IncA/C plasmid encoding blaCTX-M-55 in non-O1 Vibrio cholerae isolates from the edible river fish Mastacembelus sp.

Extended-spectrum ß-lactamase-producing non-O1 Vibrio cholerae was isolated from edible Mastacembelus sp. in Vietnam. The genome sequence was sequenced using DNBSEQ-G400 and MinION Mk1b. A plasmid of approximately 183-kb encoding blaCTX-M-55 and blaTEM-1 was detected.

Detection of chromosome-mediated blaNDM-1-carrying Aeromonas spp. in the intestinal contents of fresh water river fish in Ho Chi Minh City, Vietnam.

The spread of antibiotic-resistant bacteria is a global problem that should be addressed through the perspective of the "one health" concept. The purpose of this study was to determine the contamination rate of antibiotic-resistant Aeromonas spp. in fresh water river fish purchased from a fish market in Vietnam. We then defined the pattern of antibiotic resistance to assess antibiotic-resistant contamination. Antibiotic-resistant Aeromonas spp. were detected in the intestinal contents of 32 of 80 fish. blaNDM-1 was detected in seven strains. Extended-spectrum ß-lactamase and AmpC ß-lactamase-related genes were detected in 28 strains, including blaCTX-M-55, blaCTX-M-15, blaCTX-M-1, and blaDHA,blaFOX, and blaMOX. The blaNDM-1 detected in the seven Aeromonas spp. strains were found chromosomally. This finding suggests that the blaNDM gene is stable in the natural environment and may spread widely into animals and humans via Aeromonas spp. with a transposon. Our results suggest the importance of continuing to monitor carbapenemase genes in Aeromonas spp. to evaluate the possibility that they may spread in other Enterobacterales, and to elucidate the mechanism of spread.

IncHI2 Plasmid Encoding blaCTX-M-55 and mcr-1.1 in Salmonella enterica SE20-C72-2 and Escherichia coli EC20-C72-1 Isolates from the Edible River Fish Anabas testudineus.

Salmonella enterica SE20-C72-2 and Escherichia coli EC20-C72-1 were isolated from the edible fish Anabas testudineus in Vietnam. The chromosomes and plasmids from both strains were sequenced using Oxford Nanopore and Illumina sequencing. Plasmids approximately 250 kbp long, encoding blaCTX-M-55 and mcr-1.1, were detected in both strains.

ESBL-producing Vibrio vulnificus and V. alginolyticus harbour a plasmid encoding ISEc9 upstream of blaCTX-M-55 and qnrS2 isolated from imported seafood.

In recent years, trade liberalisation has led to the spread of antibiotic-resistant bacteria (ARB) in food products. Because ARB has reportedly been found in imported foods, the spread of plasmid-mediated ARB through food products is a concern. Here, we report the complete genome sequences of ESBL-producing Vibrio vulnificus and V. alginolyticus strains harbouring a plasmid isolated from imported seafood. First, V. vulnificus and V. alginolyticus were isolated from purchased frozen and thawed Litopenaeus vannamei shrimp, and genome extraction and sequencing were performed. Hybrid genome assemblies were performed using Unicycler and annotated using DFAST. Then genome analysis was performed using BRIG. Plasmid comparisons showed that the plasmids carried by both Vibrios are remarkably similar and encode the same antibiotic-resistance genes. The 270-310 kb region specific to both Vibrios were isolated in this study and encodes the antibiotic-resistance genes blaCTX-M and qnr. Furthermore, the mobile genetic factors ISEc9, ISVch4, and ISVpa4 are located upstream and downstream of these genes. This is the first report of ESBL-producing V. vulnificus and V. alginolyticus harbouring a common plasmid encoding ISEc9 upstream of blaCTX-M-55 and qnrS2 isolated from imported seafood.

Carbapenem-Resistant Citrobacter freundii and Escherichia coli Harboring a Common IncC-Type Plasmid Encoding IS26 Upstream of blaNDM-1, sul1, aph(3')-VI, and qacE Isolated from Edible Mastacembelidae Fish.

