Tn1546-like Elements Research Articles

Mechanisms of acquisition of the vanA operon among vancomycin-resistant Staphylococcus aureus genomes: The tip of the iceberg?

BackgroundVancomycin is frequently used as a last line of defence against infections due to multidrug-resistant Staphylococcus aureus (S. aureus). A recent finding described the acquisition of vancomycin-resistant S. aureus strains by the integration of an enterococcal plasmid containing the vanA operon into the S. aureus chromosome via homologous recombination involving a specific integration site called locus L2. MethodsTo characterise all mechanisms of acquisition of vanA, this study analysed the 15 706 S. aureus genomes to look for vanA and described its genetic environment. ResultsA complete vanA operon was found in 25 S. aureus strains isolated from 12 patients, including nine co-isolated with vancomycin-resistant Enterococcus strains. VanA was found within transposon Tn1546-like elements on 17 plasmids and eight chromosomes. VanA might be acquired through conjugation of enterococcal and staphylococcal plasmids, transposition of Tn1546 carrying vanA and plasmid integration into the chromosome. Further, L2 was detected in 2087 genomes (13.3%) of S. aureus strains across different continents. Six potential chromosomal hotspots for integration of the entire vanA-containing enterococcal plasmid were identified by homologous recombination via L2. ConclusionsThese findings suggest that the recently described scenario in a New York patient could be reproduced anywhere. Surveillance of this possibility is mandatory, especially in patients with vancomycin-resistant Enterococcus infection or colonisation.

High Incidence of Multiple-Drug-Resistant Pheromone-Responsive Plasmids and Transmissions of VanA-Type Vancomycin-Resistant Enterococcus faecalis between Livestock and Humans in Taiwan.

A total of seventy VanA-type vancomycin-resistant enterococci (VRE) isolates obtained in Taiwan in the early 2000s were retrospectively characterized. Forty isolates were obtained from human patients and thirty from livestock. Of these VRE isolates, twenty-three (57.5%) of the human VRE and thirty (100%) of the livestock VRE were Enterococcus faecalis, and the remaining seventeen (42.5%) of the human VRE were E. faecium. Of the 53 E. faecalis isolates, twenty-two (96%) of the human VRE and thirty (100%) of the livestock VRE exhibited a high level of resistance to vancomycin and sensitivity to teicoplanin. They also had three amino acid substitutions in the N-terminal region of the deduced VanS sequence. The vancomycin resistance of all of the 22 human isolates, and 20 of the 30 livestock isolates, transferred to E. faecalis FA2-2 at a frequency of 10-5 to 10-3 per donor cell in broth. Each of the transconjugants responded to E. faecalis pheromone (i.e., E. faecalis FA2-2 culture filtrate), indicating that the conjugative plasmids were pheromone-responsive plasmids. Three of the conjugative plasmids originated from human isolates, and five plasmids from livestock isolates were corresponded and classified as type A plasmid. Two plasmids originated from human isolates and six plasmids from livestock isolates were corresponded and classified as type B plasmid. E. faecalis FA2-2 containing either the type A or type B plasmid responded to the synthetic pheromone cAD1. The type A and type B plasmids transferred between E. faecalis FA2-2 and JH2SS at a frequency of about 10-2 per donor cell and conferred vancomycin, bacitracin, and erythromycin resistances. The complete DNA sequence of the representative type A plasmid pTW9 (85,068 bp) showed that the plasmid carried a Tn1546-like element encoding vanA-type resistance, erythromycin resistance (ermB), and bacitracin resistance (bcrABDR). The plasmid contained the regulatory region found in the pheromone-responsive plasmid and encoded the genes traA, traD and iad1, which are the key negative regulatory elements, and traE1, a key positive regulator of plasmid pAD1, indicating that plasmid pTW9 was pAD1-type pheromone-responsive plasmid. PFGE analysis of SmaI-digested chromosomal DNAs showed that several E. faecalis strains harboring an identical type A pheromone-responsive plasmid were indistinguishable, and that these were identified both in human and livestock isolates, indicating the transmissions of the VRE strains between livestock and humans. These data showed that the multiple-drug-resistant pheromone-responsive conjugative plasmids have been widely spread in both human and livestock VRE, and there was high potential for transfers of VRE from food animals to humans in Taiwan in the early 2000s.

