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.     

Characterization of the First VanB Vancomycin-Resistant <Emphasis Type="Italic">Enterococcus faecium</Emphasis> Isolated in a Spanish Hospital

We report the detection and characterization of the first vancomycin-resistant VanB-type Enterococcus faecium to be isolated in a Spanish hospital. Sequence analysis of the vanB gene showed that this isolate belonged to subtype vanB2. Moreover, PCR amplification analysis indicated that the vanB gene cluster was linked to a Tn5382-like transposon.     

A six amino acid deletion, partially overlapping the VanSB G2 ATP-binding motif, leads to constitutive glycopeptide resistance in VanB-type Enterococcus faecium

Enterococcus faecium clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal vanB cluster, including the vanSB/vanRB two-component system regulatory genes. Enterococcus faecium strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The ddl gene of BM4525 had a 2 bp insertion leading to an impaired d-alanine:d-alanine ligase. Sequencing of the vanB operon in BM4525 also revealed an 18 bp deletion in the vanSB gene designated vanSBDelta. The resulting six amino acid deletion partially overlapped the G2 ATP-binding domain of the VanSBDelta histidine kinase leading to constitutive expression of the resistance genes. Sequence analysis indicated that the deletion occurred between two tandemly arranged heptanucleotide direct repeats, separated by 11 base-pairs. The VanSB, VanSBDelta and VanRB proteins were overproduced in Escherichia coli and purified. In vitro autophosphorylation of the VanSB and VanSBDelta histidine kinases and phosphotransfer to the VanRB response regulator did not differ significantly. However, VanSBDelta was deficient in VanRB phosphatase activity leading to accumulation of phosphorylated VanRB. Increased glycopeptide resistance in E. faecium BM4525 was therefore a result of the lack of production of d-alanyl-d-alanine ending pentapeptide and to constitutive synthesis of d-alanyl-d-lactate terminating peptidoglycan precursors, following loss of d-alanine:d-alanine ligase and of VanSB phosphatase activity respectively. We suggest that the heptanucleotide direct repeat in vanSB may favour the appearance of high level constitutively expressed vancomycin resistance through a 'slippage' type of genetic rearrangement in VanB-type strains.     

A Multicentre Hospital Outbreak in Sweden Caused by Introduction of a vanB2 Transposon into a Stably Maintained pRUM-Plasmid in an Enterococcus faecium ST192 Clone

The clonal dissemination of VanB-type vancomycin-resistant Enterococcus faecium (VREfm) strains in three Swedish hospitals between 2007 and 2011 prompted further analysis to reveal the possible origin and molecular characteristics of the outbreak strain. A representative subset of VREfm isolates (n = 18) and vancomycin-susceptible E. faecium (VSEfm, n = 2) reflecting the spread in time and location was approached by an array of methods including: selective whole genome sequencing (WGS; n = 3), multi locus sequence typing (MLST), antimicrobial susceptibility testing, virulence gene profiling, identification of mobile genetic elements conferring glycopeptide resistance and their ability to support glycopeptide resistance transfer. In addition, a single VREfm strain with an unrelated PFGE pattern collected prior to the outbreak was examined by WGS. MLST revealed a predominance of ST192, belonging to a hospital adapted high-risk lineage harbouring several known virulence determinants (n≥10). The VREfm outbreak strain was resistant to ampicillin, gentamicin, ciprofloxacin and vancomycin, and susceptible to teicoplanin. Consistently, a vanB2-subtype as part of Tn1549/Tn5382 with a unique genetic signature was identified in the VREfm outbreak strains. Moreover, Southern blot hybridisation analyses of PFGE separated S1 nuclease-restricted total DNAs and filter mating experiments showed that vanB2-Tn1549/Tn5382 was located in a 70-kb sized rep _17/pRUM plasmid readily transferable between E. faecium. This plasmid contained an axe-txe toxin-antitoxin module associated with stable maintenance. The two clonally related VSEfm harboured a 40 kb rep _17/pRUM plasmid absent of the 30 kb vanB2-Tn1549/Tn5382 gene complex. Otherwise, these two isolates were similar to the VREfm outbreak strain in virulence- and resistance profile. In conclusion, our observations support that the origin of the multicentre outbreak was caused by an introduction of vanB2-Tn1549/Tn5382 into a rep _17/pRUM plasmid harboured in a pre-existing high-risk E. faecium ST192 clone. The subsequent dissemination of VREfm to other centres was primarily caused by clonal spread rather than plasmid transfer to pre-existing high-risk clones.     