Carbapenem-resistant Citrobacter freundii CF20-4P-1 and Escherichia coli EC20-4B-2 were isolated from edible Mastacembelidae in Vietnam. We present the draft genome sequences, and the complete plasmid genome sequencing was also performed by hybrid assembly sequencing of Oxford Nanopore and Illumina. The 137-kbp plasmid encoding the assembled blaNDM-1 was detected in both strains.

Frequent contamination of edible freshwater fish with colistin-resistant Escherichia coli harbouring the plasmid-mediated mcr-1 gene.

The threat of antimicrobial resistance is increasing. Microbial food contamination poses a serious public health risk; however, there are only a few studies on the prevalence of colistin-resistant Escherichia coli (COL-E) contamination in freshwater fish. This study aimed to characterise the antibiotic resistance genes and antibiotic susceptibility profiles of COL-E in freshwater fish in Vietnam. In total, 103 fish were collected and 63 COL-E were isolated. COL-E was investigated by genotyping mcr and AmpC/extended-spectrum ß-lactamase (ESBL)-related genes. The results show that COL-E and AmpC/ESBL-producing COL-E were confirmed in 24.3 % and 14.6 % of the fish, respectively. Multiplex PCR for mcr-1-9 showed that all 63 COL-E harboured mcr-1, while mcr-3 was detected in 7.9 % of COL-E. The minimum inhibitory concentration of colistin ranged from 2 to 256 µg/mL. Meanwhile, antibiotic susceptibility results show that all COL-E were resistant to ampicillin, streptomycin, and chloramphenicol.

Genome Sequence of Carbapenemase-Producing Enterobacter cloacae 0102-4P-1 Harboring the IncC-Type Plasmid with a Multidrug Resistance Site Encoding blaNDM-1, Isolated from Commercially Imported Shrimp.

A carbapenem-resistant Enterobacter cloacae 0102-4P-1 strain was isolated from commercially imported shrimp in Japan. Here, we present a draft genome sequence. The complete plasmid sequence was also determined by hybrid assembly sequencing using Oxford Nanopore and Illumina methods. The assembled whole genome and plasmid were 5,164,033 bp and 162,852 bp long, respectively.

Abundance of colistin-resistant Escherichia coli harbouring mcr-1 and extended-spectrum ß-lactamase-producing E. coli co-harbouring blaCTX-M-55 or -65 with blaTEM isolates from chicken meat in Vietnam.

Although the spread of plasmid-mediated antibiotic-resistant bacteria is a public health concern, food contamination with plasmid-mediated antibiotic-resistant Escherichia coli in Vietnam has not been well investigated. This study aimed to describe the prevalence of colistin-resistant, carbapenem-resistant, and endemic blaCTX-M in extended-spectrum ß-lactamase (ESBL) producing E. coli isolates. Colistin and carbapenem-resistant ESBL-producing E. coli were isolated from chickens in Vietnam and Japan. Colistin-resistant and AmpC/ESBL-producing E. coli (52% and 93%, respectively) were detected in chickens from Vietnam, in comparison to 52.7%, AmpC/ESBL-producing E. coli found in chicken from Japan. Carbapenem-resistant E. coli has not been isolated in Vietnam and Japan. Genotyping revealed that colistin-resistant E. coli harboured mcr-1, and most of the AmpC/ESBL-related genes were blaCTX-M-55 and blaCTX-M-65 together with blaTEM in Vietnamese chickens and blaCMY-2 in Japanese chickens. Multi-drug resistance analysis showed that ESBL-producing E. coli isolates had greater resistance to quinolones, streptomycin, and chloramphenicol than colistin-resistant E. coli isolates from Vietnam, suggesting the selection of multiple antibiotic resistance genes in ESBL-producing E. coli. In conclusion, colistin-resistant E. coli was detected in approximately half of the chicken samples, the majority of which harboured mcr-1. The high prevalence of ESBL-producing E. coli has remained constant in the last 5 years. The predominant blaCTX-M in ESBL-producing E. coli was blaCTX-M-55 or blaCTX-M-65, with the coexistence of blaTEM in Vietnam. These results can be implemented in monitoring systems to overcome the development of antimicrobial resistance.

Untargeted Phylogenetic Group III of Multi-drug-Resistant Bacillus cereus Isolated Using Fraser Medium from Retail Chickens in Ho Chi Minh City.

