The homogeneity of vanB gene cluster among enterococcal isolates in Iran

Aims:  This study aimed to analyse the diversity of the vanB gene cluster in enterococcal species isolated from sewage treatment plants (STP) in Tehran, Iran.
Methods and Results:  The enterococcal isolates were collected from three sewage treatment plants in Tehran, Iran, during 2005. A total of 203 enterococcal isolates, collected over six rounds of sampling from three STPs were tested for the presence of vanB gene. Long-PCR showed that amongst the isolates, three Enterococcus faecium , one Enterococcus gallinarum and one Enterococcus casseliflavus harboured the complete vanB gene cluster.
Restriction fragment length polymorphism (RFLP) of the vanB1 gene cluster (5900 bp) from the isolates showed an identical pattern to a standard strain of Enterococcus faecalis (V583). None of the isolates were able to transfer the vanB gene in conjugation experiments. Different pulsed-field gel electrophoresis patterns were obtained for the three E. faecium isolates with vanB gene clusters .
Conclusions:  Our results indicated that the dissemination of vanB is not widespread in Tehran. Although only a few vanB positive isolates were detected, vanB was found in several enterococcal species.
Significance and Impact of the study:  In view of the lack of information on vanB resistance genes and their diversity in Iran, knowledge of the global dissemination of vanB genes in Enterococcus spp. is noteworthy.     

Characterization of the vanD glycopeptide resistance gene cluster from Enterococcus faecium BM4339.

VanD-type resistance to glycopeptides in Enterococcus faecium BM4339 is due to constitutive synthesis of D-alanyl-D-lactate-terminating peptidoglycan precursors (B. Périchon, P. Reynolds, and P. Courvalin, Antimicrob. Agents Chemother. 41:2016-2018, 1997). The sequence of a 5,780-bp fragment was determined and revealed six open reading frames. The 3' distal part encoded the VanHD dehydrogenase, the VanD ligase, and the VanXD DD-dipeptidase, which were highly similar to the corresponding proteins in VanA and VanB types of resistance. The deduced VanYD protein was homologous to penicillin-binding proteins that display DD-carboxypeptidase activity. The 5' end coded for the putative VanRD-VanSD two-component regulatory system. Due to a frameshift mutation in the chromosomal ddl gene, BM4339 produced an impaired D-alanine:D-alanine ligase. However, since expression of the resistance genes is constitutive, growth of E. faecium BM4339 was not dependent on the presence of glycopeptides in the culture medium.     

Identification and characterization of enterococci from bryndza cheese

AIMS: To identify enterococci isolated from sheep milk cheese--bryndza, and to compare differences in the composition of enterococcal microflora affected by the season, and to evaluate the potential presence of vancomycin resistance and virulence determinants. METHODS AND RESULTS: Bacterial strains were isolated during analysis of bryndza cheese and identified on the genus and species level by phenotypic methods and with commercial biochemical sets. The identification of the species, Enterococcus faecium, Ent. durans and Ent. faecalis, was confirmed by PCR using species-specific primers for ddl genes. PCR was also used for assessment of presence of vanA and vanB genes and virulence determinants gelE, agg and cytolysin genes namely: cylL(L), cylL(S), cylM, cylB and cylA. Among 308 Enterococcus sp. strains, 177 isolates were proved to be Ent. faecium, 59 to be Ent. durans and 41 to be Ent. faecalis. Vancomycin resistance genes vanA and vanB were not detected. Agar plate testing confirmed their absence. Gene gelE, however, was found in 20 Ent. faecalis isolates, but only 13 of them showed gelatinase-positive phenotype. Seven isolates had five cytolysin genes, but none of the isolates exhibited a positive haemolytic phenotype. Four isolates possessed the agg gene. The prevalence of Ent. faecium species was highest in samples from the winter season harvest. CONCLUSIONS: Ent. faecium is the dominant enterococcal species in bryndza cheese and the most prevalent in the winter season product. None of the Enterococcus sp. strains was proved to have vanA or vanB genes and the vancomycin resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of enterococcal microflora in bryndza cheese and its evaluation for the presence of vanA and vanB genes as well as virulence determinants.     

