Masato Akiba

CmeR Functions as a Transcriptional Repressor for the Multidrug Efflux Pump CmeABC in Campylobacter jejuni

ABSTRACT CmeABC, a resistance-nodulation-division (RND) type of efflux pump, contributes to Campylobacter resistance to a broad spectrum of antimicrobial agents and is also essential for Campylobacter colonization of the animal intestinal tract by mediation of bile resistance. As one of the main systems for Campylobacter adaptation to different environments, CmeABC is likely subject to control by regulatory elements. We describe the identification of a transcriptional repressor for CmeABC. Insertional mutagenesis of cmeR, an open reading frame immediately upstream of the cmeABC operon, resulted in overexpression of cmeABC, as determined by transcriptional fusion (PcmeABC-lacZ) and immunoblotting with CmeABC-specific antibodies. Overexpression of the efflux pump was correlated with a moderate increase in the level of resistance of the cmeR mutant to several antimicrobials. In vitro, recombinant CmeR bound specifically to the promoter region of cmeABC, precisely, to the inverted repeat sequences in the cmeABC promoter. A single nucleotide deletion between the two half sites of the inverted repeat reduced the level of CmeR binding to the promoter sequence and resulted in overexpression of cmeABC. Together, these findings indicate that cmeR encodes a transcriptional repressor that directly interacts with the cmeABC promoter and modulates the expression of cmeABC. Mutation either in CmeR or in the inverted repeat impedes the repression and leads to enhanced production of the MDR efflux pump.

Contamination of Salmonella in retail meats and shrimps in the Mekong Delta, Vietnam.

From March 2000 to September 2001, 608 samples of retail meat (136 pork, 70 beef, 202 chicken, and 200 ducks) and 110 samples of retail shrimp from six provinces of the Mekong Delta in Vietnam were collected individually and examined for the prevalence of Salmonella. Of the 718 samples examined, 243 (33.8%) were Salmonella positive. Salmonella was isolated from 69.9% of the pork samples, 48.6% of the beef samples, 21.0% of the chicken meat samples, 22.3% of the duck meat samples, and 24.5% of the shrimp samples. From 261 Salmonella isolates, 24 different serovars were identified. The predominant serovars of the isolates were Salmonella Derby, Salmonella Weltevreden, and Salmonella London in pork; Salmonella Weltevreden, Salmonella London, and Salmonella Dessau in beef; Salmonella Emek, Salmonella Typhimurium, and Salmonella Dessau in chicken meat; Salmonella Lexington, Salmonella Derby, and Salmonella Dessau in duck meat; and Salmonella Weltevreden, Salmonella Tennessee, and Salmonella Dessau in shrimps. Salmonella Bovismorbificans, Salmonella Derby, Salmonella Dessau, and Salmonella Weltevreden were the most common serovars in all the samples examined. These results indicate a high rate of contamination by Salmonella in retail meats and shrimps in the Mekong Delta, Vietnam.

Molecular Typing and Epidemiological Study of Salmonella enterica Serotype Typhimurium Isolates from Cattle by Fluorescent Amplified-Fragment Length Polymorphism Fingerprinting and Pulsed-Field Gel Electrophoresis

ABSTRACT One hundred twenty Salmonella entericaserotype Typhimurium strains, including 103 isolates from cattle gathered between 1977 and 1999 in the prefecture located on the northern-most island of Japan, were analyzed by using fluorescent amplified-fragment length polymorphism (FAFLP) and pulsed-field gel electrophoresis (PFGE) to examine the genotypic basis of the epidemic. Among these strains, there were 17 FAFLP profiles that formed four distinct clusters (A, B, C, and D). Isolates that belonged to cluster A have become increasingly common since 1992 with the increase of bovine salmonellosis caused by serotype Typhimurium. PFGE resolved 25 banding patterns that formed three distinct clusters (I, II, and III). All the isolates that belonged to FAFLP cluster A, in which all the strains of definitive phage type 104 examined were included, were grouped into PFGE cluster I. Taken together, these results indicate that clonal exchange of serotype Typhimurium has taken place since 1992, and they show a remarkable degree of homogeneity at a molecular level among contemporary isolates from cattle in this region. Moreover, we have sequenced two kinds of FAFLP markers, 142-bp and 132-bp fragments, which were identified as a polymorphic marker of strains that belonged to clusters A and C, respectively. The sequence of the 142-bp fragment shows homology with a segment of P22 phage, and that of the 132-bp fragment shows homology with a segment of traG, which is an F plasmid conjugation gene. FAFLP is apparently as well suited for epidemiological typing of serotype Typhimurium as is PFGE, and FAFLP can provide a source of molecular markers useful for studies of genetic variation in natural populations of serotype Typhimurium.