The prevalence of food-borne bacteria in developing countries is less well understood than in developed countries. The ISO11290-1 isolation method is commonly used to study Listeria contamination in chicken; however, all isolates are identified as untargeted Bacillus cereus. This study aimed to determine the classification, antibiotic susceptibility, and virulence genes of B. cereus isolated from retail chickens in Vietnam. Bacterial isolation using the ISO11290-1 method yielded 12 strains of B. cereus from seven out of 60 chickens. For determining bacterial diversity, panC and multilocus sequence typing (MLST) analyses were performed. PanC analysis showed that all seven strains belong to the phylogenetic group III, to which the highest risk of foodborne illnesses was associated. MLST analysis showed that most strains contained a ST205 complex; further, all strains were found to be resistant to ampicillin, ciprofloxacin, and tetracycline. Virulence genes were also investigated. ces, a cereulide-related gene, was detected in 50% of the isolated strains, followed by cytK, nheA, and hblA enterotoxins in 41.7%, 16.7%, and 25% of the strains, respectively. In conclusion, B. cereus may be erroneously detected when attempting to detect Listeria in food using the ISO11290-1 method. Further study of the prevalence of B. cereus in Vietnamese food is needed to improve food safety.

Prevalence and mechanisms of fluoroquinolone-resistant Escherichia coli among sheltered companion animals.

To better understand the prevalence of fluoroquinolone-resistant Escherichia coli among sheltered companion animals, we conducted a screening study of 38 dogs and 78 cats and investigated the resistance mechanisms and characteristics of the isolates. Fluoroquinolone-resistant E. coli was detected in 18 dogs (47.4 %) and 14 cats (17.9 %). The isolates carried one to four mutations in the gyrA, parC and parE genes of the quinolone resistance-determining region, and the number of mutations was proportional to the MIC for ciprofloxacin. For plasmid-mediated quinolone resistance, aac-(6')-Ib-cr was detected in nine isolates, qnrS in five isolates and qnrB in one isolate. A relationship between the presence of these genes and MIC for ciprofloxacin was not apparent. Statistical analysis indicated that fluoroquinolone-resistant E. coli was widely distributed among sheltered companion animals with various attributes. This may relate to the wide dissemination of fluoroquinolone resistance among humans and other animals in Japan.

Collagen adhesion gene is associated with bloodstream infections caused by methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) causes hospital- and community-acquired infections. It is not clear whether genetic characteristics of the bacteria contribute to disease pathogenesis in MRSA infection. We hypothesized that whole genome analysis of MRSA strains could reveal the key gene loci and/or the gene mutations that affect clinical manifestations of MRSA infection. METHODS: Whole genome sequences (WGS) of MRSA of 154 strains were analyzed with respect to clinical manifestations and data. Further, we evaluated the association between clinical manifestations in MRSA infection and genomic information. RESULTS: WGS revealed gene mutations that correlated with clinical manifestations of MRSA infection. Moreover, 12 mutations were selected as important mutations by Random Forest analysis. Cluster analysis revealed strains associated with a high frequency of bloodstream infection (BSI). Twenty seven out of 34 strains in this cluster caused BSI. These strains were all positive for collagen adhesion gene (cna) and have mutations in the locus, those were selected by Random Forest analysis. Univariate and multivariate analysis revealed that these gene mutations were the predictor for the incidence of BSI. Interestingly, mutant CNA protein showed lower attachment ability to collagen, suggesting that the mutant protein might contribute to the dissemination of bacteria. CONCLUSIONS: These findings suggest that the bacterial genotype affects the clinical characteristics of MRSA infection.

Prevalence and genetic characterization of cephalosporin-resistant Enterobacteriaceae among dogs and cats in an animal shelter.