Vancomycin Resistance in Enterococcus faecium from the Dallas, Texas, Area Is Conferred Predominantly on pRUM-Like Plasmids.

Vancomycin-resistant E. faecium (VREfm) is a significant public health concern because of limited treatment options. Genomic surveillance can be used to monitor VREfm transmission and evolution. Genomic analysis of VREfm has not been reported for the Dallas/Fort Worth/Arlington, TX, area, which is currently the 4th largest metropolitan area in the United States. Our study aimed to address this gap in knowledge by analyzing the genomes of 46 VREfm strains and 1 vancomycin-sensitive comparator collected during routine fecal surveillance of high-risk patients upon admission to a Dallas, TX, hospital system (August to October 2015). Thirty-one complete and 16 draft genome sequences were generated. The closed VREfm genomes possessed up to 12 extrachromosomal elements each. Overall, 251 closed putative plasmid sequences assigned to previously described and newly defined rep family types were obtained. Phylogenetic analysis identified 10 different sequence types (STs) among the isolates, with the most prevalent being ST17 and ST18. Strikingly, all but three of the VREfm isolates encoded vanA-type vancomycin resistance within Tn1546-like elements on a pRUM-like (rep17) plasmid backbone. Relative to a previously reported typing scheme for the vanA-carrying Tn1546, new variants of the Tn1546 were identified that harbored a combination of 7 insertion sequences (IS), including 3 novel IS elements reported here (ISEfa16, ISEfa17, and ISEfa18). We conclude that pRUM-like plasmids are important vectors for vancomycin resistance in the Dallas, TX, area and should be a focus of plasmid surveillance efforts. IMPORTANCE Vancomycin is an antibiotic used to treat infections caused by multidrug-resistant Gram-positive bacteria. Vancomycin resistance is common in clinical isolates of the Gram-positive pathogen Enterococcus faecium. Among E. faecium strains, vancomycin resistance genes can be disseminated by plasmids with different host ranges and transfer efficiencies. Surveillance of resistance plasmids is critical to understanding antibiotic resistance transmission. This study analyzed the genome sequences of VREfm isolates collected from the Dallas, TX, area, with particular focus on the mobile elements associated with vancomycin resistance genes. We found that a single plasmid family, the pRUM-like family, was associated with vancomycin resistance in the majority of isolates sampled. Our work suggests that the pRUM-like plasmids should continue to be studied to understand their mechanisms of maintenance, transmission, and evolution in VREfm.

Novel linear plasmids carrying vanA cluster drives the spread of vancomycin resistance in Enterococcus faecium in India

The genetic basis for the spread of vancomycin resistance in Enterococcus faecium is largely unexplored in India. The present study aimed to investigate the plasmid diversity and variation of Tn1546 associated with vanA harbouring VREfm isolates. A total of 122 VREfm isolates collected from blood cultures were included in this study. MLST analysis was performed on all isolates, and they were also screened for the presence of vanA and vanB genes. Whole genome sequencing was performed for a subset of fifteen VREfm isolates belonging to ST1643. All of the 122 VREfm isolates carried the vanA gene. Twenty-four different sequence types were seen; of these, ST1643, ST80 and ST17 were predominant. Whole genome sequencing was performed on 15 VREfm isolates belonging to ST1643. For eight isolates the vanA gene was found on pRUM-like circular plasmids, and for the remaining seven isolates, the vanA gene was found on the linear plasmids. Novel Tn1546 variants carrying vanA were found on both circular and linear plasmids. Interestingly, co-presence of vanA and optrA were seen in the backbone of three linear plasmids. Multiple vanA-carrying plasmids and Tn1546-like elements were involved in the dissemination of vancomycin resistance in VREfm. The co-occurrence of Tn1546 carrying vanA and Tn554 family transposon carrying optrA on the backbone of plasmids is worrisome. The dissemination of such plasmids may pose treatment and infection control challenges.

Genetic Characteristics of Multiple Copies of Tn1546-Like Elements in ermB-Positive Methicillin-Resistant Staphylococcus aureus From Mainland China.