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.     

Genetic Linkage and Cotransfer of a Novel, vanB-Containing Transposon (Tn5382) and a Low-Affinity Penicillin-Binding Protein 5 Gene in a Clinical Vancomycin-Resistant Enterococcus faecium Isolate

Mechanisms for the intercellular transfer of VanB-type vancomycin resistance determinants and for the almost universal association of these determinants with those for high-level ampicillin resistance remain poorly defined. We report the discovery of Tn5382, a ca. 27-kb putative transposon encoding VanB-type glycopeptide resistance in Enterococcus faecium. Open reading frames internal to the right end of Tn5382 and downstream of the vanX_B dipeptidase gene exhibit significant homology to genes encoding the excisase and integrase of conjugative transposon Tn916. The ends of Tn5382 are also homologous to the ends of Tn916, especially in regions bound by the integrase enzyme. PCR amplification experiments indicate that Tn5382 excises to form a circular intermediate in E. faecium. Integration of Tn5382 in the chromosome of E. faecium C68 has occurred 113 bp downstream of the stop codon for the pbp5 gene, which encodes high-level ampicillin resistance in this clinical isolate. Transfer of vancomycin, ampicillin, and tetracycline resistance from C68 to an E. faecium recipient strain occurs at low frequency in vitro and is associated with acquisition of a 130- to 160-kb segment of DNA that contains Tn5382, the pbp5 gene, and its putative repressor gene, psr. The interenterococcal transfer of this large chromosomal element appears to be the primary mechanism for vanB operon spread in northeast Ohio. These results expand the known family of Tn916-related transposons, suggest a mechanism for vanB operon entry into and dissemination among enterococci, and provide an explanation for the nearly universal association of vancomycin and high-level ampicillin resistance in clinical E. faecium strains.     

Cloning of resistance gene analogs located on the alien chromosome in an addition line of wheat-Thinopyrum intermedium

Homology-based gene/gene-analog cloning method has been extensively applied in isolation of RGAs (resistance gene analogs) in various plant species. However, serious interference of sequences on homoeologous chromosomes in polyploidy species usually occurred when cloning RGAs in a specific chromosome. In this research, the techniques of chromosome microdissection combined with homology-based cloning were used to clone RGAs from a specific chromosome of Wheat-Thinopyrum alien addition line TAi-27, which was derived from common wheat and Thinopyrum intermedium with a pair of chromosomes from Th. intermedium. The alien chromosomes carry genes for resistance to BYDV. The alien chromosome in TAi-27 was isolated by a glass needle and digested with proteinase K. The DNA of the alien chromosome was amplified by two rounds of Sau3A linker adaptor-mediated PCR. RGAs were amplified by PCR with the degenerated primers designed based on conserved domains of published resistance genes (R genes) by using the alien chromosome DNA, genomic DNA and cDNA of Th. intermedium, TAi-27 and 3B-2 (a parent of TAi-27) as templates. A total of seven RGAs were obtained and sequenced. Of which, a constitutively expressed single-copy NBS-LRR type RGA ACR3 was amplified from the dissected alien chromosome of TAi-27, TcDR2 and TcDR3 were from cDNA of Th. intermedium, AcDR3 was from cDNA of TAi-27, FcDR2 was from cDNA of 3B-2, AR2 was from genomic DNA of TAi-27 and TR2 was from genomic DNA of Th. intermedium. Sequence homology analyses showed that the above RGAs were highly homologous with known resistance genes or resistance gene analogs and belonged to NBS-LRR type of R genes. ACR3 was recovered by PCR from genomic DNA and cDNA of Th. intermedium and TAi-27, but not from 3B-2. Southern hybridization using the digested genomic DNA of Th. intermedium, TAi-27 and 3B-2 as the template and ACR3 as the probe showed that there is only one copy of ACR3 in the genome of Th. intermedium and TAi-27, but it is absent in 3B-2. The ACR3 could be used as a specific probe of the R gene on the alien chromosome of TAi-27. Results of Northern hybridization suggested that ACR3 was constitutively expressed in Th. intermedium and TAi-27, but not 3B-2, and expressed higher in leaves than in roots. This research demonstrated a new way to clone RGAs located on a specific chromosome. The information reported here should be useful to understand the resistance mechanism of, and to clone resistant genes from, the alien chromosome in TAi-27.     