PURPOSE: The prevalence of antimicrobial-resistant bacteria, especially cephalosporin-resistant Enterobacteriaceae, is a major concern for human and animal health. We investigated the prevalence of cephalosporin-resistant Enterobacteriaceae among sheltered dogs and cats with various backgrounds. METHOD: Faecal samples or rectal swabs were collected from 151 dogs and 182 cats, and screened for the presence of antimicrobial-resistant bacteria. Isolates were characterized phenotypically and genotypically by pulsed-field gel electrophoresis, multi-locus sequence typing and phylogenetic grouping. The animal attributes related to bacterial carriage were statistically analysed. RESULTS: Cephalosporin-resistant Enterobacteriaceae was detected in 22 dogs (14.6%) and 20 cats (11.0%): 21 were extended-spectrum ß-lactamase (ESBL)-producing, 20 were AmpC-producing, and 1 was both ESBL- and AmpC-producing. Their ß-lactamase genes were varied and associated with humans, animals or other origins. The genes CTX-M-14 (n=9) and CMY-2 (n=9) were dominant, but CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-15, CTX-M-24, CTX-M-27, CTX-M-55 and DHA-1 genes were also detected. Genotyping of isolates revealed that ß-lactamase-producing Enterobacteriaceae had high genetic diversity. Relationships between animals harbouring cephalosporin-resistant Enterobacteriaceae and individual attributes, such as sex and nutrition type, were detected, but there was no correlation between history of human association and the presence of the bacterium in either dogs or cats. CONCLUSION: We found several types of cephalosporin-resistant Enterobacteriaceae distributed among companion animals with a range of individual attributes and histories in Osaka, Japan. Companion animals may play a bridging role in the circulation of antimicrobial-resistant bacteria from humans and from other origins.

Longitudinal genotyping surveillance of Mycobacterium tuberculosis in an area with high tuberculosis incidence shows high transmission rate of the modern Beijing subfamily in Japan.

Tuberculosis (TB) is a severe and wide-spread infectious disease worldwide. The modern Beijing subfamily, one lineage of M. tuberculosis, reportedly has high pathogenicity and transmissibility. This study used a molecular epidemiological approach to investigate the transmissibility of the modern Beijing subfamily in the Airin area of Osaka City, Japan. During 2006-2016, we collected 596 M. tuberculosis clinical isolates in the Airin area, Osaka city, Japan. We analyzed the 24-locus variable number of tandem repeats typing optimized for the Beijing family of isolates, M. tuberculosis lineage, and patient epidemiological data. The proportion of the modern Beijing subfamily was significantly higher not only than previously obtained data for the Airin area: it was also higher than the nationwide in Japan. The rate of recent clusters, defined as a variable number of tandem repeats profile identified within two years, of the modern Beijing subfamily was significantly higher than that the rate of recent clusters of the ancient Beijing subfamily. Results suggest that TB control measures formulated with attention to the modern Beijing subfamily might be an important benchmark to understanding recent TB transmission in the area.

Molecular and epidemiological characterization of staphylococcal foodborne outbreak of Staphylococcus aureus harboring seg, sei, sem, sen, seo, and selu genes without production of classical enterotoxins.

Staphylococcal food poisoning is the result of consumption of food contaminated with staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus. To date, 23 SEs and SE-like enterotoxins (SEls) have been described in the literature. They are divided into classical SEs (SEA-SEE) and new SE/SEls (SEG-SElX). Some have proved to be foodborne-inducible, but others remain unidentified. In May 2016, at an elderly group home in Osaka city, Japan, an outbreak from foodborne pathogens occurred among lunch party participants. Within 2h 30min to 4h 40min, 15 of 53 participants presented gastrointestinal symptoms of vomiting, diarrhea, and nausea. A subsequent laboratory investigation detected S. aureus from most stool samples from patients, several left-over food items, a kitchen swab, and hand swabs from two food handlers. Classical SEs was not detected from S. aureus isolates or left-over food items. From examination for the presence of SE/SEl genes of 20 kinds by PCR, seg, sei, sem, sen, seo, and selu genes were detected in almost all isolates. These isolates exhibited identical or closely related types by coagulase type (type VII), Sma I digested pulsed-field gel electrophoresis analysis and multi-locus sequence typing (MLST-CC45 lineage). These results suggest that the foodborne outbreak was caused by S. aureus harboring seg, sei, sem, sen, seo, and selu genes without production of classical SEs. Additionally, some S. aureus isolates from human nasal swabs and healthy human feces harboring seg, sei, sem, sen, seo, and selu genes without production of classical SEs were classified into CC45 lineage using MLST. These findings suggest new SE/SEls as a potential cause of foodborne outbreaks.

