Tryptanthrin Reduces Campylobacter jejuni Colonization in the Chicken Gut by a Bactericidal Mechanism.

Campylobacter jejuni is a leading cause of foodborne bacterial gastroenteritis worldwide, and raw or undercooked chicken meat is considered the major source of human campylobacteriosis. In this study, we identified 36 compounds that showed inhibitory effects on C. jejuni growth at low concentrations by screening a chemical compound library. Three of the 36 compounds were herbal compounds, including tryptanthrin (TRP), an indoloquinazoline alkaloid. TRP has been reported to have a variety of biological properties, such as antimicrobial, anti-inflammatory, and antitumor activities, but there was previously no information about its anti-C. jejuni activity. We further conducted in vitro and in vivo experiments to evaluate the potential of TRP for the control of C. jejuni in chicken farms. The MIC of TRP for C. jejuni was much lower than that of 13 other herbal compounds that were previously reported to have anti-C. jejuni activities. Time-kill assays under growing and nongrowing conditions demonstrated that TRP has bactericidal activity against C. jejuni. In addition, TRP showed a narrow-spectrum antimicrobial effect against C. jejuni, and there was little potential for the development of TRP-resistant C. jejuni during serially passaged culture. In chick infection experiments, the administration of TRP in drinking water significantly reduced the cecal colonization of C. jejuni when TRP was used either before or after C. jejuni infection. These data suggest that TRP is effective for the control of C. jejuni in chicken farms. IMPORTANCE Campylobacter is a widespread pathogen in the food chain of chickens. Once chickens become infected, large numbers of Campylobacter cells are excreted in their feces. The development of an effective material for reducing the amount of Campylobacter in the chicken intestinal tract will make it possible to reduce the contamination of the food chain with Campylobacter and to produce safe and secure chicken meat. In the present study, in vivo experiments revealed that the use of an herbal compound, tryptanthrin, significantly reduced the number of Campylobacter cells in the chicken gut by a bactericidal mechanism. Furthermore, our in vitro experiments demonstrated that, compared with the other herbal compounds, tryptanthrin achieved antimicrobial activity against C. jejuni at the lowest concentration. The use of tryptanthrin may lead to the development of a novel control measure for reducing the colonization of C. jejuni in the food chain.

Influence of SOS-inducing agents on the expression of ArtAB toxin gene in Salmonella enterica and Salmonella bongori.

Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) definitive phage type 104 (DT104), S. enterica subspecies enterica serovar Worthington (S. Worthington) and S. bongori produce ArtA and ArtB (ArtAB) toxin homologues, which catalyse ADP-ribosylation of pertussis toxin-sensitive G protein. ArtAB gene (artAB) is encoded on prophage in DT104 and its expression is induced by mitomycin C (MTC) and hydrogen peroxide (H2O2) that trigger the bacterial SOS response. Although the genetic regulatory mechanism associated with artAB expression is not characterized, it is thought to be associated with prophage induction, which occurs when the RecA-mediated SOS response is triggered. Here we show that subinhibitory concentration of quinolone antibiotics that are SOS-inducing agents, also induce ArtAB production in these Salmonella strains. Both MTC and fluoroquinolone antibiotics such as enrofloxacin-induced artA and recA transcription and artAB-encoding prophage (ArtAB-prophage) in DT104 and S. Worthington. However, in S. bongori, which harbours artAB genes on incomplete prophage, artA transcription was induced by MTC and enrofloxacin, but prophage induction was not observed. Taken together, these results suggest that SOS response followed by induction of artAB transcription is essential for ArtAB production. H2O2-mediated induction of ArtAB prophage and efficient production of ArtAB was observed in DT104 but not in S. Worthington and S. bongori. Therefore, induction of artAB expression with H2O2 is strain-specific, and the mode of action of H2O2 as an SOS-inducing agent might be different from those of MTC and quinolone antibiotics.

Salmonella Genomic Island 3 Is an Integrative and Conjugative Element and Contributes to Copper and Arsenic Tolerance of Salmonella enterica.

