Prevalence of vancomycin-resistant enterococci in hospitalized patients and those living in the community in the Czech Republic

Between July 1, 2002 and December 31, 2003, rectal swabs from both hospitalized patients and community subjects in the Czech Republic were taken to ascertain the prevalence of vancomycin-resistant enterococci (VRE). The swabs were used for isolating and identifying enterococci and their susceptibility to antibiotics. Vancomycin resistance phenotypes were verified by PCR detection of vanA, vanB, vanC1 and vanC2 genes. A molecular biology analysis was performed in Enterococcus faecium VanA strains. During the observed period, 2691 rectal swabs from the hospitalized patients and 6529 rectal swabs from the subjects in community setting were examined. In total, 31 VRE of hospital origin and 13 community-population strains were isolated. The prevalence of VRE in the gastrointestinal tract was 1.9% in the hospitalized patients and 0.4% in the community subjects. The prevailing strains were Enterococcus faecium VanA (61.3%) in the VRE of hospital origin and Enterococcus gallinarum VanC (46.2%) in the community VRE. Mutual comparison between the hospital and community Enterococcus faecium VanA strains showed no similarity.     

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.     

The first molecular analysis of clinical isolates of VanA-type vancomycin-resistant Enterococcus faecium strains in Mainland China

AIMS: The aim of this study was to examine two VanA-type vancomycin-resistant Enterococcus faecium (VRE) strains that had been isolated from patients resident in mainland China. This is the first molecular analysis of clinical VRE strains being isolated in mainland China. METHODS AND RESULTS: Two VanA-type VRE isolates were isolated from in-patients at hospitals located in the Chinese cities Beijing and Dalian and were designated C264 and I125. The plasmids pC264V (40 kbp) and pI125V (370 kbp) that were isolated from C264 and I125, respectively, carried a Tn1546-like element encoding VanA resistance. The vancomycin-resistant plasmids pC264V and pI125V were transferred by filter mating at frequencies of 10(-7) and 10(-4) respectively. Sequence analysis of pC264V revealed that two IS1216V sequences and an IS1542 sequence were present within the Tn1546-like element. pI125V had two IS1216V insertions in the Tn1546-like element. CONCLUSIONS: The two VanA-type vancomycin-resistant E. faecium (VRE) strains C264 and I125 were isolated from in-patients in Chinese hospitals. The vancomycin-resistant conjugative plasmids pC264V and pI125V plasmids isolated from these strains carried the Tn1546-like element. The Tn1546-like element was found to contain the insertion sequences IS1216V and IS1542. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first molecular analysis of VanA-type VRE strains from patients resident in mainland China.     

VanA-Type Vancomycin-Resistant Enterococci in Equine and Swine Rectal Swabs and in Human Clinical Samples

Vancomycin-resistant enterococci (VRE) in healthy people and in food-producing animals seems to be quite common in Europe. The existence of this community reservoir of VRE has been associated with the massive use of avoparcin in animal husbandry. Eight years after the avoparcin ban in Europe, we investigated the incidence of VanA enterococci, their resistance patterns, and the mobility of their glycopeptide-resistance determinants in a sampling of animal rectal swabs and clinical specimens. A total of 259 enterococci isolated from equine, swine, and clinical samples were subcultured on KF-streptococcus agar (Difco Laboratories, Detroit, MI) supplemented with vancomycin and teicoplanin; 7 (6.7%), 10 (16%), and 8 (8.6%) respectively were found to be glycopeptides resistant (VanA phenotype). Slight differences in antimicrobial resistance patterns resulted among VRE recovered from the different sources. Polymerase chain reaction amplification demonstrated the presence of the vanA gene cluster and its extrachromosomal location in VRE plasmid DNA. VanA resistance was transferred in 7 out of 25 mating experiments, 4 with clinical, 2 with swine, and only 1 with equine donors. The conjugative plasmids of animal strains showed a high homology in the restriction profiles, unlike plasmids of clinical microrganisms. Our observations confirmed the possible horizontal transfer of VanA plasmids across different strains and, consequently, the diffusion of the vancomycin-resistance determinants.     

Transposase-dependent formation of circular IS256 derivatives in Staphylococcus epidermidis and Staphylococcus aureus

IS256 is a highly active insertion sequence (IS) element of multiresistant staphylococci and enterococci. Here we show that, in a Staphylococcus epidermidis clinical isolate, as well as in recombinant Staphylococcus aureus and Escherichia coli carrying a single IS256 insertion on a plasmid, IS256 excises as an extrachromosomal circular DNA molecule. First, circles were identified that contained a complete copy of IS256. In this case, the sequence connecting the left and right ends of IS256 was derived from flanking DNA sequences of the parental genetic locus. Second, circle junctions were detected in which one end of IS256 was truncated. Nucleotide sequencing of circle junctions revealed that (i) either end of IS256 can attack the opposite terminus and (ii) the circle junctions vary significantly in size. Upon deletion of the IS256 open reading frame at the 3' end and site-directed mutageneses of the putative DDE motif, circular IS256 molecules were no longer detectable, which implicates the IS256-encoded transposase protein with the circularization of the element.     

