Taketoshi Iwata

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.

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.

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.

Distribution and Relationships of Antimicrobial Resistance Determinants among Extended-Spectrum-Cephalosporin-Resistant or Carbapenem-Resistant Escherichia coli Isolates from Rivers and Sewage Treatment Plants in India

ABSTRACT To determine the distribution and relationship of antimicrobial resistance determinants among extended-spectrum-cephalosporin (ESC)-resistant or carbapenem-resistant Escherichia coli isolates from the aquatic environment in India, water samples were collected from rivers or sewage treatment plants in five Indian states. A total of 446 E. coli isolates were randomly obtained. Resistance to ESC and/or carbapenem was observed in 169 (37.9%) E. coli isolates, which were further analyzed. These isolates showed resistance to numerous antimicrobials; more than half of the isolates exhibited resistance to eight or more antimicrobials. The blaNDM gene was detected in 14/21 carbapenem-resistant E. coli isolates: blaNDM-1 in 2 isolates, blaNDM-5 in 7 isolates, and blaNDM-7 in 5 isolates. The blaCTX-M gene was detected in 112 isolates (66.3%): blaCTX-M-15 in 108 isolates and blaCTX-M-55 in 4 isolates. We extracted 49 plasmids from selected isolates, and their whole-genome sequences were determined. Fifty resistance genes were detected, and 11 different combinations of replicon types were observed among the 49 plasmids. The network analysis results suggested that the plasmids sharing replicon types tended to form a community, which is based on the predicted gene similarity among the plasmids. Four communities each containing from 4 to 17 plasmids were observed. Three of the four communities contained plasmids detected in different Indian states, suggesting that the interstate dissemination of ancestor plasmids has already occurred. Comparison of the DNA sequences of the blaNDM-positive plasmids detected in this study with known sequences of related plasmids suggested that various mutation events facilitated the evolution of the plasmids and that plasmids with similar genetic backgrounds have widely disseminated in India.

Colistin-Resistant mcr-1-Positive Pathogenic Escherichia coli in Swine, Japan, 2007-2014.

To the Editor: Colistin is an old-generation antimicrobial agent; however, because it is one of the few agents that remain effective against multidrug-resistant gram-negative bacteria (e.g., carbapenem-resistant Pseudomonas aeruginosa and Enterobacteriaceae), its clinical usefulness is being increasingly recognized (1). Previous reports have described the mechanisms of colistin resistance (2) as being chromosomally mediated and not associated with horizontal gene transfer. However, from 2011 through 2014, a plasmid-encoded colistin-resistance gene, mcr-1, was identified in colistin-resistant Escherichia coli isolated in China, particularly from animals. Specifically, mcr-1–positive isolates were found in 21% of healthy swine at slaughter, 15% of marketed pork and chicken meat, and 1% of hospitalized human patients (3). A study of E. coli isolated from healthy cattle, swine, and chickens in Japan during 2000–2014 found only 2 (0.02%) of 9,308 isolates positive for mcr-1 (4). We report the rates at which mcr-1 was detected in our stored collection of E. coli isolates from diseased swine (swine with diarrhea or edema disease), hereafter referred to as swine-pathogenic E. coli.

Characteristics of Salmonella enterica Serovar 4,[5],12:i:- as a Monophasic Variant of Serovar Typhimurium

Salmonella enterica subspecies enterica serovar 4,[5],12:i:- (S. 4,[5]12:i:-) is believed to be a monophasic variant of S. enterica serovar Typhimurium (S. Typhimurium). This study was conducted to corroborate this hypothesis and to identify the molecular and phenotypic characteristics of the S. 4,[5]12:i:- isolates in Japan. A total of 51 S. 4,[5]12:i:- isolates derived from humans, cattle, swine, chickens, birds, meat (pork), and river water in 15 prefectures in Japan between 2000 and 2010 were analyzed. All the S. 4,[5],12:i:- isolates were identified as S. Typhimurium by two different polymerase chain reactions (PCR) for identification of S. Typhimurium. Of the 51 S. 4,[5],12:i:- isolates, 39 (76.5%) harbored a 94-kb virulence plasmid, which is known to be specific for S. Typhimurium. These data suggest that the S. 4,[5],12:i:- isolates are monophasic variants of S. Typhimurium. The flagellar phase variation is induced by three adjacent genes (fljA, fljB, and hin) in the chromosome. The results of PCR mapping of this region and comparative genomic hybridization analysis suggested that the deletion of the fljAB operon and its flanking region was the major genetic basis of the monophasic phenotype of S. 4,[5],12:i:-. The fljAB operon and hin gene were detectable in eight of the S. 4,[5],12:i:- isolates with common amino acid substitutions of A46T in FljA and R140L in Hin. The introduction of these mutations into S. Typhimurium isolates led to the loss of selectability of isolates expressing the phase 2 H antigen. These data suggested that a point mutation was the genetic basis, at least in part, of the S. 4,[5],12:i:- isolates. The results of phenotypic analysis suggested that the S. 4,[5],12:i:- isolates in Japan consist of multiple distinct clones. This is the first detailed characterization of the S. 4,[5],12:i:- isolates derived from various sources across Japan.