The 3Ns chromosome of Psathyrostachys huashanica carries the gene(s) underlying wheat stripe rust resistance

Stripe rust (Puccinia striiformis tritici (Pst)) is one of the most destructive diseases of wheat in the world. Exploiting and utilizing stripe rust resistance genes of wild species has become an essential strategy for resistance breeding. Psathyrostachyshuashanica Keng ex Kuo is a wild species in Triticeae that has been used for wheat improvement because of its high resistance or immunity to stripe rust. In this study, 9 wheat-P. huashanica addition lines were characterized by Giemsa C-banding, genomic in situ hybridization (GISH), and disease resistance evaluation. Giemsa C-banding and GISH demonstrated that lines 163-5, 165-1, 183-5, 240-3, and 240-4 are P. huashanica 3Ns chromosome monosomic addition lines; lines 183-1 and 183-20 are P. huashanica 3Ns chromosome disomic addition lines; line 165-20 is a P. huashanica 3Ns and 4Ns chromosomes double disomic addition line, and line 219-1 is a P. huashanica 1Ns and 3Ns/5A chromosomes double disomic addition-substitution line. All these addition lines with P. huashanica 3Ns chromosome(s) expressed high resistance or immunity to stripe rust. By comparing the series of wheat-P. huashanica chromosome addition lines, we concluded that the P. huashanica 3Ns chromosome carries the gene(s) for resistance or immunity to stripe rust. These addition lines can be used as a donor source of novel stripe rust resistance to wheat breeding programs.     

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.     

Mapping the Flv locus controlling resistance to flaviviruses on mouse chromosome 5.

Genetically determined resistance to flaviviruses in mice is a dominant trait conferred by alleles at a single autosomal locus designated Flv, but no gene products have been associated with this locus and the mechanism of resistance is not well understood. To further characterize this model of genetic resistance, we conducted mapping studies to determine the chromosomal location of Flv. Because of evidence suggesting that the Flv locus is on chromosome 5, three-point backcross linkage analyses were used to define the location of Flv relative to previously assigned chromosome 5 markers. The results confirm the chromosome 5 location of Flv and indicate a map position between the anchor loci rd and Gus-s. The chromosomal localization of Flv is the first step in the production of a detailed linkage map of the Flv region, which may open approaches to positional cloning of the resistance gene.     

Identification of an alien chromosome in the common wheat line multi 6R

The chromosome of Agropyron intermedium (Host) Beauv. substituting chromosome 6D has been identified in the karyotype of the Triticum aestivum L. line Multi 6R with the use of C-banding. The alien chromosome, temporarily designated 6Ag1, contains gene(s) of the resistance to the Saratov population of Puccinia recondita Rob. ex. Desm. f. tritici. It has been demonstrated that the resistance gene(s) is(are) highly efficient and that chromosome 6Ag1 is preferably transferred through gametes.     

Tn903 induces inverted duplications in the chromosome of bacteriophage lambda

Specialized transducing strains of bacteriophage lambda have been isolated that carry the transposable kanamycin resistance element, Tn903. Tn903 carries an inverted duplication of 1130 base-pairs flanking the kanamycin resistance gene. Often, when λ::Tn903 particles are infected into bacterial cells, the lambda chromosome is rearranged into a defective lambda plasmid which replicates with the bacterial cell. The formation of the defective plasmids (called Tn903λdv) is most likely induced by the Tn903 insertion itself. This follows from the fact that the novel DNA sequence found in these plasmids, with respect to the ancestral λTn903 chromosome, is always adjacent to the Tn903 element. Physical chromosomal mapping of these plasmids shows that they contain large inverted duplications of lambda sequences situated about the Tn903 insertion. The formation of the Tn903λdv plasmids from the ancestral λTn903 is not dependent on the recombination functions provided through the phage red gene or the host recA gene.     