Characterization of glycopeptides, aminoglycosides and macrolide resistance among Enterococcus faecalis and Enterococcus faecium isolates from hospitals in Tehran

The prevalence of glycopeptides, aminoglycosides and erythromycin resistance among Enterococcus faecalis and Enterococcus faecium was investigated. The susceptibility of 326 enterococcal hospital isolates to amikacin, kanamycin, netilmicin and tobramycin were determined using disk diffusion method. The minimum inhibitory concentration (MIC) of vancomycin, teicoplanin, gentamicin, streptomycin, and erythromycin were determined by microbroth dilution method. The genes encoding aminoglycoside modifying enzymes described as AMEs genes, erythromycin-resistant methylase (erm) and vancomycin-resistant were targeted by multiplex-PCR reaction. High level resistance (HLR) to gentamicin and streptomycin among enterococci isolates were 52% and 72% respectively. The most prevalent of AMEs genes were aac (6')-Ie aph (2") (63%) followed by aph (3')-IIIa (37%). The erythromycin resistance was 45% and 41% of isolates were positive for ermB gene. The ermA gene was found in 5% of isolates whereas the ermC gene was not detected in any isolates. The prevalence of vancomycin resistant enterococci (VRE) was 12% consisting of E. faecalis (6%) and E. faecium (22%) and all of them were VanA Phenotype. The results demonstrated that AMEs, erm and van genes are common in enterococci isolated in Tehran. Furthermore our results show an increase in the rate of vancomycin resistance among enterococci isolates in Iran.     

Amino acid substitutions in the VanS sensor of the VanA-type vancomycin-resistant Enterococcus strains result in high-level vancomycin resistance and low-level teicoplanin resistance

The vancomycin-resistant enterococci GV1, GV2 and GV3, which were isolated from droppings from broiler farms in Japan have been characterized as VanA-type VRE, which express high-level vancomycin resistance (256 or 512 microg ml(-1), MIC) and low-level teicoplanin resistance (1 or 2 microg ml(-1), MIC). The vancomycin resistances were encoded on plasmids. The vancomycin resistance conjugative plasmid pMG2 was isolated from the GV2 strain. The VanA determinant of pMG2 showed the same genetic organization as that of the VanA genes encoded on the representative transposon Tn1546, which comprises vanRSHAXYZ. The nucleotide sequences of all the genes, except the gene related to the vanS gene on Tn1546, were completely identical to the genes encoded on Tn1546. Three amino acid substitutions in the N-terminal region of the deduced VanS were detected in the nucleotide sequence of vanS encoded on pMG2. There were also three amino acid substitutions in the vanS gene of the GV1 and GV3 strains in the same positions as in the vanS gene of pMG2. Vancomycin induced the increased teicoplanin resistance in these strains.     

Identification of an unusual VanA element in glycopeptide-resistant Enterococcus faecium in Brazil following international transfer of a bone marrow transplant patient

A vancomycin-resistant Enterococcus (VRE) was isolated from a blood culture of a patient in a Brazilian hospital who had a treatment history of a bone marrow transplant in the USA. The organism was identified as Enterococcus faecium, which exhibited an MIC (minimum inhibitory concentration) >or= 256 microg/mL for vancomycin. This was confirmed by E-test and the vanA gene was detected by PCR. Overlapping PCR revealed a left IR deletion and an additional 1.5 kb fragment between vanSH genes. DdeI digestion of vanRSHAX genes showed the determinant to be a T type variant, and the element was cloned and sequenced. These results revealed an IS1251 downstream of nucleotide 5820 of the VanA element. Insertions like this have not been reported previously in Brazil, but have been detected in the USA. The genotype and association with a patient previously treated in the USA suggest that this VRE was introduced from abroad, probably through inter-hospital strain spread.     

