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Colistin (polymyxin E) is increasingly used as a last-resort antibiotic for the treatment of severe infections with multidrug-resistant Gram-negative bacteria. In contrast to human medicine, colistin is also used in veterinary medicine for metaphylaxis. Our objective was to decipher common colistin resistance mechanisms in Klebsiella pneumoniae isolates from animals. In total, 276 veterinary K. pneumoniae isolates, derived from companion animals or livestock, and 12 isolates from human patients were included for comparison. Six out of 276 veterinary isolates were colistin resistant (2.2%). Human isolates belonging to high-risk clonal lineages (e.g., ST15, ST101, ST258), displayed multidrug-resistant phenotypes and harboured many resistance genes compared to the veterinary isolates. However, the common colistin resistance mechanism in both human and animal K. pneumoniae isolates were diverse alterations of MgrB, a critical regulator of lipid A modification. Additionally, deleterious variations of lipopolysaccharide (LPS)-associated proteins (e.g., PmrB P95L, PmrE P89L, LpxB A152T) were identified. Phylogenetic analysis and mutation patterns in genes encoding LPS-associated proteins indicated that colistin resistance mechanisms developed independently in human and animal isolates. Since only very few antibiotics remain to treat infections with MDR bacteria, it is important to further analyse resistance mechanisms and the dissemination within different isolates and sources.

Here, we report a case of fatal sepsis resulting from an intra-abdominal infection caused by a Bacteroides fragilis strain containing a CfiA4 metallo-β-lactamase and an upstream insertion sequence (IS) element. Meropenem was used as empiric therapy for septic shock as a result of the intra-abdominal infection, although two rounds of carbapenem treatment had been administered previously. B. fragilis was isolated from two anaerobic blood culture bottles 4 days after the onset of septic shock. Susceptibility testing revealed that the isolate was non-susceptible to all tested agents except metronidazole and tigecycline. The isolate gave a positive result in ethylenediaminetetraacetic acid and carbapenem inactivation tests, but a negative result in a double-disk synergy test using sodium mercaptoacetate. Next-generation whole-genome sequencing indicated the presence of the cfiA4 , emrG and emrF genes. PCR indicated the presence of an IS element upstream of the cifA4 gene. Although carbapenem-resistant B. fragilis isolates have previously been reported, clinical sepsis by this organism is considered rare. In Japan, as in most countries worldwide, routine susceptibility testing and the detection of metallo-β-lactamases is not carried out in anaerobic organisms, including B. fragilis. The emergence of carbapenem resistance during therapy should be monitored, as B. fragilis strains containing the cfiA gene show decreased sensitivity during carbapenem therapy. Therefore, susceptibility testing and appropriate antibiotic stewardship are required in cases of anaerobic bacterial infections. [ABSTRACT FROM AUTHOR]

Abstract: Oenococcus oeni is responsible for the malolactic fermentation of wines. Genomic diversity has been recently established in the species and extensive attention is now being given to the genomic bases of strain-specific differences. We explored the role of insertion sequences (IS), which are considered as driving forces for novel genotypic and phenotypic variants in prokaryotes. The present study focuses on members of the IS30 family, which are widespread among lactic acid bacteria. An in silico analysis of the three available genomes of O. oeni in combination with the use of an inverse PCR strategy targeting conserved IS30-related sequences indicated the presence of seven IS30 copies in the pangenome of O. oeni. A primer designed to anneal to the conserved 3′ end of the IS30 element was paired with each of the seven primers selected to bind to unique sequences upstream of each of the seven mobile elements identified. The study presents an overview of the abundance, and the genomic environment of IS30 elements in the O. oeni pangenome and shows that the two existing genetic sub-populations previously described in the species through multilocus sequence typing analysis (MLST) differ in their IS30 content. Possible IS30 impacts on bacterial adaptation are discussed. [Copyright &y& Elsevier]