Rapid identification of Salmonella enterica serovars, Typhimurium, Choleraesuis, Infantis, Hadar, Enteritidis, Dublin and Gallinarum, by multiplex PCR

Salmonella enterica subsp. enterica poses a threat to both human and animal health, with more than 2500 serovars having been reported to date. Salmonella serovars are identified by slide and tube agglutination tests using O and H antigen-specific anti-sera, although this procedure is both labor intensive and time consuming. Establishment of a method for rapid screening of the major Salmonella serovars is therefore required. We have established multiplex polymerase chain reaction (m-PCR) assays for identification of seven serovars of Salmonella, i.e., Typhimurium, Choleraesuis, Infantis, Hadar, Enteritidis, Dublin and Gallinarum. Three serovar-specific genomic regions (SSGRs) of each serovar were selected using an approach in comparative genomics. The Salmonella-specific invA gene was used to confirm the genetic background of the organisms. The isolates tested were identified as a target serovar when the three selected SSGRs and invA were all positive for amplification. The specificity of each m-PCR assay was investigated using 118 serovars of Salmonella and 12 species of non-Salmonella strains. Although a small number of false-positive results were observed in the m-PCR assays used to identify Typhimurium, Choleraesuis, Enteritidis and Dublin for closely related serovars, false-negative results were not observed in any assays. These assays had sufficient specificity to identify the seven Salmonella serovars, and therefore, have the potential for use as rapid screening methods.

Molecular Characterization of a Prophage of Salmonella enterica Serotype Typhimurium DT104

ABSTRACT Isolates of the Salmonella enterica serotype Typhimurium definitive phage type (DT104) were found to contain the same prophage (designated phage ST104). The complete sequence of the DNA genome of prophage ST104 was determined. The entire DNA sequence consisted of 41,391 bp, including 64 open reading frames, and exhibited high similarity to P22 and to phage type conversion phage ST64T.

Characterization of Salmonella enterica Serovar Typhimurium Isolates Harboring a Chromosomally Encoded CMY-2 β-Lactamase Gene Located on a Multidrug Resistance Genomic Island

ABSTRACT Since 2004, extended-spectrum cephalosporin (ESC)-resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) isolates have been detected from cattle in the northern major island of Japan, Hokkaido. Resistance to ESCs was found to be mediated by CMY-2 type β-lactamase among 22 epidemiologically unrelated isolates showing indistinguishable pulsed-field gel electrophoresis patterns. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that the CMY-2 β-lactamase gene (blaCMY-2) was integrated in a 2.5-Mb chromosomal fragment. Genetic analysis of S. Typhimurium isolate L-3553 indicated that blaCMY-2 was located on a unique 125-kb genomic island, GI-VII-6, which consists of 140 open reading frames. Pairwise alignment of GI-VII-6 and Escherichia coli plasmid pAR060302 (size, 167 kb) revealed that a large proportion of GI-VII-6 (99%) shows a high sequence similarity (>99%) with pAR060302. GI-VII-6 contains 11 antimicrobial resistance genes including sul1, qacEΔ1, aadA2, and dfrA12 in the aadA2 region; sugE1 and blaCMY-2 in the blaCMY-2 region; and sul2, strA, strB, tet(A), and floR in the floR region. Two directly repeated IS26 copies were present at both ends of GI-VII-6. Junction regions of GI-VII-6 were flanked by an 8-bp direct repeat, indicating that GI-VII-6 was acquired by transposition involving IS26 transposase. PCR scanning revealed that the overall structure of GI-VII-6 was almost identical in the 22 isolates. Phylogenetic analysis suggested that S. Typhimurium isolates harboring GI-VII-6 belong to a different genomic lineage than other whole-genome-sequenced S. Typhimurium strains. These data indicate that a particular clone of S. Typhimurium harboring GI-VII-6 has spread among the cattle population in Hokkaido, Japan.

Fluoroquinolone resistance mechanisms in an Escherichia coli isolate, HUE1, without quinolone resistance-determining region mutations.