Contents of Ag and other metals in food-contact plastics with nanosilver or Ag ion and their migration into food simulants.

Six nanosilver-labelled products and five silver ion (Ag(+))-labelled products were investigated to measure the migration of Ag from food-contact plastics, including nanosilver into various food simulants. The products were obtained in Japanese markets in 2012. Zinc (Zn), another major antimicrobial agent, and three harmful metals, cadmium (Cd), lead (Pb) and arsenic (As), were also examined. Ag and Zn were detected in all six nanosilver products at concentrations of 21-200 and 8.4-140 mg kg(-1), respectively. These metals were also detected in all five Ag(+) products at the same level as nanosilver products. Cd, Pb and As were not detected in any sample. Migrations of Ag and Zn were highest in 4% acetic acid, but also observed in water and 20% ethanol. Big differences were not observed in the migration ratio between nanosilver products and Ag(+) products. The ultrafiltration experiments suggested that the Ag that migrated from nanosilver products into 4% acetic acid was in its ionic form, while that into water and 20% ethanol was in its nanoparticle form.

Phylogenetic assignment of Mycobacterium tuberculosis Beijing clinical isolates in Japan by maximum a posteriori estimation.

Intra-species phylogeny of Mycobacterium tuberculosis has been regarded as a clue to estimate its potential risk to develop drug-resistance and various epidemiological tendencies. Genotypic characterization of variable number of tandem repeats (VNTR), a standard tool to ascertain transmission routes, has been improving as a public health effort, but determining phylogenetic information from those efforts alone is difficult. We present a platform based on maximum a posteriori (MAP) estimation to estimate phylogenetic information for M. tuberculosis clinical isolates from individual profiles of VNTR types. This study used 1245 M. tuberculosis clinical isolates obtained throughout Japan for construction of an MAP estimation formula. Two MAP estimation formulae, classification of Beijing family and other lineages, and classification of five Beijing sublineages (ST11/26, STK, ST3, and ST25/19 belonging to the ancient Beijing subfamily and modern Beijing subfamily), were created based on 24 loci VNTR (24Beijing-VNTR) profiles and phylogenetic information of the isolates. Recursive estimation based on the formulae showed high concordance with their authentic phylogeny by multi-locus sequence typing (MLST) of the isolates. The formulae might further support phylogenetic estimation of the Beijing lineage M. tuberculosis from the VNTR genotype with various geographic backgrounds. These results suggest that MAP estimation can function as a reliable probabilistic process to append phylogenetic information to VNTR genotypes of M. tuberculosis independently, which might improve the usage of genotyping data for control, understanding, prevention, and treatment of TB.

Associations between co-detected respiratory viruses in children with acute respiratory infections.

Viruses are the major etiological agents of acute respiratory infections (ARIs) in young children. Although respiratory virus co-detections are common, analysis of combinations of co-detected viruses has never been conducted in Japan. Nineteen respiratory viruses or subtypes were surveyed using multiplex real-time PCR on 1,044 pediatric (patient age < 6 years) ARI specimens collected in Osaka City, Japan between January 2010 and December 2011. In total, 891 specimens (85.3%) were virus positive (1,414 viruses were detected), and 388 of the virus-positive specimens (43.5%, 388/891) were positive for multiple viruses. The ratio of multiple/total respiratory virus-positive specimens was high in children aged 0-35 months. Statistical analyses revealed that human bocavirus 1 and human adenovirus were synchronously co-detected. On the other hand, co-detections of human parainfluenza virus type 1 (HPIV-1) with HPIV-3, HPIV-3 with human metapneumovirus (hMPV), hMPV with respiratory syncytial virus A (RSV A), hMPV with influenza virus A (H1N1) 2009 (FLUA (H1N1) 2009), RSV A with RSV B, and human rhinovirus and FLUA (H1N1) 2009 were exclusive. These results suggest that young children (<3 years) are highly susceptible to respiratory viruses, and some combinations of viruses are synchronously or exclusively co-detected.

Distribution of Capnocytophaga canimorsus in dogs and cats with genetic characterization of isolates.