Objective To characterize a unique tandem blaCMY-2 gene arrangement found in two non-identical clinical strains of Escherichia coli. Methods Both plasmid and chromosomal DNA were evaluated using PFGE, restriction digest analysis, plasmid profiling and Southern hybridization. blaCMY-2 gene expression and gene copy number were evaluated by real-time PCR. Susceptibilities to selected β-lactam antibiotics were determined by agar dilution. Results A tandem arrangement for blaCMY-2 was identified in both isolates and was the only arrangement for blaCMY-2 observed. These isolates had distinct PFGE and plasmid profiles. Each strain exhibited 2-fold higher blaCMY-2 mRNA expression and up to 8-fold lower β-lactam susceptibility compared with a strain with a single copy of blaCMY-2. Conclusion This is the first report of IS5 being associated with tandem blaCMY-2. IS5 has previously been associated with antibiotic resistance through tandem gene amplification. The unique tandem arrangement provides a mechanism for increased blaCMY-2 expression. [ABSTRACT FROM PUBLISHER]

A new IS-like element (604 bp) was revealed in the genome of several Acidithiobacillus ferrooxidans strains isolated from diverse biotopes. It includes 26-bp imperfectly matched terminal inverted repeats (TIRs), similar in structure to the TIRs of the ISAfel insertion element. The 60-bp DNA fragment adjacent to the right TIR (TIRR) exhibits pronounced homology with the similarly located DNA fragments in ISAfel and IST445, as well as with the internal fragment of ISAfel encoding the transposase gene (nucleotides from 254 to 311 bp). The central section of ISAfe600 is unique and exhibits no homology with any prokaryotic DNA. A duplication of 8 bp of the target DNA was found in the ISAfe600 insertion site. One to four copies of ISAfe600 were revealed by Southern hybridization in the genome of A. ferrooxidans strains studied. The number of ISAfe600 copies varies depending on the growth conditions (energy substrate). Since open reading frames big enough to encode transposase are not presert in the structure of ISAfe600, it may be a deficient IS element; its translocation is possibly achieved under control of the ISAfel transposase. [ABSTRACT FROM AUTHOR]

Abstract: We have investigated the genetic diversity of Tn1546 among 17 vancomycin-resistant enterococci (VRE) isolates of Enterococcus faecium. Most of these multidrug-resistant strains harboured plasmids of 2kb to >300kb in size. The vancomycin resistance marker vanA was located on both the plasmid and the chromosomal DNA. VRE isolates 18 and 22 failed to amplify the orf1-IRR and orf2-IRR but contained the orf1 and orf2. VRE3 failed to amplify the orf1, orf2, vanR and vanS, but still yielded a larger than expected (4.4kb vs. 2.3kb) vanSH amplicon. VRE9, 10, 21 and 22 also yielded larger (5.5kb) vanSH amplicons; all others yielded 4.0kb vanSH amplicons. Sequence analysis of the vanSH amplicons from VRE9, 10, 21 and 22 revealed the presence of IS1251 between the vanS and vanH genes in these isolates. The observed vanSH amplicon from VRE3 contained orf31, orf30 and orf29 of the plasmid pRUM followed by the vanHAXYZ region of Tn1546. Translocation of Tn1546 to a pRUM-like plasmid in VRE3 resulted in the loss of its orf1, orf2, vanR and vanS elements and a loss of the orf32 of pRUM, leading to a unique structural arrangement of vanA elements that is hitherto unknown. [Copyright &y& Elsevier]