Salmonella genomic island 3 (SGI3) was first described as a chromosomal island in Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium. The SGI3 DNA sequence detected from Salmonella 4,[5],12:i:- isolated in Japan was identical to that of a previously reported one across entire length of 81 kb. SGI3 consists of 86 open reading frames, including a copper homeostasis and silver resistance island (CHASRI) and an arsenic tolerance operon, in addition to genes related to conjugative transfer and DNA replication or partitioning, suggesting that the island is a mobile genetic element. We successfully selected transconjugants that acquired SGI3 after filter-mating experiments using the S. enterica serovars Typhimurium, Heidelberg, Hadar, Newport, Cerro, and Thompson as recipients. Southern blot analysis using I-CeuI-digested genomic DNA demonstrated that SGI3 was integrated into a chromosomal fragment of the transconjugants. PCR and sequencing analysis demonstrated that SGI3 was inserted into the 3' end of the tRNA genes pheV or pheR The length of the target site was 52 or 55 bp, and a 55-bp attI sequence indicating generation of the circular form of SGI3 was also detected. The transconjugants had a higher MIC against CuSO4 compared to the recipient strains under anaerobic conditions. Tolerance was defined by the cus gene cluster in the CHASRI. The transconjugants also had distinctly higher MICs against Na2HAsO4 compared to recipient strains under aerobic conditions. These findings clearly demonstrate that SGI3 is an integrative and conjugative element and contributes to the copper and arsenic tolerance of S. enterica.

Transcriptomic analysis of Campylobacter jejuni grown in a medium containing serine as the main energy source.

Campylobacter jejuni is one of the most important causes of food-borne diseases in industrialized countries. Amino acids are an important nutrient source for this pathogen because it lacks enzymes related to glycolysis. However, the metabolic characteristics of C. jejuni grown in a nutrient-restricted medium with specific amino acids have not been fully elucidated. This study shows that C. jejuni NCTC 11168 grows well in a nutrient-restricted medium containing serine, aspartate, glutamate, and proline. Subtracting serine significantly reduced growth, but the removal of the three other amino acids did not, suggesting that serine is a priority among the four amino acids. A transcriptomic analysis of C. jejuni NCTC 11168 grown in a medium with serine as the main energy source was then performed. Serine seemed to be sensed by some chemoreceptors, and C. jejuni reached an adaptation stage with active growth in which the expression of flagellar assembly components was downregulated and the biosyntheses of multiple amino acids and nucleotide sugars were upregulated. These data suggest that C. jejuni NCTC 11168 requires serine as a nutrient.

Phylogenetic Characterization of Salmonella enterica Serovar Typhimurium and Its Monophasic Variant Isolated from Food Animals in Japan Revealed Replacement of Major Epidemic Clones in the Last 4 Decades.

Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) and its monophasic variant (Salmonella 4,[5],12:i:-) are the major causes of gastroenteritis in both humans and animals. Pulsed-field gel electrophoresis and multilocus variable-number tandem-repeat analysis have been used widely as subtyping methods for these pathogens in molecular epidemiological analyses, but the results do not precisely reflect phylogenetic information. In this study, we performed a phylogenetic analysis of these serovars using whole-genome sequencing data and identified nine distinct genotypic clades. Then, we established an allele-specific PCR-based genotyping method detecting a clade-specific single nucleotide polymorphism to rapidly identify the clade of each isolate. Among a total of 815 isolates obtained from cattle in Japan between 1977 and 2017, clades 1, 7, and 9 contained 77% of isolates. Obvious replacement of the dominant clone was observed five times in this period, and clade 9, which mostly contains Salmonella 4,[5],12:i:-, is currently dominant. Among 140 isolates obtained from swine in Japan between 1976 and 2017, clades 3 and 9 contained 64% of isolates. Clade 9 is the latest clone as is the case in cattle isolates. Clade 9 is similar to an epidemic clone from Europe, which is characterized by sequence type 34 (ST34), chromosomal Salmonella genomic island 3, and a composite transposon containing antimicrobial resistance genes. The increased prevalence of clade 9 among food animals in Japan might be a part of the pandemic of the European Salmonella 4,[5],12:i:- clone.

In vivo effect of a TLR5 SNP (C1205T) on Salmonella enterica serovar Typhimurium infection in weaned, specific pathogen-free Landrace piglets.