Identification of a QTL conferring seedling and adult plant resistance to eyespot on chromosome 5A of Cappelle Desprez

Eyespot is an economically important fungal disease of wheat and other cereals caused by two fungal species: Oculimacula yallundae and Oculimacula acuformis. However, only two eyespot resistance genes have been characterised and molecular markers made available to plant breeders. These resistances are Pch1, introduced into wheat from the relative Aegilops ventricosa, and Pch2, originally identified in the cultivar Cappelle Desprez (CD). There are drawbacks associated with both resistances; Pch1 is linked to deleterious traits carried on the Ae. ventricosa introgression and Pch2 has been shown to have limited effectiveness. An additional resistance has been reported on chromosome 5A of CD that confers resistance to eyespot in adult plants. In the present study, we demonstrate that resistance on this chromosome is effective against both O. yallundae and O. acuformis eyespot pathogens and confers resistance at both seedling and adult plant stages. This resistance was mapped in both seedling bioassays and field trials in a 5A recombinant population derived from a cross between CD and a CD single chromosome substitution line carrying 5A from the susceptible line Bezostaya. The resistance was also mapped using seedling bioassays in a 5A recombinant population derived from a cross between the susceptible line Chinese Spring (CS) and a single chromosome substitution line carrying 5A from CD. A single major QTL on the long arm of chromosome 5A was detected in all experiments. Furthermore, the SSR marker Xgwm639 was found to be closely associated with the resistance and could be used for marker-assisted selection of the eyespot resistance by plant breeders.     

A new vector for insertion of any DNA fragment into the chromosome of transformable Neisseriae

A useful method for inserting any DNA fragment into the chromosome of Neisseriae has been developed. The method relies on recombination-proficient vector plasmid pNLE1, a pUC19 derivative containing (1) genes conferring resistance to ampicillin and erythromycin, as selectable markers; (2) a chromosomal region necessary for its integration into the Neisseria chromosome; (3) a specific uptake sequence which is required for natural transformation; (4) a promoter capable of functioning in Neisseria; and (5) several unique restriction sites useful for cloning. pNLE1 integrates into the leuS region of the neisserial chromosome at high frequencies by transformation-mediated recombination. The usefulness of this vector has been demonstrated by cloning the tetracycline-resistance gene (tet) and subsequently inserting the tet gene into the meningococcal chromosome.     

A method for construction of E. coli strains with multiple DNA insertions in the chromosome

A system for construction of E. coli strains with multiple DNA insertions in the chromosome, based on elements of modules for site specific recombination of Tn1545 and phage λ, has been developed. Circular non-replicating DNA fragments containing the transposon attachment site (attTn), an excisable cassette with a selectable marker, and a gene of interest integrate randomly into the chromosome of a host E. coli strain when provided with transposon integrase, Int-Tn (the host strain was obtained by insertion of the fragment containing transposon int-Tn gene coding for Int-Tn into the chromosome). Integration of these fragments into the chromosome of int-Tn⁺ cells gives rise to a collection of antibiotic-resistant clones with single insertions at different locations in the chromosome. These insertions are transferred subsequently by P1 transduction into one strain and selected for antibiotic resistance provided by the cassette with the selectable marker. After transduction of each copy, a helper plasmid bearing phage λ xis and int genes is introduced into the cells to excise the drug resistance gene flanked with the λattL and λattR sites from the chromosome. Cells cured of the helper plasmid can undergo the next cycle of P1 transduction/drug resistance gene excision. Each cycle adds another chromosomal copy of the foreign gene. To show the utility of the system, we constructed an E. coli strain bearing several chromosomal copies of lacZ at different locations.     

Characterization and properties of very large inversions of the E. coli chromosome along the origin-to-terminus axis