肠球菌的临床感染与耐药性分析

目的了解温州医学院附属第一医院临床分离主要肠球菌的分布及其对常用抗菌药物的耐药现状,以指导临床合理用药。方法对2008年至2011年临床分离的635株粪肠球菌和屎肠球菌的标本来源和药敏结果进行回顾性分析。结果各种临床标本中两种肠球菌的分布比例存在差异,总体以尿液标本所占比例最多,且屎肠球菌的总体分离率高于粪肠球菌。粪肠球菌对利奈唑胺、氨苄西林、万古霉素、呋喃妥因和替考拉宁的耐药率都在5．0％以下,对莫西沙星和青霉素G的耐药率也仅为7．0％和6．7％;屎肠球菌对莫西沙星、左旋氧氟沙星、环丙沙星、氨苄西林、青霉素G和红霉素的耐药率都在90．0％以上,对利奈唑胺、万古霉素、替考拉宁和奎奴敏感。粪肠球菌的多重耐药株占总数的26．4％,屎肠球菌的多重耐药株占总数的78．2％。结论粪肠球菌和屎肠球菌对15种抗菌药物的耐药情况不同,屎肠球菌具有更高的耐药率和更广的耐药谱。临床应根据药敏试验的结果合理选择抗菌药物,以防止耐药菌株的产生和播散。     

Identification of a novel insertion sequence in vanB2-containing Enterococcus faecium

AIMS: The characterization of a novel insertion sequence (IS) in vanB2-containing Enterococcus faecium was conducted. METHODS AND RESULTS: Direct PCR amplification of ORFC region of Tn5382 from DNA extracted from vanB2-containing E. faecium, and sequence analysis were performed. A novel IS was identified. It is 1418 bp in length and contains one putative open reading frame that is similar to transposase. There exists inverted terminal repeats of 12 bp, but direct repeats are not present. According to high similarity to putative transposases of IS3 members, such as, IS150, IS861, IS1077 and IS911, we designated it ISEnfa3. SIGNIFICANCE AND IMPACT OF STUDY: Since ISEnfa3 was detected in all vanB2-containing strains examined so far, it could be used as a tool for epidemiological study.     

Comparative analysis of the first complete Enterococcus faecium genome

Vancomycin-resistant enterococci (VRE) are one of the leading causes of nosocomial infections in health care facilities around the globe. In particular, infections caused by vancomycin-resistant Enterococcus faecium are becoming increasingly common. Comparative and functional genomic studies of E. faecium isolates have so far been limited owing to the lack of a fully assembled E. faecium genome sequence. Here we address this issue and report the complete 3.0-Mb genome sequence of the multilocus sequence type 17 vancomycin-resistant Enterococcus faecium strain Aus0004, isolated from the bloodstream of a patient in Melbourne, Australia, in 1998. The genome comprises a 2.9-Mb circular chromosome and three circular plasmids. The chromosome harbors putative E. faecium virulence factors such as enterococcal surface protein, hemolysin, and collagen-binding adhesin. Aus0004 has a very large accessory genome (38%) that includes three prophage and two genomic islands absent among 22 other E. faecium genomes. One of the prophage was present as inverted 50-kb repeats that appear to have facilitated a 683-kb chromosomal inversion across the replication terminus, resulting in a striking replichore imbalance. Other distinctive features include 76 insertion sequence elements and a single chromosomal copy of Tn1549 containing the vanB vancomycin resistance element. A complete E. faecium genome will be a useful resource to assist our understanding of this emerging nosocomial pathogen.     