ObjectiveTo determine the genetic structure of ermB-positive Tn1546-like mobile elements in methicillin-resistant Staphylococcus aureus (MRSA) from mainland China.MethodsA total of 271 erythromycin-resistant MRSA isolates were isolated from Sir Run Run Shaw Hospital (SRRSH) from 2013 to 2015. Whole-genome sequencing was performed for the ermB-positive strains, and the genetic environment of the ermB genes was analyzed. Southern hybridization analysis and transformation tests were performed to confirm the location of the ermB gene.ResultsA total of 64 isolates (64/271, 23.6%) were ermB-positive strains, with 62 strains (62/64, 96.9%) belonging to the CC59 clone. The other two strains, SR130 and SR231, belonging to CC5-ST965, both harbored 14,567 bp ermB-positive Tn1546-like elements and displayed multidrug-resistant profiles. PFGE followed by Southern blot demonstrated that the ermB genes were located on the plasmids of both SR130 and SR231, while two copies of ermB were located on the chromosome of SR231. Further sequencing demonstrated that SR231 carried one Tn1546-ermB elements in the plasmid and two identical copies integrated on the chromosome, which had 99.99% identity to the element in the plasmid of SR130. The Tn1546-ermB elements were highly similar (100% coverage, >99.9% identity) to the element Tn6636 reported in a previous study from Taiwan. The plasmids (pSR130 and pSR231) harboring ermB-positive Tn1546-like elements were also identical to the mosaic plasmid pNTUH_5066148. However, conjugation of ermB-carrying plasmids of SR130 and SR231 were failed after triple repeats.ConclusionMultiple copies of ermB-positive Tn1546-like mobile elements were found in CC5-ST965 MRSA from mainland China, showing the wide dissemination of these Enterococcus faecium-originated ermB-positive Tn1546-like elements. Molecular epidemiological study of Tn1546-like elements is essential to avoid the spreading of resistant determinants.

Emergence of vanA-Type Vancomycin-Resistant Enterococcus faecium ST 78 Strain with a rep2-Type Plasmid Carrying a Tn1546-Like Element Isolated from a Urinary Tract Infection in China.

PurposeThe emergence of vancomycin-resistant enterococci (VRE) dramatically narrows therapeutic options. Although the prevalence of VRE in China has maintained a low level, VRE outbreaks have been reported in some tertiary hospitals in the developed areas of China. The clonal background of vanA-positive Enterococcus faecium strains has not been well characterized in China. Here, we report the whole-genome sequence of a vanA-type vancomycin-resistant E. faecium belonging to sequence type (ST) 78 isolated from a urinary tract infection in China.Patients and MethodsA vancomycin-resistant E. faecium was isolated from a 66-year-old male patient diagnosed with brainstem hemorrhage. Antibiotic susceptibility assays were performed according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). Complete genome sequencing was performed using both the HiSeqTM 4000 platform and the MinION platform. Plasmid, genomic and phylogenetic relationship analysis were further performed.ResultsE. faecium VRE1 was resistant to all antimicrobials tested except for tetracyclines and oxazolidinones. The whole genome of E. faecium VRE1 was composed of one chromosomal DNA and four plasmids. Two virulence genes and five antimicrobial resistance genes were identified. In silico multilocus sequence typing (MLST) showed that it belonged to ST78 (clonal complex CC17), a well-known epidemic clone that is widespread in Europe and the United States. Three antimicrobial resistance genes, including aminoglycoside resistance genes ant(6)-Ia and aph(3ʹ)-III; and glycopeptide resistance gene vanA, were located on a rep2-type plasmid carrying a Tn1546-like element that has not been reported. The most closely related strain harboring a similar plasmid backbone was recovered from fodder sample in China that differed by 178 cgMLST loci.ConclusionOur study characterizes the genomic feature of a vancomycin-resistant E. faecium ST78 strain harboring a vanA-carrying plasmid in China. The ST78 clonal group possessed the potential to emerge as a successful vanA-carrying epidemic lineage in China.

Phenotypic and Genotypic Traits of Vancomycin-Resistant Enterococci from Healthy Food-Producing Animals.