Summary Suppression of a dnaA46 mutation by integration of plasmid R100.1 derivatives in the termination region of chromosome replication in E. coli results in medium dependence, the suppressed bacteria being sensitive to rich medium at 42° C. Derivatives of such bacteria have been selected for growth at 42° C in rich medium and we have analyzed representatives of the most frequently observed type: bacteria displaying, once cured of the suppressor plasmid, both rich-medium sensitivity and temperature sensitivity. We found, in all cases, that the chromosome had undergone a major inversion event between two inverted IS5's. One is located at 29.2 min on the chromosome map and the other at either one of two positions between 69 and 80 min. The consequences of such inversions for cell growth are discussed. Some of them result from the fact that the replication terminator T2 is located, in inverted chromosomes, close to oriC in the orientation which allows its functioning as a terminus (de Massy et al. in press). Our observations allow an estimation of the frequency of inversions arising from recombination between pairs of inverted chromosomal IS, which could be as high as 10^-2 per cell per generation. We also found that inversion reversal occurs frequently after Hfr conjugational transfer of one of the IS5's, in its wild-type location. This led us to propose a new mechanisms of recombination, in which the incoming DNA strands serve as guides to favor recombination between the resident sequences.Abbreviations Sin suppresive integration - Rms/Rmr rich medium sensitivity/resistance - Ts/Tr temperature sensitivity/resistance - Ap^r ampicillin resistance - Nal^r nalidixic acid resistance - Sp^r spectinomycin resistance - St^r streptomycin resistance - Tc^r tetracycline resistance     

Construction of a bacterial artificial chromosome (BAC) library and identification of overlapping BAC clones with chromosome 4-specific RFLP markers in rice

 To facilitate construction of physical map of the rice genome, a bacterial artificial chromosome (BAC) library of IR64 genomic DNA was constructed. It consists of 18 432 clones and contains 3.28 rice genomic equivalents. The insert size ranged from 37 to 364 kb with an average of 107 kb. We used 31 RFLP markers on chromosome 4 to screen the library by colony hybridization. Sixty eight positive clones were identified with 2.2 positive clones per RFLP marker. The positive clones were analyzed to generate 29 contigs whose sizes ranged from 50 to 384 kb with an average of 145.6 kb. Chromosome walking was initiated for ten contigs linked to resistance genes. Thirty eight BAC clones were obtained and two contigs were integrated. Altogether, they covered 5.65 Mb (15.1%) of chromosome 4. These contigs may be used as landmarks for physical mapping of chromosome 4, and as starting points for chromosome walking towards the map-based cloning of disease resistance genes which were located nearby. Received: 15 November 1996 / Accepted: 24 January 1997     

Assignment of a human DNA double-strand break repair gene (XRCC5) to chromosome 2

The Chinese hamster ovary (CHO-K1) cell mutant XRS-6 is defective in rejoining of DNA double-strand breaks and is hypersensitive to X-rays, γ-rays, and bleomycin. Radiation resistance or sensitivity of somatic cell hybrids constructed from the fusion of XRS-6 cells with primary human fibroblasts strongly correlated with the retention of human chromosome 2 isozyme and molecular markers. Discordancies between some chromosome 2 markers and the radiation resistance phenotype in some of the hybrid cells suggested the location of the X-ray repair cross complementing 5 (XRCC5) gene on the p arm of chromosome 2. Introduction of human chromosome 2 by microcell-mediated chromosome transfer into the radiation-sensitive XRS-6 cells resulted in hybrid cells in which the radiation sensitivity was complemented. The chromosome 2p origin of the complementing human DNA in the microcell hybrids was supported by fluorescent in situ hybridization analysis of human metaphases using human DNA amplified from the hybrids by inter-Alu-PCR as chromosome-painting probes. XRCC5 is therefore provisionally assigned to human chromosome 2p.     

Exclusion of vanA, vanB and vanC type glycopeptide resistance in strains of Lactobacillus reuteri and Lactobacillus rhamnosus used as probiotics by polymerase chain reaction and hybridization methods

Strains of Lactobacillus reuteri and Lact. rhamnosus are used as probiotics in man and animal. The aim of this study was to determine whether the glycopeptide resistance in these lactobacilli has a similar genetic basis as in enterococci. Five Lact. reuteri strains and one Lact. rhamnosus, as well as four Enterococcus control strains, were probed for the vanA gene cluster, the vanB gene and the vanC gene by PCR and Southern hybridization, and DNA/DNA hybridization. Their resistance and plasmid patterns were also investigated. All Lactobacillus strains were resistant to vancomycin but susceptible to a broad range of antibiotics. Four of the Lactobacillus strains (including the Lact. rhamnosus strain) did not harbour any plasmid and two of them contained five and 6 plasmid bands respectively. None of the Lactobacillus strains possessed the vanA, vanB or vanC gene. These findings indicate that the glycopeptide resistance of the Lactobacillus strains analysed is different from the enterococcal type. The study provides reassurance on the safety of the Lactobacillus strains used as probiotics with regard to their vancomycin resistance.