Toll-like receptor 5 is a pattern-recognition receptor for bacterial flagellin. We previously reported that a single nucleotide polymorphism (SNP) of swine TLR5, C1205T, impairs recognition of Salmonella typhimurium (ST) flagellin and ethanol-killed Salmonella Choleraesuis (SC). In the present study, weaned, specific pathogen-free (SPF) Landrace piglets with CC, CT or TT genotypes were orally infected with ST (L-3569 strain) to determine the effect of this specific SNP on ST infection in vivo. Eighteen ST-infected piglets (six each with CC, CT, or TT) exhibited fever and diarrhea for 1 week after infection. TT piglets had the longest duration of fever. TT piglets had the greatest mean diarrhea score during the experimental period, followed by CT and CC piglets. Fecal ST shedding was greater in CT and TT pigs than CC pigs from 2 days after infection. Serum haptoglobin concentration increased in ST-infected piglets and to greater extents in CT and TT pigs than CC pigs. Daily weight gain was lower in infected pigs, particularly TT piglets, than control pigs. To the best of our knowledge, this study is the first to demonstrate that impairment of TLR recognition affects pig susceptibility to disease in vivo. Thus, piglets with the T allele of swine TLR5 (C1205T) exhibit impaired resistance to ST infection. Furthermore, elimination of the T allele of this SNP from Landrace pigs would lead to enhancement of their resistance to ST infection.

Seasonal movement of trace-element discharge in a typical south-Indian suburban community.

Four conventional activated sludge sewage treatment plants (STPs) in Southern India were chosen to investigate the occurrence of major ions and trace metals in dissolved fraction of the wastewater. Samples were collected from inlet and outlet of STPs during the pre-monsoon and monsoon seasons. Except for a few elements (Mn, Mo and Ni), the concentrations of the rest increased at the outlet of STPs, suggesting that there is an addition of these elements during the treatment process. The increase in concentrations at the STP outlet ranged from 1.25 times for Mn to 3,254 times for Ag during the pre-monsoon. In the monsoon, the increase ranged from 1.75 (Fe) to 1,510 (Ag). This suggests that there is a substantial anthropogenic input of these elements as they pass through the treatment process. Removal rates of elements in STPs also varied. 59% of Mn was removed during the treatment process during pre-monsoon as compared to 67% removal during monsoon.

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.

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.

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

EnterotoxigenicEscherichia coli(ETEC) and Shiga toxin-producingE. coli(STEC) are important causes of diarrhea and edema disease in swine. The majority of swine-pathogenicE. colistrains 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-pathogenicE. coli In the present study, we determined the O serogroups of 967E. coliisolates collected between 1991 and 2014 from diseased swine in Japan, and we found that O139, O149, O116, and OSB9 (O serogroup ofShigella boydiitype 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.

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.

Spatio-temporal distribution, source, and genotoxic potential of polycyclic aromatic hydrocarbons in estuarine and riverine sediments from southern India.

In order to categorize the distribution, source, and effects of polycyclic aromatic hydrocarbons (PAHs) in aquatic systems of southern India, chemical and toxicological analyses were performed on surface and core sediments, collected from Adyar river, Cooum river, Ennore estuary, and Pulicat lake near Chennai city. The total PAH concentration in surface sediment ranged from 13 to 31,425ng/g with a mean value of 4320ng/g; the concentration was markedly higher in Cooum river compared to that at other sites. The historical PAH dissemination in core samples in the Cooum river, Ennore estuary, and Pulicat lake ranged from 30 to 31,425ng/g, from 8.6 to 910ng/g, and from 62 to 546ng/g, respectively. Surface sediments were predominantly contaminated with low molecular weight (LMW) PAHs. Historical profiles suggest that PAH contamination in the area is now greater than it had been in the past. PAH accumulation in Pulicat lake was distinct from that at other locations where high molecular weight (HMW) PAHs were predominant. DNA damage in HepG2 cells treated with sediment extracts from different locations showed a good correlation with their respective total PAH levels. Statistical analysis revealed that 3-ring and 4-ring PAHs may synergistically contribute to the genotoxic potency compared to others in sediments. The study also showed that a majority of PAHs in the study area indicated a petrogenic origin. Based on the enrichment and toxicological assessment of PAHs in sediments, Cooum river was shown to suffer the highest biological impairment among the studied water bodies.

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.

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

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.