Abstract: A clinical strain of Enterococcus faecium ATCC 51559 exhibits heteroresistance, i.e. a high level of resistance to vancomycin (minimum inhibitory concentration (MIC)>256μg/mL) by broth dilution but sensitivity to vancomycin by Etest (MIC=1.8μg/mL). Three variants of this strain, EF1, EF2 and EF3, exhibit high levels of resistance to vancomycin both by broth dilution and Etest assays. The four strains were used to study heteroresistance by pulsed-field gel electrophoresis (PFGE), polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequence analysis of a partial region of the van operon. Minor differences between SalI and SmaI restriction profiles of the variants and the parental strain were observed by PFGE analysis. PCR analysis confirmed the presence of the vancomycin resistance marker vanA (0.73kb) and a larger than expected amplicon (8.2kb vs. 6.7kb) of the van operon in all the strains. The 8.2kb van operon was cloned for EcoRI RFLP and sequence analysis. All of the clones exhibited distinctly different RFLP profiles when grown in the presence of kanamycin or vancomycin+kanamycin. The presence of these antibiotics during overnight growth of EF1 on plates also resulted in altered SalI PFGE profiles. Sequence analysis of the van operon clones revealed a 1.5kb IS1251-like insertion element between the vanS and vanH genes in all the strains. Several novel point mutations in the vanR, vanS, vanH, vanA, vanX and vanY genes were also discovered. Some of these mutations were present in the parental strain only and included base substitutions T→C, A→G, T→A and T→C at nucleotide positions 4202, 4597, 4763 and 6207 of Tn1546, resulting in amino acid replacements I76→T and K208→E of vanR, S19→T of vanS and L64→P of vanH genes, respectively. We believe that these are responsible for the observed heteroresistance. The present study clearly shows how independent novel mutations can give rise to polymorphism, heteroresistance and clonal diversity among vancomycin-resistant enterococci strains as a result of continuous exposure to antibiotics. [Copyright &y& Elsevier]

Abstract: Genetic polymorphism within the genomes of bacterial pathogens determines their evolutionary potential during long-term interaction with their hosts. To investigate the level of genetic variation in Xanthomonas oryzae pv. oryzae (Xoo), the causative agent of rice bacterial blight disease, three DNA marker systems, including (i) restriction fragment length polymorphism (RFLP) of the avrBs3/PthA family genes (avrXa27), (ii) RFLP of insertion (IS) elements and (iii) random amplified polymorphic DNA (RAPD) markers, were used to detect polymorphism among 32 Xoo strains that differed in their virulence patterns. All these strains contained multiple avrXa27 homologs that were variable in copy number and genomic location. RFLP of six IS elements revealed that these mobile sequences were abundant in Xoo genomes, with 150 of the total of 165 discernable markers being variable. Thirty-eight decamer primers of RAPD amplified a total of 691 bands, with 100% of them being variable. In addition, analysis of molecular variance (AMOVA) of data from RFLP analysis of IS elements and from RAPD analysis showed that most of the genetic variation residues were within Xoo populations, rather than between populations. Although all three DNA marker systems supported that substantial variation was maintained in Xoo genomes, Mantel tests did not identify significant correlation between the similarity coefficients calculated from them. The results of the present study indicated that Xoo genomes contain a high level of genetic polymorphism, which greatly facilitates the evolution of this important pathogen during interaction with its host rice plant. [Copyright &y& Elsevier]

Culex pipiens quinquefasciatus Say and Culex pipiens pipiens Linnaeus are sibling species incriminated as important vectors of emerging and re-emerging infectious diseases worldwide. The two forms differ little morphologically and are differentiated mainly based upon ecological, behavioural, physiological and genetic traits. Within the North American zone of sympatry, populations of Cx. p. quinquefasciatus and Cx. p. pipiens undergo extensive introgression and hybrid forms have been reported in nature. Both Cx. p. quinquefasciatus and Cx. p. pipiens are infected with the endosymbiotic bacteria Wolbachia pipientis. Here, we report the presence of a transposable element belonging to the IS256 family (IS256wPip) associated with Wolbachia in both Cx. p. quinquefasciatus and Cx. p. pipiens populations. Using reverse transcriptase PCR and sequence analysis, we show that IS256wPip has disrupted the wspB locus, a paralogue of the Wolbachia outer membrane protein ( wspA) gene. The inactivation of the wspB appears to be specific to Cx. p. quinquefasciatus and to hybrids of the two forms, and was not observed in the surveyed Cx. p. pipiens mosquitoes. Our results support the hypothesis of a different origin of North American Cx. p. quinquefasciatus and Cx. p. pipiens populations. The flux of mobile genetic elements in the Wolbachia wPip genome could explain the high level of crossing types observed among different Culex populations. The insertion of IS256wPip into wspB may comprise a genetic candidate for discriminating Wolbachia symbionts in Culex. [ABSTRACT FROM AUTHOR]