Capnocytophaga canimorsus, which is often found in the oral cavities of dogs and cats, is sometimes transmitted to humans, causing severe infection. To elucidate the risk of C. canimorsus in humans and animals, this study was undertaken to characterize this bacterium epidemiologically and genetically. We examined the distribution of C. canimorsus in dogs and cats, and analyzed the correlation between the presence of bacteria and individual factors statistically. We also compared C. canimorsus isolates genetically using 16S rRNA gene sequence analysis and pulsed-field gel electrophoresis (PFGE). C. canimorsus was detected in 76 of 109 dogs (69.7%) and 57 of 104 cats (54.8%). A relation between C. canimorsus presence and some individual factors was detected both in dogs and cats, but the predictive factors of carrying the bacterium differed between dogs and cats. 16S rRNA gene sequences from C. canimorsus isolates in this study were combined with previously published sequences to assess their intra-specific phylogeny. Results show that C. canimorsus is classifiable into two main groups (I and II) with differing γ-glutamyl aminopeptidase activity. Strains from human patients belonged unevenly to group I, possibility suggesting that group I can be transmitted to humans and group II is indigenous only to the oral cavities of dogs and cats. PFGE genotyping showed high discriminatory power, and the dendrogram accorded with genetic segregation between isolates of group I and II. Sma I-digest PFGE developed for this study is useful as a molecular typing method for additional epidemiological and phylogenetic studies of C. canimorsus.

Frequent respiratory viral infections in a young child in a 27-month follow-up study.

INTRODUCTION: Viruses are major aetiological agents of acute respiratory infection in young children. Although many studies have reported detection and analysis of respiratory viruses in sporadic cases, there have been few follow-up studies of individuals. The purpose of this study was to investigate the frequency of respiratory viral infections in a young child and to examine the duration of viral genome detection in clinical specimens. CASE PRESENTATION: A total of 284 nasal swabs were collected during symptomatic (196 specimens) and asymptomatic (88 specimens) periods of respiratory symptoms from a young female child (from 4 months to 31 months of age, who was admitted to a nursery school at 9 months). Multiplex real-time PCR for 19 respiratory viruses or subtypes was performed. One hundred and ninety-eight of the tested specimens were virus positive (69.7 %) (symptomatic periods, 149/196, 76.0 %; asymptomatic periods, 49/88, 55.7 %). Rhinovirus was the most frequently detected (26 times). Long durations of detection were observed for human coronavirus NL63 (30 days), rhinovirus (28 days) and human bocavirus 1 (22 days). CONCLUSION: Young children living in a group context have a high risk of respiratory virus infections, especially rhinovirus. In some instances, viral genomes were detectable for about 1 month by PCR.

[Effectiveness of comprehensive variable number of tandem repeat (VNTR) analysis in areas with a low incidence of tuberculosis].

PURPOSE: To ascertain the effectiveness of variable number of tandem repeat (VNTR) analysis in areas with a low incidence of tuberculosis (TB), we examined the combination of comprehensive VNTR analyses and field epidemiological investigation results in Yamagata Prefecture, Japan, where estimated incidence of new TB cases per 100,000 population was 11.3 in 2011. METHODS: We collected Mycobacterium tuberculosis isolates from 184 (69.2%) of 266 pulmonary TB patients across the whole of Yamagata Prefecture between 2009 and 2011. Next, 24 loci [JATA (12), QUB-11a, ETR A, QUB-18, QUB3232, v3820, v4120, MIRU04, MIRU16, MIRU40, ETR C, Mtub30, Mtub39] in VNTR genotypes were determined. The relationships among TB patients derived from the respective clusters were surveyed using field epidemiological investigation results provided by the Public Health Center. RESULTS: Seventeen clusters were formed by 49 (26.6%) of the 184 isolates. We found 3 hospital infection cases, 3 family infection cases, and 1 nursing home infection case forming 6 clusters. Among these cases, two relationships among patients were revealed after additional epidemiological investigation at the Public Health Center. The VNTR pattern of the largest cluster, which was formed by 12 isolates, was identical with that of an incipient patient of a TB mass infection that occurred in 2007. DISCUSSION: In areas with a low incidence of TB, a combination of comprehensive VNTR analysis and field epidemiological investigation is useful to find unknown transmission routes, identify for new risk groups, and trace mass infections.