Food-producing animals may be a reservoir of vancomycin-resistant enterococci (VRE), potentially posing a threat to animal and public health. The aims of this study were to estimate the faecal carriage of VRE among healthy cattle (n = 362), pigs (n = 350), sheep (n = 218), and poultry (n = 102 flocks) in Switzerland, and to characterise phenotypic and genotypic traits of the isolates. VRE were isolated from caecum content of six bovine, and 12 porcine samples respectively, and from pooled faecal matter collected from 16 poultry flock samples. All isolates harboured vanA. Three different types of Tn1546-like elements carrying the vanA operon were identified. Conjugal transfer of vanA to human Enterococcus faecalis strain JH2-2 was observed for porcine isolates only. Resistance to tetracycline and erythromycin was frequent among the isolates. Our data show that VRE harbouring vanA are present in healthy food-producing animals. The vanA gene from porcine isolates was transferable to other enterococci and these isolates might play a role in the dissemination of VRE in the food production chain.

Partial Sequencing of IS1216V Transposase Gene of Staphylococcus Aureus Isolated from Food Samples

Background: Insertion sequence is a short DNA sequence encode for proteins implicated in the transposition activity. Transposase catalyzes the enzymatic reaction allowing the insertion sequence to +9*lo2 move. ;qqa;.
 Objective: To study the sequencing of transposase gene, tnp, IS1216V of S. aureus isolated from food and then compared with that documented in National Center for Biotechnology Information (NCBI).
 Methods: Food samples of animal and plant origin were collected, and screened for presence of S. aureus, IS1216V was identified in the Tn1546-like elements in the genomes of all Staphylococcus aureus isolates.
 Results: About 75% of total food samples were positive to S. aureus especially in the food of animal origin. tnp amplification showed that, 85% of isolates gave positive result. Sequencing of amplified part of IS1216V tnp of S. aureus isolates showed that, tnp gene had high identity (78-79%) with the reference strains of NCBI.
 Conclusion: High percentage of local food samples were contaminated with S. aureus especially of animal origin. Most of the S. aureus isolates showed the presence of transposase gene (tnp) of IS1216V. Sequencing showed some dissimilarity between the sequence of transposase gene (tnp) of IS1216V S. aureus isolated from local foods and strains recorded in database of NCBI.

VanA-type vancomycin-resistant Enterococcus faecium ST1336 isolated from mussels in an anthropogenically impacted ecosystem

We report the occurrence and genomic features of multidrug-resistant vancomycin-resistant Enterococcus faecium vanA belonging to a novel sequence type (designated ST1336), carrying a Tn1546-like element, in marine brown mussels (Perna perna) from anthropogenically affected coastal waters of the Atlantic coast of Brazil, highlighting a potential source of dissemination for related ecosystems, with additional consequences for seafood safety and quality.

Vancomycin-resistant Enterococcus faecium at a university hospital in Taiwan, 2002-2015: Fluctuation of genetic populations and emergence of a new structure type of the Tn1546-like element.

Vancomycin resistance increased significantly to 31.3% among Enterococcus faecium in 2006 and remained high thereafter at a university hospital in Taiwan. A longitudinal study was retrospectively conducted to characterize these vancomycin-resistant E. faecium (VRE-fm). A total of 378 non-repetitive VRE-fm blood isolates collected during 2002-2015 were studied. Multilocus sequence typing, pulsed-field gel electrophoresis, analysis of van genes and the Tn1546 structure, and conjugation experiments were performed. The majority (78.0%) of the isolates were associated with hospital-acquired infections. Molecular typing revealed nine major pulsotypes and five predominant sequence types (STs): ST17 (33.9%), ST78 (18.3%), ST414 (14.6%), ST18 (10.6%), and ST203 (7.4%). Fluctuation of these prevailing STs among the study years in association with some major pulsotypes was noted. All isolates carried vanA genes, except that in four isolates vanB genes were found. Among the vanA-carrying Tn1546-like elements, one predominant structure type (Type I, 55.9%) was noted throughout the study years. Since 2009, another predominant structure type (Type II, 40.1%) has emerged firstly in ST414 and gradually spread to other 11 STs in subsequent years. Isolates carrying these Type II Tn1546-like elements have become the most predominant population since 2014, majorly found in ST78 and ST17. Preliminary experiments indicated that plasmids carrying the Type II Tn1546-like elements demonstrated ten-fold higher efficiency than those carrying the Type I Tn1546-like elements. Dissemination of some major STs and horizontal transfer of plasmids carrying two major structure types of Tn1546-like elements may have together contributed to the increase of VRE-fm infection.