Carrot storage roots accumulate free sugars. The type of sugar accumulated is conditioned by the Rs locus so that typical carrot roots ( Rs/-) accumulate predominantly glucose and fructose while rs/ rs plants accumulate predominantly sucrose. We recently have found rs/ rs plants in one inbred line that harbor a naturally occurring insertion sequence of 2.5 kb integrated into the first intron region of acid soluble invertase isozyme II. Using these facts, three primers were designed to differentiate Rs/ Rs, Rs/ rs and rs/ rs carrot plants with simple PCR amplification. Co-dominant, PCR-based markers for acid soluble invertase isozyme II allowed genotyping of the Rs locus in 1-week-old carrot seedlings whereas mature carrot roots were needed to make this evaluation previously, and homozygous dominant plants could not be differentiated from heterozygotes without lengthy progeny testing. Marker-assisted evaluation and selection of carrot root sugar type were exercised in segregating families of diverse background and complete accuracy in predicting sugar type was realized in subsequent generations to further confirm that acid soluble invertase isozyme II conditions the Rs locus. These PCR-based markers will be useful in carrot breeding programs screening for this trait in segregating populations, for studying the distribution and origins of this trait in domestic and wild carrots, and for identifying seed mixtures as low as 10% Rs/- or 1% rs/ rs. [ABSTRACT FROM AUTHOR]

Abstract: The mobile insertion element IS5 is a relatively small but genetically compact DNA sequence of 1195bp found in variable copy number in the genome of Escherichia coli strains. This study presents a detailed transcript analysis of the population of IS5 elements present in E. coli strains MC4100 and MG1655. The findings indicate that the ins5A gene comprising 978bp is transcribed from its own promoter, which is located close to the right-hand end of the element. The two divergently transcribed genes ins5C and in5B form an operon, and this transcript is fully contained within the borders of the ins5A transcript. Although transcription out of IS5 from element-internal promoters was negligible, in the case of MG1655 a major transcript was found to extend into the insertion element. This suggests that IS5-specific transcription can be influenced by the specific location of the element in the chromosome, the orientation it adopts and the gene it interrupts. [Copyright &y& Elsevier]

The association of historical plague pandemics with Yersinia pestis remains controversial, partly because the evolutionary history of this largely monomorphic bacterium was unknown. The microevolution of Y. pestis was therefore investigated by three different multilocus molecular methods, targeting genomewide synonymous SNPs, variation in number of tandem repeats, and insertion of IS 100 insertion elements. Eight populations were recognized by the three methods, and we propose an evolutionary tree for these populations, rooted on Yersinia pseudotuberculosis. The tree invokes microevolution over millennia, during which enzootic pestoides isolates evolved. This initial phase was followed by a binary split 6,500 years ago, which led to populations that are more frequently associated with human disease. These populations do not correspond directly to classical biovars that are based on phenotypic properties. Thus, we recommend that henceforth groupings should be based on molecular signatures. The age of V. pestis inferred here is compatible with the dates of historical pandemic plague. However, it is premature to inter an association between any modern molecular grouping and a particular pandemic wave that occurred before the 20th century. [ABSTRACT FROM AUTHOR]

In characterising Mycoplasma agalactiae strains from various European countries and from Africa, a new insertion sequence (IS), ISMag1, which is related to IS of the family of IS30 insertion elements, has been identified by DNA sequence analysis and Southern blot hybridisation. ISMag1 has a size of 1515 bp, and contains inverted repeats of 3 bp and a gene encoding the putative transposase on a single open reading frame. ISMag1 is present only in the rarely isolated serotypes E, F, G and H of M. agalactiae, where it is found in 1 to approximately 30 copies. The different patterns obtained by hybridisation of a labelled probe of ISMag1 to genomic DNA cut with various restriction enzymes correlate to some extent to the different serotypes and to variations of the nucleotide sequences of the uvrC genes of the different strains. Based on uvrC sequences, the strains of M. agalactiae carrying ISMag1 form a cluster, separate from the other strains. IS patterns obtained with ISMag1 allow a fine subtyping of the serotypes E, F, G and H of M. agalactiae for epidemiological studies. The potential role of ISMag1 and of its copy numbers on virulence and persistence of the respective strains requests further studies. [Copyright &y& Elsevier]