Emergence and characterisation of vanB vancomycin-resistant Enterococcus faecalis in Rio de Janeiro, Brazil

Here we describe the detection and characterisation of three isolates of vancomycin-resistant VanB-type Enterococcus faecalis. Sequence analysis suggested that these isolates harboured the vanB1 gene. The isolates were susceptible to the majority of antimicrobial agents tested, with the exception of chloramphenicol, erythromycin and vancomycin, and showed distinct profiles of high-level resistance to aminoglycosides. Analysis of the clonal relatedness of the vanB E. faecalis isolates showed similar pulsed-field gel electrophoresis profiles. To our knowledge, this is the first report of the occurrence of enterococcal strains carrying vanB genes in Brazil.     

Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA

Vancomycin resistance in Enterococcus faecium BM4147 is mediated by vancomycin resistance proteins VanA and VanH. VanA is a D-alanine:D-alanine ligase of altered substrate specificity [Bugg, T. D. H., Dutka-Malen, S., Arthur, M., Courvalin, P., & Walsh, C. T. (1991) Biochemistry 30, 2017-2021], while the sequence of VanH is related to those of alpha-keto acid dehydrogenases [Arthur, M., Molinas, C., Dutka-Malen, S., & Courvalin, P. (1991) Gene (submitted)]. We report purification of VanH to homogeneity, characterization as a D-specific alpha-keto acid dehydrogenase, and comparison with D-lactate dehydrogenases from Leuconostoc mesenteroides and Lactobacillus leichmanii. VanA was found to catalyze ester bond formation between D-alanine and the D-hydroxy acid products of VanH, the best substrate being D-2-hydroxybutyrate (Km = 0.60 mM). The VanA product D-alanyl-D-2-hydroxybutyrate could then be incorporated into the UDPMurNAc-pentapeptide peptidoglycan precursor by D-Ala-D-Ala adding enzyme from Escherichia coli or by crude extract from E. faecium BM4147. The vancomycin binding constant of a synthetic modified peptidoglycan analogue N-acetyl-D-alanyl-D-2-hydroxybutyrate (Kd greater than 73 mM) was greater than 1000-fold higher than the binding constant for N-acetyl-D-alanyl-D-alanine (Kd = 54 microM), partly due to the disruption of a hydrogen bond in the vancomycin-target complex, thus providing a molecular rationale for high-level vancomycin resistance.     

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.     

Safety assessment of commercial Enterococcus probiotics in Korea

There have been concerns about possible pathogenicity and antimicrobial resistance in Enterococcus, which constitute more than 50% of probiotics in the worldwide market. In this study, Enterococcus in sixteen products manufactured by ten different companies was tested for the presence of six virulence genes and two vancomycin resistance genes. Results in this study showed the safety of Enterococcus on the Korean market and the importance of screening vanA, vanB, agg, cylA, esp, and gelE. Pulse-field gel electrophoresis showed that the sixteen isolates tested in this study are originated from three strains.     

Comparative study of vanA gene transfer from Enterococcus faecium to Enterococcus faecalis and to Enterococcus faecium in the intestine of mice

Vancomycin-resistant enterococci represent a large reservoir in animals because of the use of avoparcin as a growth promoter in Europe. These strains of animal origin enter the food chain and can either colonize the human gut or transfer their resistance genes to the human microbiota. In this study, we compared the transfer of vancomycin resistance from resistant animal Enterococcus faecium to sensitive human Enterococcus faecalis and E. faecium. We analysed these transfers in dibiotic mice and human faecal flora-associated mice. VanA transfer from animal E. faecium to human E. faecalis occurred in dibiotic mice. The transconjugants appeared rapidly and persisted at levels between 3.0 and 4.0 log10 colony-forming units g(-1) of faeces. In human faecal flora-associated mice, vanA gene transfer was not detected towards E. faecalis but was possible between E. faecium strains. Our experiments revealed the possibility of vanA transfer from animal E. faecium to human E. faecalis in vitro and in vivo in the intestine of dibiotic mice. However, intraspecies transfer of vanA gene seems more common than interspecies transfer among enterococci.     