Local spread of Tn1546-like element among three species of vancomycin resistant enterococci in an intensive care unit.

In an intensive care unit (ICU) of the Emergency Center in the Clinical Center of Serbia, four species of vancomycin resistant enterococci (VRE) were isolated in a 17-month period mostly from blood cultures, including E. faecalis, E. faecium, E. raffinosus and E.gallinarum. The relationship between isolates from each species was investigated by PFGE, and PCR experiments for detection of pathogenicity factor genes and van genes to determine the nature of each clone. A PCR-based method, using 10 primer pairs (p1/2-p19/20), was used to investigate the presence of the Tn1546-like structure. PFGE indicated the presence of two different E. faecium clones, while the three other enterococcal species belonged to one clone each. Transposon typing revealed that isolates of E. raffinosus (4), E. gallinarum (4) and E. faecalis (3) yielded gene sequences identical to 10 primer pairs (p1/2-p19/20), suggesting the possibility of identical transposon-like structure in these species. The results of the study indicate probable horizontal spread of Tn1546-like structure in three species of VRE obtained from the same ICU.

Novel Structure of Enterococcus faecium-Originated ermB-Positive Tn1546-Like Element in Staphylococcus aureus.

We determined the resistance determinants in 274 erythromycin-resistant methicillin-susceptible Staphylococcus aureus (MSSA) isolates during a 13-year period, 2000 to 2012. The resistance phenotypes, inducible macrolide-lincosamide-streptogramin (iMLS), constitutive MLS (cMLS), and macrolide-streptogramin (MS) resistance phenotypes, were examined by a double-disk diffusion D test. The ermB gene was more frequent (35%; 97/274) than ermC (27%; 75/274) or ermA (21%; 58/274). All 97 ermB-positive isolates harbored Tn551 and IS1216V The majority (89/97) of ermB-positive isolates displayed the cMLS phenotype and carried mobile element structure (MES)-like structures, which has been previously reported in sequence type 59 (ST59) methicillin-resistant S. aureus (MRSA). The remaining 8 ermB-carrying isolates, belonging to ST7 (n = 4), ST5 (n = 3), and ST59 (n = 1), were sasK intact and did not carry MES-like structures. Unlike a MES-like structure that was located on the chromosome, the ermB elements on sasK-intact isolates were located on plasmids by S1 nuclease pulsed-field gel electrophoresis (PFGE) analysis and conjugation tests. Sequence data for the ermB-containing region (14,566 bp) from ST59 NTUH_3874 revealed that the best match was a Tn1546-like element in plasmid pMCCL2 DNA (GenBank accession number AP009486) of Macrococcus caseolyticus Tn1546 is recognized as an enterococcal transposon and was known from the vancomycin resistance gene cluster in vancomycin-resistant Enterococcus (VRE). So far, acquisitions of Tn1546 in S. aureus have occurred in clonal complex 5 (CC5) MRSA, but not in MSSA. This is the first report that MSSA harbors an Enterococcus faecium-originated ermB-positive Tn1546-like element located on a plasmid.

Streptococcus gallolyticus subsp. gallolyticus from human and animal origins: genetic diversity, antimicrobial susceptibility, and characterization of a vancomycin-resistant calf isolate carrying a vanA-Tn1546-like element.

