Journal: Journal of Microbiological Methods (Elsevier)

Title: Accurate identification of Escherichia albertii by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Authors: Noritoshi Hatanaka, Sharda Prasad Awasthi, Atsushi Hinenoya, Osamu Ueda and Shinji Yamasaki*

Abstract: A specific identification protocol for Escherichia albertii by using a MALDI-TOF/MS method was developed. For this purpose, a novel database was established which can differentiate E. albertii from E. coli by combining the mass spectra obtained from 58 E. albertii and 36 E. coli strains.

Associate Professor M. Matsubayashi has been promoted to Professor in Laboratory of Veterinary Immunology, Graduate School of Life and Environmental Sciences, OPU.

Congratulations!!

Journal:   Emerging Infectious Diseases (CDC)

Title:  Prevalence of Escherichia albertii in raccoons (Procyon lotor) in Japan.

Authors:  Atsushi Hinenoya, Keigo Nagano, Sharda Prasad Awasthi, Noritoshi Hatanaka
and Shinji Yamasaki*

Abstract: Natural reservoirs of Escherichia albertii remain unclear. In this study, we detected E. albertii by PCR in 248 (57.7%) of 430 raccoons from Osaka, Japan, and isolated 143 E. albertii strains from the 62 PCR-positive samples. These data indicate that raccoons could be a natural reservoir of E. albertii in Japan.

Significance: We identified a potential natural reservoir of E. albertii.

Journal:  Diagnostic Microbiology and Infectious Disease (Elsevier)

Title:  Development of XRM-MacConkey agar selective medium for the isolation of Escherichia albertii.

Authors:  Atsushi Hinenoya, Keigo Nagano, Kentaro Okuno, Akira Nagita, Noritoshi Hatanaka, Sharda Prasad Awasthi and Shinji Yamasaki*

Abstract:  Escherichia albertii has increasingly been recognized as an emerging pathogen. However, lack of selective medium for E. albertii is the bottleneck for clinical and epidemiological investigations. In this study, a selective medium for E. albertii named XRM-MacConkey agar, which is modified MacConkey agar supplemented with xylose (X), rhamnose (R), and melibiose (M) instead of lactose, was developed and evaluated. All 49 E. albertii and 6 different species out of 23 grew as colorless colonies, whereas 17 remaining species grew as red colonies. Detection limit of E. albertii by this medium was 10⁵ CFU/g stool when examined with spiked healthy human stool. Furthermore, colorless colonies on XRM-MacConkey agar obtained from 7 E. albertii–positive diarrheal stools were consistently E. albertii. In contrast, 57%, 18%, and 36% colorless colonies on MacConkey, DHL, and mEA agars, respectively, were non–E. albertii. We concluded that XRM-MacConkey agar could specifically be used for isolation of E. albertii.

Conference: 54th Japan Panel Conference on Cholera and Other Bacterial Enteric Infections

Date: December 10th to 12th, 2019

Place: Alumnus Union Building of Osaka University (Icho Kaikan)

Presentation: Oral
　　　Phong Quoc Le  ‘Co-occurrence of mcr-1 and mcr-3 genes in ESBL-producing Escherichia coli isolated from retail raw food in Nha Trang, Vietnam’

Noritoshi Hatanaka ‘Chlorous acid is more bactericidal than sodium hypochlorite against
Campylobacter jejuni and Campylobacter coli‘

Atsushi Hinenoya ‘Development of detection, isolation and identification methods for Escherichia
albertii‘

Poster
　　　Rabee A. Ombarak (Lecturer at Faculty of Veterinary Medicine, Sadat City University, Egypt)

‘Occurrence of virulence genes associated with diarrheagenic and extraintestinal pathogenic Escherichia coli from bulk tank milk and dairy farm environment in Egypt’

Journal: Food Control (Elsevier)

Title: Chlorous acid is a more potent antibacterial agent than sodium hypochlorite against Campylobacter.

Authors: Noritoshi Hatanaka, Sharda Prasad Awasthi, Hisataka Goda, Hiroyuki Kawata, Yuzuru Uchino, Takahiro Kubo, Shigeru Aoki, Atsushi Hinenoya, Shinji Yamasaki*