Occurrence and the potential ecological risk of veterinary antimicrobials in swine farm wastewaters in Japan: Seasonal changes, relation to purchased quantity and after termination of oxytetracycline usage.

This study provides the first comprehensive investigation of the residual concentrations of eight classes of antimicrobial agents (AMs, 20 compounds) in 13 swine wastewater treatment facilities in Japan. These facilities implemented the aerobic activated sludge (AS) or its alternative methods. The maximum concentrations before treatment were found at the level of 7100, 6900, 6000, 3600, 3400, and 1400 µg/L for tilmicosin, oxytetracycline (OTC), chlortetracycline, lincomycin , sulfamethoxazole, and trimethoprim, respectively. The highest detection rate (96.3%) in influents was noted for the morantel, which was a feed additive. The seasonal difference in residual concentration was much greater for tetracyclines (TCs) and macrolides (MLs) when their residual concentrations were high, especially in the cold season. There was a positive correlation between the purchased quantity of TCs and fluoroquinolones (FQs) and their residue levels detected in the effluents (p < 0.01). The estimated removal rate of AMs was greater than 80%. In contrast, on a few occasions, it was diminished due to failing operating conditions, such as water temperature and AS rate in the aeration tank. The estimated ecological risks of AMs in effluents based on risk quotients (RQs) considered to enhance the selection pressure for drug resistance (RQs-AMR) were high for TCs and FQs, whereas ecotoxicological effects (RQs-ENV) to aquatic organisms were higher for sulfonamides and MLs. When OTC usage ceased, its concentration in wastewater decreased rapidly; however, it remained longer period in the effluents, probably due to OTC desorption from the AS. The concentrations (and respective RQs) of TCs were decreased by >99.8% and >92% in the influents and effluents, respectively. This data suggested that it is essential to reduce the amount used and introduce more efficient methods and operating conditions to constantly remove AMs during the treatment to reduce the risk of AM discharge from swine farms.

Characteristics of antimicrobial residues in manure composts from swine farms: Residual patterns, removal efficiencies, and relation to purchased quantities and composting methods in Japan.

Present study provides first comprehensive results on the residual levels of 19 antimicrobial (AM) residues in 12 Japanese swine manure composting facilities that use open or enclosed types of treatment methods. Tilmicosin (14000 µg/kg d.w.) and tiamulin (15000 µg/kg d.w.) were present in the highest concentrations in manure composts. Morantel (MRT) had the highest detection frequency (100%) in compost, suggesting its ubiquitous usage and resistance to degradation during composting. Sulfamethoxazole had low detection frequencies and concentrations, likely due to limited partitioning to the solid phase. A positive correlation (p < 0.05) between purchasing quantities and residue levels in manure composts was detected for fluoroquinolones (FQs). The removal efficiencies of AMs in enclosed-type facilities were lower and more inconsistent than those in open-type facilities. Tetracyclines (TCs), lincomycin, and trimethoprim were easily removed from open-type facilities, whereas FQs and MRT persisted in both facilities. After discontinuing the usage of oxytetracycline (OTC), TCs concentrations reduced drastically in input materials, remained pseudo-persistent in composts for up to 4 months, suggesting a time lag for composting and were not detected (<10 µg/kg) after 4 months of OTC withdrawal. This study emphasizes on the effectiveness of manure composting methods in reducing AM residues in swine waste.

Nationwide analysis of antimicrobial resistance in pathogenic Escherichia coli strains isolated from diseased swine over 29 years in Japan.

Pathogenic Escherichia coli strains are important causes of several swine diseases that result in significant economic losses worldwide. In Japan, the use of antimicrobials in swine is much higher than that in other farm animals every year. Antimicrobial resistance in pathogenic E. coli strains also heavily impacts the swine industry due to the limited treatment options and an increase in the potential risk of the One Health crisis. In 2016, we investigated 684 Japanese isolates of swine pathogenic E. coli belonging to four major serogroups and reported the emergence and increase in the highly multidrug-resistant serogroups O116 and OSB9 and the appearance of colistin-resistant strains. In the present study, by expanding our previous analysis, we determined the serotypes and antimicrobial resistance of 1,708 E. coli strains isolated from diseased swine between 1991 and 2019 in Japan and found recent increases in the prevalences of multidrug-resistant strains and minor serogroup strains. Among the antimicrobials examined in this study that have been approved for animal use, a third-generation cephalosporin was found to be effective against the most isolates (resistance rate: 1.2%) but not against highly multidrug-resistant strains. We also analyzed the susceptibilities of the 1,708 isolates to apramycin and bicozamycin, both which are available for treating swine in Japan, and found that the rates of resistance to apramycin and bicozamycin were low (6.7% and 5.8%, respectively), and both antimicrobials are more effective (resistance rates: 2.7% and 5.4%, respectively) than third-generation cephalosporins (resistance rate: 16.2%) against highly multidrug-resistant strains.