The aim of this work was to characterize the antibiotic susceptibility and genetic diversity of 41 Streptococcus gallolyticus subsp. gallolyticus isolates: 18 isolates obtained from animals and 23 human clinical isolates. Antibiotic susceptibility was determined by the semiautomatic Wider system and genetic diversity by pulsed-field gel electrophoresis (PFGE) with SmaI. Animal isolates grouped separately in the PFGE analysis, but no statistical differences in antimicrobial resistance were found between the two groups. The LMG 17956 sequence type 28 (ST28) strain recovered from the feces of a calf exhibited high levels of resistance to vancomycin and teicoplanin (MIC, ≥256 mg/liter). Its glycopeptide resistance mechanism was characterized by Southern blot hybridization and a primer-walking strategy, and finally its genome, determined by whole-genome sequencing, was compared with four closely related S. gallolyticus subsp. gallolyticus genomes. Hybridization experiments demonstrated that a Tn1546-like element was integrated into the bacterial chromosome. In agreement with this finding, whole-genome sequencing confirmed a partial deletion of the vanY-vanZ region and partial duplication of the vanH gene. The comparative genomic analyses revealed that the LMG 17956 ST28 strain had acquired an unusually high number of transposable elements and had experienced extensive chromosomal rearrangements, as well as gene gain and loss events. In conclusion, S. gallolyticus subsp. gallolyticus isolates from animals seem to belong to lineages separate from those infecting humans. In addition, we report a glycopeptide-resistant isolate from a calf carrying a Tn1546-like element integrated into its chromosome.

National surveillance on vancomycin-resistant Enterococcus faecium in Taiwan: emergence and widespread of ST414 and a Tn1546-like element with simultaneous insertion of IS1251-like and IS1678.

Cases of bacteremia caused by vancomycin-resistant E. faecium (VRE-fm) increased significantly in Taiwan. The present multicenter surveillance study was performed to reveal the associated epidemiological characteristics. In 2012, 134 non-repetitive VRE-fm isolates were prospectively collected from 12 hospitals in Taiwan. Antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and analysis of van genes and Tn1546 structures were investigated. Two isolates carried vanB genes, while all the remaining isolates carried vanA genes. Three isolates demonstrated a specific vanA genotype - vanB phenotype. Nine (6.7%) isolates demonstrated tigecycline resistance, and all were susceptible to daptomycin and linezolid. Molecular typing revealed 58 pulsotypes and 13 sequence types (STs), all belonged to three major lineages 17, 18, and 78. The most frequent STs were ST17 (n = 48, 35.8%), ST414 (n = 22, 16.4%), and ST78 (n = 16, 11.9%). Among the vanA harboring isolates, eight structure types of the Tn1546-like element were demonstrated. Type I (a partial deletion in the orf1 and insertion of IS1251-like between the vanS - vanH genes) and Type II (Type I with an additional insertion of IS1678 between orf2 - vanS genes) were the most predominant, consisted of 60 (45.5%) and 62 (47.0%) isolates, respectively. The increase of VRE-fm bacteremia in Taiwan may be associated with the inter- and intra-hospital spread of some major STs and horizontal transfer of vanA genes mostly carried on two efficient Tn1546-like elements. The prevailing ST414 and widespread of the Type II Tn1546-like elements are an emerging problem that requires continuous monitoring.

Comparison of vanA gene mRNA levels between vancomycin-resistant Enterococci presenting the VanA or VanB phenotype with identical Tn1546-like elements.

During the routine screening of vancomycin-resistant Enterococci faecium (VREfm) we found that VanA phenotype-vanA genotype and VanB phenotype-vanA genotype isolates had an identical Tn1546-like element structure. This study aimed to evaluate the genetic background and vanA gene expression to identify the mechanisms of the development of the VanB phenotype-vanA genotype VREfm. Twelve VREfm isolates were collected from a 1500-bed tertiary-care teaching hospital in Beijing. Genetic variations of the Tn1546-like element were determined by an overlapping polymerase chain reaction assay and sequencing. The genetic background wasdetermined by pulsed-field gel electrophoresis and mutilocus sequence typing. vanA gene expression was evaluated using a TaqMan quantitative real-time polymerase chain reaction. For the 12 isolates, six isolates with the VanA phenotype-vanA genotype and six with the VanB phenotype-vanA genotype were identified. According to the structure analysis of the Tn1546-like elements, our isolates were divided into two types. In the four isolates of type A, IS1542 and IS1216V were inserted into the orf2-vanR and vanX-vanY regions, respectively. In the eight isolates of type D, a similar insertion as type A occurred, except for an ISEfa4 insertion into IS1542. A significant difference in vanA gene expression was observed between the VanA and VanB phenotype isolates in type A, but not in type D. Mutilocus sequence typing and pulsed-field gel electrophoresis analysis showed that these isolates have a different genetic background. Our results indicated that the occurrence of the VanB phenotype-vanA genotype might not completely depend on the structure of Tn1546-like elements and vanA gene expression.