Nucleotide sequence analysis of IS256 from the Staphylococcus aureus gentamicin-tobramycin-kanamycin-resistance transposon Tn4001

Resistance to the aminoglycosides gentamicin, tobramycin and kanamycin (GmTmKmR) in Australian clinical strains of Staphylococcus aureus is commonly carried on the composite transposon Tn4001. The resistance gene aacA-aphD of Tn4001, which encodes a bifunctional AAC(6')-APH(2") modifying enzyme, is flanked by two 1324-bp inverted repeats, IS256L and IS256R, that are identical in sequence. Analysis of the IS256 sequence revealed structural features characteristic of IS elements including 26-bp imperfect terminal inverted repeats and a single open reading frame with coding capacity for a 45.6 kDa protein. The nucleotide sequence of IS256 described here, together with the sequence of the aacA-aphD gene reported previously [Rouch et al., J. Gen. Microbiol. 133 (1987) 3039-3052], completes the entire sequence of Tn4001, which totals 4566 bp.     

Glycopeptide-resistance transferability from vancomycin-resistant enterococci of human and animal source to Listeria spp

AIMS: The glycopeptide-resistance transferability from vancomycin-resistant enterococci (VRE) of clinical and animal origin to different species of Listeria was investigated. METHODS AND RESULTS: Of 36 matings, performed on membrane filter, the glycopeptide resistance was successfully transferred in six attempts, five with donors of animal origin and only one with donors from clinical source. The acquired glycopeptide resistance in Listeria transconjugants was confirmed by the presence of the conjugative plasmid band and by the amplification of the 732-bp fragment of vanA gene in transferred plasmids. CONCLUSIONS: Despite the lower number of bacteria used in this study, the source of enterococci influenced the outcome of mating. Moreover transferred VanA plasmid induced a different expression in Listeria transconjugants, suggesting that gene expression might be influenced by species affiliation of recipients. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data strengthen the opinion that enterococci are an important source of resistance genes for Listeria via the transfer of movable genetic elements. As these strains are commonly found in the same habitats, a horizontal spread of glycopeptide resistance in Listeria spp. could be possible.     

Evaluation of clonality in enterococci isolated in Brazil carrying Tn1546-like elements associated with vanA plasmids

Fifty-one vancomycin-resistant enterococci samples isolated from different geographic regions in Brazil were studied. All the isolates harboured the vanA gene as demonstrated by PCR analysis, and in a majority of strains the gene was associated with a transferable plasmid of 70 kb. A single variant of the prototype Tn1546 associated with common transferable vanA-containing plasmids has spread among the enterococcal strains circulating in Brazil. The VanA element integrity in these enterococci strains and the different pulsed-field gel electrophoresis patterns suggest horizontal transmission of the vancomycin resistance transposon in Brazilian strains.     

Detection and characterization of IS256, an insertion sequence in Staphylococcus aureus

Resistance to the aminoglycosides gentamicin (Gmr), tobramycin (Tmr) and kanamycin (Kmr) in strains of Staphylococcus aureus isolated in Australia is mediated by the transposon Tn4001. The 1.35 kb inverted repeat of this transposon exhibits many of the characteristics of an insertion sequence and has consequently been designated IS256. Tandem duplication of IS256 contiguous with Tn4001 results in an increase in the level of GmrTmrKmr, thereby implying that the element possesses strong promoter sequences. Both contiguous and independent insertions of IS256 into the staphylococcal chromosome have been observed, the latter suggesting that the element may play a role in molecular rearrangements of the genome.     

肠球菌万古霉素耐药基因簇遗传特性

万古霉素耐药肠球菌自20世纪80年代后期被发现以来,已逐渐发展成为重要的医院感染病原菌。此类耐药肠球菌携带的万古霉素耐药基因簇编码产物可催化合成与万古霉素、替考拉宁等糖肽类抗生素亲和力极低的细胞壁前体导致耐药。目前已在肠球菌中发现的万古霉素耐药基因簇根据基因序列及构成不同分为9个型别;依据它们编码的连接酶合成产物不同又可分为D-Ala:D-Lac连接酶基因簇(VanA、VanB、VanD及VanM型)和D-Ala:D-Ser连接酶基因簇(VanC、VanE、VanG、VanL和VanN型)。这些耐药基因簇介导的耐药水平及其传播模式各有特点。文章综述了肠球菌中万古霉素耐药基因簇的类型、基因构成及传播特性。     

Vancomycin-resistant enterococci in humans and imported chickens in Japan

The phenotypes and genotypes of 22 VanA-type vancomycin-resistant enterococci that had been isolated in Japan were examined. The VanA resistance determinant was plasmid mediated in each of the 22 strains. Of the 22 strains, 8 were isolated from different patients and 11 and 3 were obtained from different samples of chickens imported from Thailand and France, respectively. Three of the strains that were isolated from patients and the 11 strains isolated from the Thai chickens showed high-level vancomycin resistance (MICs, 512 to 1,024 micro g/ml) and low-level teicoplanin resistance (MICs, 0.5 to 4 micro g/ml). Each of these strains had three amino acid substitutions in the N-terminal region of the deduced VanS sequence. L50 was converted to V, E54 was converted to Q, and Q69 was converted to H compared to the vanS gene sequence of Tn1546.     