Characterization of Salmonella enterica serovar Typhimurium isolates harboring a chromosomally encoded CMY-2 beta-lactamase gene located on a multidrug resistance genomic island.

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 ß-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 ß-lactamase gene (bla(CMY-2)) was integrated in a 2.5-Mb chromosomal fragment. Genetic analysis of S. Typhimurium isolate L-3553 indicated that bla(CMY-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 bla(CMY-2) in the bla(CMY-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.

Molecular epidemiology of Salmonella enterica serovar typhimurium isolates from cattle in hokkaido, Japan: evidence of clonal replacement and characterization of the disseminated clone.

The molecular epidemiology of 545 Salmonella enterica serovar Typhimurium isolates collected between 1977 and 2009 from cattle in Hokkaido, Japan, was investigated using pulsed-field gel electrophoresis (PFGE). Nine main clusters were identified from 116 PFGE patterns. Cluster I comprised 248 isolates, 243 of which possessed a sequence specific to definitive phage type 104 (DT104) or U302. The cluster I isolates were dominant in 1993 to 2003, but their numbers declined beginning in 2004. Beginning in 2002, an increase was observed in the number of cluster VII isolates, consisting of 21 PFGE patterns comprising 165 isolates. A total of 116 isolates representative of the 116 PFGE profiles were analyzed by multilocus variable-number tandem-repeat analysis (MLVA). Other than two drug-sensitive isolates, 19 isolates within cluster VII were classified in the same cluster by MLVA. Among the cluster VII isolates, an antibiotic resistance type showing resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, kanamycin, cefazolin, and sulfamethoxazole-trimethoprim and a resistance type showing resistance to ampicillin, streptomycin, sulfonamides, tetracycline, and kanamycin were found in 23 and 125 isolates, respectively. In the 19 isolates representative of cluster VII, the bla(TEM-1) gene was found on a Salmonella serotype Typhimurium virulence plasmid, which was transferred to Escherichia coli by electroporation along with resistance to two to four other antimicrobials. Genomic analysis by subtractive hybridization and plasmid analysis suggested that the bla(TEM-1)-carrying virulence plasmid has a mosaic structure composed of elements of different origin. These results indicate an emerging multidrug-resistant S. Typhimurium clone carrying a virulence-resistance plasmid among cattle in Hokkaido, Japan.

Interaction between PKD1L3 and PKD2L1 through their transmembrane domains is required for localization of PKD2L1 at taste pores in taste cells of circumvallate and foliate papillae.

The polycystic kidney disease 1-like 3 (PKD1L3) and polycystic kidney disease 2-like 1 (PKD2L1) proteins have been proposed to form heteromers that function as sour taste receptors in mammals. Here, we show that PKD1L3 and PKD2L1 interact through their transmembrane domains, and not through the coiled-coil domain, by coimmunoprecipitation experiments using a series of deletion mutants. Deletion mutants lacking the critical interaction region were not transported to the cell surface and remained in the cytoplasm, whereas PKD1L3 and PKD2L1 proteins were expressed at the cell surface when both are transfected. Calcium imaging analysis revealed that neither the coiled-coil domain nor the EF-hand domain located in the C-terminal cytoplasmic tail of PKD2L1 was required for response on stimulation with an acidic solution. Finally, PKD2L1 did not localize to the taste pore but was distributed throughout the cytoplasm in taste cells of circumvallate and foliate papillae in PKD1L3(-/-) mice, whereas it localized to the taste pore in wild-type mice. Collectively, these results suggest that the interaction between PKD1L3 and PKD2L1 through their transmembrane domains is essential for proper trafficking of the channels to the cell surface in taste cells of circumvallate and foliate papillae and in cultured cells.