Fluoroquinolone resistance can cause major clinical problems. Here, we investigated fluoroquinolone resistance mechanisms in a clinical Escherichia coli isolate, HUE1, which had no mutations quinolone resistance-determining regions (QRDRs) of DNA gyrase and topoisomerase IV. HUE1 demonstrated MICs that exceeded the breakpoints for ciprofloxacin, levofloxacin, and norfloxacin. HUE1 harbored oqxAB and qnrS1 on distinct plasmids. In addition, it exhibited lower intracellular ciprofloxacin concentrations and higher mRNA expression levels of efflux pumps and their global activators than did reference strains. The genes encoding AcrR (local AcrAB repressor) and MarR (MarA repressor) were disrupted by insertion of the transposon IS3-IS629 and a frameshift mutation, respectively. A series of mutants derived from HUE1 were obtained by plasmid curing and gene knockout using homologous recombination. Compared to the MICs of the parent strain HUE1, the fluoroquinolone MICs of these mutants indicated that qnrS1, oqxAB, acrAB, acrF, acrD, mdtK, mdfA, and tolC contributed to the reduced susceptibility to fluoroquinolone in HUE1. Therefore, fluoroquinolone resistance in HUE1 is caused by concomitant acquisition of QnrS1 and OqxAB and overexpression of AcrAB–TolC and other chromosome-encoded efflux pumps. Thus, we have demonstrated that QRDR mutations are not absolutely necessary for acquiring fluoroquinolone resistance in E. coli.

Porcine Toll-like receptors: recognition of Salmonella enterica serovar Choleraesuis and influence of polymorphisms.

Salmonella enterica serovar Choleraesuis (SC) is a highly invasive pathogen that causes enteric and septicemic diseases in pigs. Although there have been some reports on gene expression profiles in the course of infection with SC in pigs, little is known about the genes involved in the infection. By measuring activation, as represented by nuclear factor-κB activity, after stimulation by the pathogen, we showed the involvement of Toll-like receptor (TLR) 5 and the TLR2-TLR1 heterodimer in the recognition of SC. We previously found single nucleotide polymorphisms (SNPs) in the TLRs of various pig populations. Here we demonstrated that the polymorphisms resulting in amino acid changes TLR5(R148L), TLR5(P402L), and TLR2(V703M) attenuated the responses to SC by the cells transfected with the TLR genes. Each of these three SNPs was differently restricted in distribution among breeds. These results suggest that there are differences in resistance to salmonellosis among breeds; these differences may be of great importance for the pig industry in terms of breeding and vaccine development.

Comparison of Salmonella enterica serovar Abortusequi isolates of equine origin by pulsed-field gel electrophoresis and fluorescent amplified-fragment length polymorphism fingerprinting

Equine paratyphoid is caused by Salmonella enterica serovar Abortusequi, and manifests mainly as abortion in the mare. We compared S. Abortusequi strains isolated in Japan and other countries using pulsed-field gel electrophoresis (PFGE) and fluorescent amplified-fragment length polymorphism (FAFLP) analysis. PFGE analysis of S. Abortusequi strains gave 21-27 fragments ranging in size from 33 to 602kb. Although two PFGE profiles were observed among the 20 S. Abortusequi isolates in Japan, the restriction fragments originating from the chromosome were common between the two profiles. The similarity index of the two profiles was 90.9%, while those between Japanese and five other S. Abortusequi strains were 29.8-37.5%. On the other hand, FAFLP analysis of S. Abortusequi strains generated 64-67 amplified fragments ranging in size from 100 to 400bp. One polymorphic fragment was observed among the 20 S. Abortusequi isolates in Japan. These data indicate the close relation of this agent in Japan. S. Abortusequi strains sharing a common ancestry might have been conserved in Japan.

Impact of wastewater from different sources on the prevalence of antimicrobial-resistant Escherichia coli in sewage treatment plants in South India.

The sewage treatment plant (STP) is one of the most important interfaces between the human population and the aquatic environment, leading to contamination of the latter by antimicrobial-resistant bacteria. To identify factors affecting the prevalence of antimicrobial-resistant bacteria, water samples were collected from three different STPs in South India. STP1 exclusively treats sewage generated by a domestic population. STP2 predominantly treats sewage generated by a domestic population with a mix of hospital effluent. STP3 treats effluents generated exclusively by a hospital. The water samples were collected between three intermediate treatment steps including equalization, aeration, and clarification, in addition to the outlet to assess the removal rates of bacteria as the effluent passed through the treatment plant. The samples were collected in three different seasons to study the effect of seasonal variation. Escherichia coli isolated from the water samples were tested for susceptibility to 12 antimicrobials. The results of logistic regression analysis suggest that the hospital wastewater inflow significantly increased the prevalence of antimicrobial-resistant E. coli, whereas the treatment processes and sampling seasons did not affect the prevalence of these isolates. A bias in the genotype distribution of E. coli was observed among the isolates obtained from STP3. In conclusion, hospital wastewaters should be carefully treated to prevent the contamination of Indian environment with antimicrobial-resistant bacteria.