Vancomycin-Resistant Enterococci in a Chinese Hospital

In investigating the prevalence of vancomycin-resistant enterococci (VREs) in a hospital in China, 338 enterococci were detected by using multiplex polymerase chain reaction. Eleven VREs were found, including one VanA E. faecalis, one VanB E. faecium, three VanB E. faecalis, five VanC₁ E. gallinarum, and one VanC₂ E. flavescens. VITEK 2, microbroth dilution, and E-test were used to determine the susceptibilities of the VREs to certain antimicrobial agents; multiple-drug resistance of the 11 VREs was distinct. Compared with phenotypic methods, multiplex PCR rapidly detected 11 VREs and provided van genotype information. This is the first study that sequenced van genes of VRE isolates in China to date and found type VanB in China. Because of the relatively low isolating rate, early detection of VRE is vital.     

Identification and molecular characterization of Van A-type vancomycin-resistant Enterococcus faecalis in Northeast of Brazil

The isolation of vancomycin resistant enterococci (VRE) in Brazil has rapidly increased, following the world wide tendency. We report in the present study the first isolation of vancomycin resistant Enterococcus faecalis (VRE) in the Northeast of Brazil. The four VRE isolates were characterized for antimicrobial susceptibility, genotypic typing by macro restriction of chromosomal DNA followed by pulsed-field gel electrophoresis and for characterization of the Tn1546-like element and plasmid contents. The isolates showed resistance to multiple antibiotics and a single genotype profile, suggesting the dissemination of a single clone among the patients. Tn1546 associated to genetic elements as plasmids shows the importance of infection control measures to avoid the spreading of glycopeptide resistance by conjugative transfer of VanA elements.     

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.     

Determination and characterization of IS4Bsu1-insertion loci and identification of a new insertion sequence element of the IS256 family in a natto starter

The insertion sequence IS4Bsu1 frequently causes Bacillus subtilis starters for the production of Japanese fermented soybean pasts (natto) to lose the ability to produce poly-gamma-glutamate, the viscous material characteristic of natto. Bacillus subtilis NAFM5, a derivative of a natto starter, has six IS4Bsu1 copies on its chromosome. In this study, we determined all six insertion loci of the insertion sequence (IS). One was located in the coding region of yktD, a putative gene involved in polyketide synthesis. Four were located in non-coding regions between iolR and iolA, between tuaA and lytC, between rapI and orf1 (a potential gene of unknown function), and between ynaE and orf3 (a putative gene similar to thiF), and one resided in an intergenic region between divergent possible orf4 and orf5 genes of unknown function. Here we describe the structural features of these loci and discuss the effects of the IS4Bsu1 insertions on the functions of the target gene and the expression of the downstream genes. In addition, we found that strain NAFM5 and commercial natto starters possess eight to 10 loci of another IS of the IS256 family (designated IS256Bsu1) on their chromosomes. IS256Bus1 appeared active in transposition, potentially causing phenotypic alterations in natto starters like those induced by IS4Bsu1.     

Detection and distribution of insertion sequence 1 (IS1)-containing bacteria in the freshwater environment(1)

The distribution of insertion sequence 1 (IS1)-containing bacteria was investigated in Windermere (Cumbria, UK), a freshwater body impacted by treated sewage discharge and run-off from the surrounding catchment. Culturable IS1-containing bacteria were recovered from the water column at three depths in Windermere North Basin (WNB) and South Basin (WSB), and from sediment at both sites (at the sediment surface in WSB and to a depth of 12-13 cm in WNB). Polymerase chain reaction amplification of IS1 and the Escherichia coli/Shigella sp. specific gene uidA, from community DNA from shallow sediments, extended the detection limit beyond that of culture at both sites. This detection was extended further into deep sediment extracted from WNB as IS1 and uidA were detected in sub-samples to a depth of 4.7 and 2.3 m, respectively. Analysis of a representative subset of 90 IS1-carrying isolates recovered from water and sediment at both sites demonstrated 21 heterogeneous IS1 profiles with estimated copy numbers ranging from 1 to 16. Identification of the host bacteria showed that the element was confined mainly to Enterobacter spp. However, this study showed IS1 to be present in Citrobacter freundii for the first time. Plasmids were carried by 75.3% of enterobacterial isolates and four plasmids (2.6%) carried IS1. DNA sequence analysis of five IS1 clones demonstrated that IS1 isoforms from this study were similar (>89% nucleotide identity) to known IS1 isoforms. Two isoforms of IS1 from a single Enterobacter cloacae isolate differed by 6.7% at the nucleotide level suggesting that they had been acquired independently.