Pulsed-field gel electrophoresis profile changes resulting from spontaneous chromosomal deletions in enterohemorrhagic Escherichia coli O157:H7 during passage in cattle

ABSTRACT A total of 905 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates that were recovered from experimentally infected cattle, in addition to the inoculated strain, were analyzed by pulsed-field gel electrophoresis (PFGE). Twelve PFGE profiles other than that of the inoculated strain were observed. We successfully identified five distinct chromosomal deletions that affected the PFGE profiles using whole-genome PCR scanning and DNA sequencing analysis. The changes in PFGE profiles of EHEC O157:H7 isolates after passage through the intestinal tract of cattle were partially generated by deletion of chromosomal regions.

Lineage-specific distribution of insertion sequence excision enhancer in enterotoxigenic Escherichia coli isolated from swine.

ABSTRACT Insertion sequences (ISs) are the simplest transposable elements and are widely distributed in bacteria; however, they also play important roles in genome evolution. We recently identified a protein called IS excision enhancer (IEE) in enterohemorrhagic Escherichia coli (EHEC) O157. IEE promotes the excision of IS elements belonging to the IS3 family, such as IS629, as well as several other families. IEE-mediated IS excision generates various genomic deletions that lead to the diversification of the bacterial genome. IEE has been found in a broad range of bacterial species; however, among sequenced E. coli strains, IEE is primarily found in EHEC isolates. In this study, we investigated non-EHEC pathogenic E. coli strains isolated from domestic animals and found that IEE is distributed in specific lineages of enterotoxigenic E. coli (ETEC) strains of serotypes O139 or O149 isolated from swine. The iee gene is located within integrative elements that are similar to SpLE1 of EHEC O157. All iee-positive ETEC lineages also contained multiple copies of IS629, a preferred substrate of IEE, and their genomic locations varied significantly between strains, as observed in O157. These data suggest that IEE may have been transferred among EHEC and ETEC in swine via SpLE1 or SpLE1-like integrative elements. In addition, IS629 is actively moving in the ETEC O139 and O149 genomes and, as in EHEC O157, is promoting the diversification of these genomes in combination with IEE.

Emergence of a Multidrug-Resistant Shiga Toxin-Producing Enterotoxigenic Escherichia coli Lineage in Diseased Swine in Japan

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing E. coli (STEC) are important causes of diarrhea and edema disease in swine. The majority of swine-pathogenic E. coli strains belong to a limited range of O serogroups, including O8, O138, O139, O141, O147, O149, and O157, which are the most frequently reported strains worldwide. However, the circumstances of ETEC and STEC infections in Japan remain unknown; there have been few reports on the prevalence or characterization of swine-pathogenic E. coli. In the present study, we determined the O serogroups of 967 E. coli isolates collected between 1991 and 2014 from diseased swine in Japan, and we found that O139, O149, O116, and OSB9 (O serogroup of Shigella boydii type 9) were the predominant serogroups. We further analyzed these four O serogroups using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing, and virulence factor profiling. Most of the O139 and O149 strains formed serogroup-specific PFGE clusters (clusters I and II, respectively), whereas the O116 and OSB9 strains were grouped together in the same cluster (cluster III). All of the cluster III strains belonged to a single sequence type (ST88) and carried genes encoding both enterotoxin and Shiga toxin. This PFGE cluster III/ST88 lineage exhibited a high level of multidrug resistance (to a median of 10 antimicrobials). Notably, these bacteria were resistant to fluoroquinolones. Thus, this lineage should be considered a significant risk to animal production due to the toxigenicity and antimicrobial resistance of these bacteria.

Peptidoglycan Acetylation of Campylobacter jejuni Is Essential for Maintaining Cell Wall Integrity and Colonization in Chicken Intestines

ABSTRACT Peptidoglycan (PG) acetylation of Gram-positive bacteria confers lysozyme resistance and contributes to survival in the host. However, the importance of PG acetylation in Gram-negative bacteria has not been fully elucidated. The genes encoding putative PG acetyltransferase A (PatA) and B (PatB) are highly conserved in Campylobacter jejuni, the predominant cause of bacterial diarrhea worldwide. To evaluate the importance of PatA and PatB of C. jejuni, we constructed patA and patB isogenic mutants and compared their phenotypes with those of the parental strains. Although transmission electron microscopy did not reveal morphological changes, both mutants exhibited decreased motility and biofilm formation in vitro. The extent of acetylation of the PG purified from the patA and patB mutants was significantly lower than the PG acetylation in the parental strains. Both mutants exhibited decreased lysozyme resistance and intracellular survival in macrophage cells. In a chick colonization experiment, significant colonization deficiency was observed for both mutants. These results suggest that PatA and PatB of C. jejuni play important roles in maintaining cell wall integrity by catalyzing PG O-acetylation and that the loss of these enzymes causes decreased motility and biofilm formation, thus leading to colonization deficiency in chicken infection. IMPORTANCE The importance of peptidoglycan (PG) acetylation in Gram-negative bacteria has not been fully elucidated. The genes encoding putative PG acetyltransferase A (PatA) and B (PatB) are highly conserved in Campylobacter jejuni, the predominant cause of bacterial diarrhea worldwide. We evaluated the importance of these enzymes using isogenic mutants. The results of this study suggest that PatA and PatB of C. jejuni play important roles in maintaining cell wall integrity. The loss of these factors caused multiple phenotypic changes, leading to colonization deficiency in chicken infection. These data should be useful in developing novel control measures to prevent chicken colonization by C. jejuni. Inhibitors of the PG acetylation enzymes PatA and PatB might serve as potent anti-C. jejuni agents.