Sources and pathways of spread of vancomycin-resistant enterococci in hemato-oncological patients

The presented study aims at analyzing an increasing prevalence of vancomycin-resistant enterococci (VRE) isolated from various kinds of clinical material obtained from patients in the Department of Hemato-oncology (DHO), University Hospital in Olomouc, Czech Republic. Between January 1 and March 31, 2005, enterococci were isolated by standard microbiological procedures using both clinical material obtained from hospitalized patients and samples from the department environment. Resistance to vancomycin and teicoplanin was determined by a standardized microdilution method. Phenotype determination of resistance to vancomycin was verified by PCR detection of vanA and vanB genes. In VanA Enterococcus faecium, macrorestriction analysis was performed by pulsed-field gel electrophoresis. During the monitored period, a total of 128 Enterococcus sp. strains were isolated, of which 38 (30 %) isolates from 22 different patients were determined as VRE. Dominating were Enterococcus faecium VanA (63 %) and Enterococcus casseliflavus VanC (16 %) strains. At the same time, one Enterococcus faecium VanA strain was acquired from a bed-side table used by a patient in whom a similar strain had been isolated repeatedly from various clinical materials including a rectal swab taken in 2004. Based on the macrorestriction analysis of genome DNA in 24 vancomycin-resistant Enterococcus faecium VanA strains isolated from the patients' clinical material, one strain from the bed-side table surface and one strain isolated from stools in 2004, 8 unique restriction profiles with similarity ranging from 90 % to 100 % were identified, which could be classified into 3 clonal types. Thus, we can assume not only the endogenous origin of the VRE in hemato-oncological patients and their potential selection caused by therapy with broad-spectrum antibiotics but also the ability of the strains to survive in a hospital setting and, subsequently, to be spread clonally by various vectors.     

Drug resistance of 100 clinical strains of Enterococcus spp

The aim of this study was to evaluate the drug susceptibility of 100 Enterococcus spp. strains isolated from patients hospitalized in State Clinical Hospital No 1 in Warsaw. All strains were identified (API 20 STREP) and their susceptibility to antibiotics was tested (ATB STREP) in automatic ATB system. Additionally, PYRase activity, beta-lactamase production (in nitrocefin test), MICs for vancomycin and teicoplanin (E test), HLAR--high level aminoglycoside resistance and susceptibility to vancomycin, teicoplanin, piperacillin and piperacillin/tazobactam (disc diffusion method) were determined. E. faecalis ATCC 29212 was used as the control strain. Fifty E. faecalis, 45 E. faecium, 2 E. casseliflavus, 2 E. durans and 1 E. avium strain were cultured. All strains were PYRase-positive and beta-lactamase-negative. Ten isolates demonstrated intermediate susceptibility to vancomycin (6--E. faecalis and 4--E. faecium). One E. faecalis strain was intermediately susceptible to both glycopeptides. One E. casseliflavus strain showed low-level resistance to vancomycin, but this strain was susceptible to teicoplanin--phenotype Van C. HLAR strains were found among 31 E. faecalis and 40 E. faecium strains. 48 E. faecalis strains were susceptible to piperacillin and 49 to piperacillin/tazobactam. Whereas, 41 E. faecium were resistant to both these drugs. Thirty six per cent of isolates were resistant to penicillin and ampicillin, 73% to erythromycin, 87% to tetracycline, 89% to lincomycin and 56% to nitrofurantoin. Some discrepancies were noticed between the results of different methods applied for susceptibility testing--ATB system, E test and disc diffusion. These discrepancies concerned HLAR detection and susceptibility to glycopeptides determination. The best methods were: disc-diffusion for HLAR detection and E test for determination of resistance to vancomycin and teicoplanin. Increasing resistance to antimicrobial agents is observed in clinical Enterococcus spp. isolates cultured in our laboratory, especially in E. faecium strains. It is necessary to control the dissemination of multiresistant Enterococcus spp. strains in hospital wards.     