Cell surface (S)-layer protein synthesis in Bacillus stearothermophilus PV72/p6 is blocked when cells are grown at elevated temperature. From a culture exhibiting the S-layer-negative phenotype, the S-layer deficient mutant T5 (SbsA–) was isolated. Genetic analysis of the S-layer-encoding gene (sbsA) of mutant T5 revealed an insertion element (IS4712) integrated into the upstream regulatory region of the S-layer gene, thereby blocking sbsA transcription. The insertion element consists of 1371 base pairs which are flanked by two perfect inverted terminal repeats. Sequence similarity to other transposases of the IS4 family was detected. DNA-DNA hybridizations demonstrated that multiple homologues of IS4712 were also present within the genomes of several other thermophilic bacillus isolates. Attempts to isolate SbsA+ revertants failed. Instead, cells with altered surface proteins were detected. The synthesis of the altered S-layer proteins was correlated with the presence of IS4712 along with the occurrence of deletions in the sbsA coding region. Furthermore imprecise excision of IS4712 was detected. This work demonstrated that B. stearothermophilus is able to express at least four different S-layer proteins and that blocking of sbsA transcription by the insertion element IS4712 is associated with the expression of altered surface proteins. [ABSTRACT FROM AUTHOR]

An IS element, termed IS Cg2, was identified in the chromosome of Corynebacterium glutamicum ATCC 13032. After screening a cosmid library of the C. glutamicum ATCC 13032 genome, six copies of IS Cg2 including their flanking regions were sequenced and analyzed. IS Cg2 is 1636 bp in length and has 26-bp imperfect inverted repeats flanked by 3-bp direct repeats. By comparisons with other IS elements, IS Cg2 was classified as a member of the IS 30 family of insertion sequences. The six copies of IS Cg2 were identical at the nucleotide level and were located in intergenic, AT-rich regions of the chromosome. The regions in which the six copies of IS Cg2 were inserted displayed significant similarities. This similarity extends over a region of 65 bp, which was assumed to be the target region for IS Cg2. Interestingly, five of the six copies of IS Cg2 were located adjacent to genes that may be involved in aspartate and glutamate metabolism or its regulation. Investigation of the distribution of IS Cg2 showed that the IS element is restricted to certain C. glutamicum strains. Analysis of various integration regions indicates active transposition of IS Cg2 in C. glutamicum. [ABSTRACT FROM AUTHOR]

DNA sequences homologous to the T-DNA region of the octopine Ti plasmid from Agrobacterium tumefaciens are found in various fast-growing Rhizobium fredii strains. The largest fragment ( BamHI fragment 2) at the right-boundary region of the 'core' T-DNA hybridizes to more than one plasmid present in R. fredii. However, one smaller fragment ( EcoRI fragment 19a) adjacent to the 'core' T-DNA shows homology only with the plasmid carrying the symbiotic nitrogen-fixation genes ( pSym). Hybridization data obtained with digested R. fredii USDA193 pSym DNA suggests that the homology is mainly with two HindIII fragments, 1.7 kb and 8.8 kb in size, of the plasmid. The 1.7 kb HindIII fragment also hybridizes to two regions of the virulence plasmid of A. tumefaciens, pAL1819, a deletion plasmid derived from the octopine Ti plasmid, pTiAch5. Hybridization studies with an insertion element IS66 from A. tumefaciens indicate that the 1.7 kb HindIII fragment of R. fredii plasmid, homologous to the T-DNA and the virulence region of Ti plasmid, is itself an IS66 homologue. [ABSTRACT FROM AUTHOR]