Isolation of Tn1546-like elements in vancomycin-resistant Enterococcus faecium isolated from wood frogs: an emerging risk for zoonotic bacterial infections to humans

Isolation and characterization of vancomycin-resistant enterococci (VRE), mainly Enterococcus faecium, from the faecal pellet of wood frogs (Rana sylvatica). The frog VRE isolates were tested for their susceptibility to various antibiotics and were found resistant to ampicillin (Am), chloramphenicol (Cm), erythromycin (Em), gentamicin (Gm), tetracycline (Tc), teicoplanin (Tp) and vancomycin (Vn). The linkage of multiple antibiotic resistances to Em, Tc, Tp and Vn was observed in 84% of resistant Ent. faecium. Inducible antibiotic resistance (MIC ≥ 512 μg ml(-1) ) to Vn was also detected in these isolates. PCR analysis revealed the presence of vanA in all strains, and none of the strains were positive for vanB, indicating the existence of vanA phenotype. Furthermore, the PCR-RFLP analysis of the frog vanA amplicon with PstI, BamHI and SphI generated identical restriction patterns similar to Tn1546-like elements found in human VRE isolates. DNA homoduplex analysis also confirmed that vanA from the frog VRE has DNA sequence homology with the vanA of Tn1546-like elements of human and animal isolates. Blastx analysis of frog vanA sequence showed similarities with protein sequences generated from protein database of Vn-resistant Ent. faecium, Baccilus circulans, Paenibacillus apiarius and Oerskovia turbata isolates. Horizontal transfer of Vn resistance was not detected in frog isolates as revealed by filter mating conjugal experiment. In summary, our results demonstrated that wood frogs carry Vn-resistant bacteria, and resistance genes (vanA) are located on Tn1546-like elements. This study highlights a previously less recognized role of amphibians as sentinels for multidrug-resistant bacteria and alerts the public health workers for an emerging risk of zoonotic bacterial infections to humans.

Spread of vancomycin-resistant Enterococcus faecium in two haematological centres in Russia

This paper describes the clonal diversity of vancomycin-resistant Enterococcus faecium isolated from patients with haematological malignancies in Russia. Pulsed-field gel electrophoresis (PFGE) typing of 129 vanA-positive E. faecium strains revealed 23 independent restriction profiles with two predominant clonal types. Multilocus sequence typing (MLST) of 16 strains selected from two predominant PFGE types showed that they belong to the epidemic clonal complex (CC) 17. Tn 1546-like elements of isolates were compared with the prototype element from E. faecium BM4147 by polymerase chain reaction (PCR). Four different Tn 1546 types were distinguished according to structural alternations. Polymorphism in the orf1 and vanSH genes was detected. However, a significant prevalence of the prototype Tn 1546 was revealed. Tn 1546-like elements with the same structures were observed in strains of different PFGE types. The virulence genes esp, gelE and hyl were detected by PCR in 118 isolates (91%), 87 isolates (67%) and 35 isolates (27%), respectively. In contrast, agg and cylA genes were not found. The detection frequency of esp was higher in epidemic strains than in sporadic ones (100% vs. 56%; P < 0.05). This study describes a genetically variable population of vancomycin-resistant E. faecium in two Russian haematological centres. The spread of vancomycin resistance was mostly due to the distribution of the two subclones of E. faecium CC17, enriched with the virulence marker esp. At the same time, dissemination of an altered Tn 1546 also occurred.