Vancomycin-resistant Leuconostoc mesenteroides and Lactobacillus casei synthesize cytoplasmic peptidoglycan precursors that terminate in lactate.

The emergence of acquired high-level resistance among Enterococcus species has renewed interest in mechanisms of resistance to glycopeptide antibiotics in gram-positive bacteria. In Enterococcus faecalis and Enterococcus faecium, resistance is encoded by the van gene cluster and is due to the production of a peptidoglycan precursor terminating in D-alanyl-D-lactate, to which vancomycin does not bind. Most Leuconostoc and many Lactobacillus species are intrinsically resistant to high levels of glycopeptide antibiotics, but the mechanism of resistance has not been elucidated. To determine whether the mechanisms of resistance are similar in intrinsically resistant bacteria, cytoplasmic peptidoglycan precursors were isolated from Leuconostoc mesenteroides and Lactobacillus casei and analyzed by mass spectrometry, revealing structures consistent with UDP-N-acetylmuramyl-L-Ala-D-Glu-L-Lys-(L-Ala)-D-Ala-D-lactate and UDP-N-acetylmuramyl-L-Ala-D-Glu-L-Lys-D-Ala-D-lactate, respectively.     

Antimicrobial susceptibility of<Emphasis Type="Italic">Enterococcus</Emphasis> species isolated from slovak bryndza cheese

Three hundred and ten enterococcal isolates (178 Enterococcus faecium, 68 E. durans, 49 E. faecalis, 8 E. italicus, 3 E. gallinarum, 3 E. casseliflavus, and 1 E. hirae) from Slovak Bryndza cheese were evaluated for susceptibility to nine antimicrobial agents (vancomycin, teicoplanin, ampicillin, streptomycin, gentamicin, erythromycin, rifampicin, nitrofurantoin, and ciprofloxacin). All enterococcal isolates from Bryndza cheese were susceptible to ampicillin, streptomycin, gentamicin, vancomycin, and teicoplanin as determined by the disk diffusion method. Vancomycin resistance genes vanA and vanB were not detected. Resistance rates of enterococcal isolates to rifampicin, erythromycin, ciprofloxacin, and nitrofurantoin were 24, 26, 2, and 1 %, respectively. Thirty-six % of E. faecium isolates and 22 % of the E. faecalis isolates were resistant to erythromycin. Resistance to rifampicin was similar in E. faecium (31 %) and E. faecalis (29 %). Both E. faecium and E. faecalis strains showed the same resistance to ciprofloxacin (2 %). E. durans isolates showed low levels of resistance to rifampicin, erythromycin, ciprofloxacin, and nitrofurantoin (1-4 %). Forty-eight (30 %) of the E. faecium isolates, two (3 %) of the E. durans isolates, and six (12 %) of the E. faecalis isolates exhibited multidrug resistance. The highest frequency of resistant enterococci was observed in Bryndza produced in winter season.     

Influence of growth media on vancomycin resistance of Enterococcus isolates and correlation with resistance gene determinants

The effect of Mueller-Hinton (MH), MH+blood or brain heart infusion medium (agar or broth) on 13 Enterococcus isolates was determined, when testing their antibiotic susceptibility. Disk diffusion and Vitek methods were used to determine vancomycin resistance, while broth dilution and E-test methods were used to measure the minimum inhibitory concentration. The data were correlated with the presence of vancomycin resistance genes. A definite correlation pattern could not be established between the presence of van genes and vancomycin resistance in any plating medium, when tested by the disk diffusion assay. The broth dilution, irrespective of the plating medium, and Vitek methods were more reliable than the E-test method in testing isolates with vanA or vanB genes. However, for vanC2/C3 genotypes, the E-test method, irrespective of the plating medium, tested better than the broth dilution assay.