of virulence factors among 30 E. italicus isolates originating mainly from different Italian cheeses were tested. Although not all 30 isolates showed unique genotypes, PCR fingerprinting evidenced a notable genotypic diversity among the E. italicus collection under study. All isolates were susceptible to vancomycin, gentamicin, erythromycin, ampicillin, chloramphenicol and bacitracin. Five isolates corresponding to three unique genotypes exhibited phenotypic resistance to tetracycline with MICs ranging from 64-256mug/ml. By PCR-based detection, the genetic basis of the Tet(R) phenotype in these strains was linked to the tet(S) gene whereas detection of tet(L) and tet(M) genes and the integrase element int of the Tn916/Tn1545 family of transposons were negative. Likewise, none of the strains appeared to contain any of the tested virulence genes gelE, asaI, cpd, agg, cylA, cylB, cylM, ace and hyl(Efm). The results of this study warrant further research into the environmental dissemination of Tet(R)E. italicus and into the potential transferability of its tet(S) genes.     

An evolutionary strategy for isobutanol production strain development in Escherichia coli

Higher alcohols such as isobutanol possess several physical characteristics that make them attractive as biofuels such as higher energy densities and infrastructure compatibility. Here we have developed a rapid evolutionary strategy for isolating strains of Escherichia coli that effectively produce isobutanol from glucose utilizing random mutagenesis and a growth selection scheme. By selecting for mutants with the ability to grow in the presence of the valine analog norvaline, we obtained E. coli NV3; a strain with improved 24-h isobutanol production (8.0 g/L) in comparison with a productivity of 5.3 g/L isobutanol obtained with the parental wild type strain. Genomic sequencing of NV3 identified the insertion of a stop codon in the C-terminus of the RNA polymerase σ^s-factor, RpoS. Upon repair of this inhibitory mutation (strain NV3r1), a final isobutanol titer of 21.2 g/L isobutanol was achieved in 99 h with a yield of 0.31 g isobutanol/g glucose or 76% of theoretical maximum. Furthermore, a mutation in ldhA, encoding d-lactate dehydrogenase, was identified in NV3; however, repair of LdhA in NV3r1 had no affect on LdhA activity detected from cell extracts or on isobutanol productivity. Further study of NV3r1 may identify novel genotypes that confer improved isobutanol production.     

Unique Colony Housing the Coexisting Escherichia coli and Dictyostelium discoideum

Two well-characterized and phylogenetically diverse species, Escherichiacoli and Dictyostelium discoideum, were used as the modelorganisms. When the two species were mixed and allowed to grow onminimal agar plates at 22 ^°C, instead of the predator Dictyostelium exterminating E.coli, the two species remarkablyachieved a state of stable coexistence in about two weeks. In addition, theemerged colonies housing the coexisting species have a mucoidal naturethat is distinctive from its origin. The simplicity of the system and the shorttime span for the two species to develop the coexistence state, that isproven stable and reproducible on laboratory conditions, hence, providesa new model system for the study of symbiosis, particularly with referenceto the initial stages.     

Comparison of the effects of Enterococcus faecium and aromatic oils from sage and oregano on growth performance and diarrhoeal diseases of weaned pigs

The influence of a probiotic additive and aromatic oils from medicinal and spicy plants on the basic production parameters and on the incidence of intestinal disorders of weaned pigs (weaning at the age of 30–32 days) was compared in two trials in a commercial pig fattening farm (Michalovce, Slovakia). In the first trial, an experimental (n _ex = 38) and a control group (n _co = 32) were used. A commercial probiotic additive containing Enterococcus faecium M-74 (6 × 10⁸ CFU/g) and IgY antibodies (920 mg/g dried activated egg matter) against pig Rotavirus and different strains of Escherichia coli (K88, K99, 987P, F18) were added to feed mixture of the experimental group from 30^th to 40^th day (12 × 10⁸ CFU/g) and subsequently from 40^th to 50^th day (6 × 10⁸ CFU/g) of age. The second trial was performed with three groups of pigs (n _ex1 = 28, n _ex2 = 24, n _co = 28). Aromatic oils isolated from sage (Salvia officinalis L., Labiateae) or oregano (Origanum vulgare L., Lamiaceae) were added to feed mixtures of the 1^st (90 mL/100 kg) and 2^nd experimental group (35 mL/100 kg), respectively, from 32^th to 53^th day of age. A statisticaly non-significant decrease of the incidence of diarrhoeal diseases was observed in the experimental groups of both trials as follows: in the 1^st trial by 43.41%, whereas in the 2^nd trial by 20.03% (1^st exp. group) and 6.72% (2^nd exp. group) in comparison with the control groups. The daily weight gains increased in the 1^st trial by 42.95 g/day, while in the 2^nd trial by 69.52 g/day (1^st exp. group) and 105.23 g/day (2^nd exp. group; p < 0.05) compared with controls. Presented at the Second Probiotic Conference, Košice, 15–19 September 2004, Slovakia.     

Drug resistance in Indian isolates of enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis>

Five different strains of enteropathogenic Escherichia coli (EPEC) and one non-pathogenic strain of E. coli were studied for the determination of resistance to various antibiotics by disc inhibition test. Our results demonstrated multiple drug resistance among the selected E. coli isolates with all of them including the non-pathogenic control strain showing high resistance towards ampicillin. Our results from conjugation test clearly showed the presence of most of the antibiotic markers on the transferred plasmids. Simultaneously, the plasmid profile of conjugants also indicated the presence of more than one plasmid along with the expected megaplasmid of ~90 Kb present in them. Thus our results amply demonstrate that these drug markers are associated with these mobile plasmids.     

Activities and regulation of the enzymes involved in the first and the third steps of the aspartate biosynthetic pathway in Enterococcus faecium

The enzymes aspartokinase and homoserine dehydrogenase catalyze the reaction at key branching points in the aspartate pathway of amino acid biosynthesis. Enterococcus faecium has been found to contain two distinct aspartokinases and a single homoserine dehydrogenase. Aspartokinase isozymes eluted on gel filtration chromatography at molecular weights greater than 250,000 and about 125,000. The molecular weight of homoserine dehydrogenase was determined to be 220,000. One aspartokinase isozyme was slightly inhibited by meso-diaminopimelic acid. Another aspartokinase was repressed and inhibited by lysine. Although the level of diaminopimelate-sensitive (DAP^s) enzyme was not much affected by growth conditions, the activity of lysine-sensitive (Lys^s) aspartokinase disappeared rapidly during the stationary phase and was depressed in rich media. The synthesis of homoserine dehydrogenase was controlled by threonine and methionine. Threonine also inhibited the specific activity of this enzyme. The regulatory properties of aspartokinase isozymes and homoserine dehydrogenase from E. faecium are discussed and compared with those from Bacillus subtilis.     

Escherichia coli strains with defined mutations which alter cellular permeability

Summary We describe the construction and analysis of an isogenic series ofEscherichia coli K12 strains that vary in their outer membrane permeability. They carry mutations that alter the amount and the type of porin present in the outer membrane. The permeability profiles of these strains suggest that both the amount and the type of porin present in the outer membrane affects permeability. Several of the strains carry mutations that extend the permeability of the outer membrane to include a variety of compounds that are normally excluded from entering the cell.