Clonal spread of CC17 vancomycin-resistant Enterococcus faecium with multilocus sequence type 78 (ST78) and a novel ST444 in Taiwan

From May 2007 to January 2008, 30 isolates of vancomycin-resistant enterococci (VRE), including 29 Enterococcus faecium (96.7%) and 1 E. faecalis (3.3%) were obtained from various clinical specimens of 30 patients treated at a university hospital in Taiwan. Among these patients, 27 had VRE infections, including urinary tract infection (n = 16), bacteremia (n = 5), wound infection (n = 5), and central nervous system infection (n = 1). Three patients had VRE colonization. All of these isolates belonged to the vanA genotype with vancomycin minimum inhibitory concentrations of 64>or=128 microg/ml. The isolate of E. faecalis had VanB phenotype-vanA genotype. All these isolates were susceptible to linezolid and were inhibited by tigecycline at 0.25 microg/ml. Multilocus sequence typing (MLST) analysis of the E. faecium isolates showed that 82.8% were ST78, which belongs to lineage C1. Transposon typing classified the 30 isolates of VRE into three types and most of the Tn1546-like elements contained an IS1251-like insertion sequence. Mating experiments showed that the vanA gene clusters were transferable at a frequency of about 10(-6) to 10(-7). Our findings indicate that nosocomial spread of VRE resulted from dissemination of lineage C1 E. faecium clones, including a novel E. faecium MLST type (ST444), and the horizontal transfer of Tn1546 elements among enterococci.

Molecular characterisation of outbreak-related strains of vancomycin-resistant Enterococcus faecium from an intensive care unit in Beijing, China

We investigated an outbreak of vancomycin-resistant Enterococcus faecium affecting 14 patients in a 20-bed intensive care unit (ICU) between September 2006 and August 2007 (incidence: 3.56 cases per 1000 ICU patient days). Eighteen isolates of vanA type E. faecium were analysed by pulsed field gel electrophoresis, which showed 14 types overall. Multilocus sequence typing (MLST) identified eight different sequence types (STs) (ST78, ST117, ST203, ST316, ST362, ST363, ST364 and ST365), including four new types (ST362, ST363, ST364 and ST365) and 17 strains belonged to clonal complexes CC17. Sixteen of these carried the esp gene. Eighteen Tn1546-like elements encoding vanA-type VRE were classified into three types (types I to III) and all of them contained both IS1216V and IS1542 insertions. Vancomycin resistance of 14 vanA type E. faecium isolates was transferred at a frequency of 1.3 x 10(-6) to 6.4 x 10(-5) between E. faecium strains during filter mating. Our findings indicate that conjugative dissemination of Tn1546-like elements among CC17 E. faecium occurred during the outbreak in this ICU.

Clonal expansion within clonal complex 2 and spread of vancomycin-resistant plasmids among different genetic lineages of Enterococcus faecalis from Portugal

The aim of this study was to assess the diversity of Enterococcus faecalis populations recovered in different regions of Portugal during the last decade (1996-2007) and to analyse their genetic elements associated with vancomycin resistance. Forty E. faecalis isolates (22 vancomycin-susceptible and 18 vancomycin-resistant) representing disseminated and/or multiresistant strains from different sources (humans, animals and the environment) were characterized by PFGE and multilocus sequence typing. Genes encoding putative virulence markers and the backbone of Tn1546 were investigated by PCR. Plasmid analysis included determination of size, content (S1 hybridization) and comparison of restriction fragment length polymorphism patterns. The 40 E. faecalis isolates (22 PFGE types) mostly clustered within the worldwide-spread clonal complexes (CCs) CC2 (13 ST6 mostly corresponding to an epidemic strain, where ST stands for sequence type), CC21 (3 ST21, 1 ST22 and 1 ST224) and ST16 (n = 7), but also comprised ST159, ST35, ST19, ST26, ST30, ST41, ST55, ST59, ST117, ST160 and ST200. CC2 and CC21 were isolated from both hospital and community settings. Similar Tn1546-like elements encoding VanA were found on related plasmids within strains belonging to different clonal lineages and recovered in distinct hospitals over several years. The predominance of E. faecalis CC2 is mainly due to the dissemination of a particular clone persistently recovered for 11 years. The presence in the community of specific strains belonging to major clonal lineages highlights the role of community-associated hosts as possible reservoirs of putative human pathogenic enterococci. Both clonal expansion and dissemination of epidemic conjugative VanA plasmids seem to join forces in the establishment of pathogenic E. faecalis strains.