Abstract:  Foodborne disease caused by campylobacters is one of the major global problems for food safety. Infection source of Campylobacter to human is mainly through contaminated meat particularly chicken. Contamination of meat with Campylobacter usually occurs during processing at slaughterhouse and to prevent such contaminations, sodium hypochlorite is commonly used. However, it is well known that bactericidal activity of sodium hypochlorite becomes weak under organic matter rich conditions. In this study, we compared the strength of bactericidal activity of chlorous acid and sodium hypochlorite against Campylobacter jejuni and Campylobacter coli strains under organic matter rich conditions. Bactericidal activity against 5 representative C. jejuni and C. coli strains in chicken juice (an organic matter rich condition) showed that minimum concentration of chlorous acid required for complete killing of C. jejuni and C. coli cells is 200–400 ppm while that of sodium hypochlorite is 2,000 to 4,000 ppm. Similar results were obtained by using Bolton broth. Furthermore, it was observed that 400 ppm of chlorous acid but not 400 ppm of sodium hypochlorite is highly effective in killing of 25 different Campylobacter strains (12 C. jejuni and 13 C. coli strains) under the same conditions. To determine whether 400 ppm of chlorous acid treatment had killed bacterial cells or induced them into viable but non-culturable (VBNC) state, live and dead cell assay using DAPI and propidium iodide fluorescent dyes was done. Such assay clearly indicated that Campylobacter cells were indeed killed and not induced to VBNC state. Moreover, SDS-PAGE analysis of whole-cell lysates of campylobacters indicated distinct effects in protein profiles of chlorous acid but not sodium hypochlorite treated cells. The results strongly suggest that chlorous acid could efficiently kill C. jejuni and C. coli cells with much lower concentration than sodium hypochlorite and the bactericidal mechanisms of chlorous acid may be due to damages of bacterial proteins. Thus, chlorous acid could be a better disinfectant in chicken slaughtering and processing to kill campylobacters and prevent contamination.

Journal: Antimicrobial Agents and Chemotherapy (American Society for Microbiology)

Title: Salmonella genomic island 3 is an integrative and conjugative element and contributes to copper and arsenic tolerance of Salmonella enterica.

Authors: Nobuo Arai, Tsuyoshi Sekizuka, Yukino Tamamura, Masahiro Kusumoto, Atsushi Hinenoya, Shinji Yamasaki, Taketoshi Iwata, Ayako Watanabe-Yanai, Makoto Kuroda and Masato Akiba*

Abstract:  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 with 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 with 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.

Journal:  Diagnostic Microbiology and Infectious Disease (Elsevier)

Title:  Development of a specific cytolethal distending toxin (cdt) gene (Eacdt)-based PCR assay for the detection of Escherichia albertii.

Authors:  Atsushi Hinenoya, Hidetoshi Ichimura, Noritomo Yasuda, Seiya Harada, Kazuhiro Yamada, Masahiro Suzuki, Yoshio Iijima, Akira Nagita, Manuel John Albert, Noritoshi Hatanaka, Sharda Prasad Awasthi and Shinji Yamasaki*

Abstract:  Many Escherichia albertii isolates, an emerging pathogen of human and birds, might have been misidentified due to the difficulty of differentiating this bacterium from Escherichia coli and Shigella spp. by routine biochemical tests, resulting in underestimation of E. albertii infections. We have developed a polymerase chain reaction (PCR) assay that targets E. albertii cytolethal distending toxin (Eacdt) genes, which include the genes previously identified as Escherichia coli cdt-II. This assay could generate a single 449-bp PCR product in each of 67 confirmed E. albertii strains but failed to produce PCR product from any of the tested non-E. albertii enteric strains belonging to 37 different species, indicating 100% sensitivity and specificity of the PCR assay. The detection limit was 10 CFU per PCR tube and could detect 10⁵ CFU E. albertii per gram of spiked healthy human stool. The Eacdt gene-based PCR could be useful for simple, rapid, and accurate detection and identification of E. albertii.

Journal: Journal of Microbiological Methods (Elsevier)

Title: Evaluation of the GeneFields® EHEC/SS PCR dipstick DNA chromatography kit for the detection of enteric bacterial pathogens in stool specimens of healthy humans.

Authors: Hiroki Maruoka, Atsushi Hinenoya, Noritomo Yasuda, Atsuyoshi Takeda, Shin Inoue, Tomoko Sumi, Kazuaki Koitabashi, Hiroshi Yasue, Kazuhiko Kogou and Shinji Yamasaki*

Abstract:  We developed a new GeneFields® EHEC/SS PCR dipstick DNA chromatography kit for the simultaneously detection of invA, ipaH, and stx genes in Salmonella enterica (56 strains), Shigella spp. (44), and enterohemorrhagic Escherichia coli (EHEC) (28), respectively and other bacteria (57), and evaluated the sensitivity and specificity by this kit. The sensitivity and specificity were 100%, respectively. The detection limit of various methods was determined using 5% (w/v) stool suspensions spiked with each bacterium. The detection limit of the GeneFields® EHEC/SS kit ranged from approximately 10²-10³ CFU/g. Additionally, the relative sensitivities and specificities of the GeneFields® EHEC/SS kit vs two commercially available real-time PCR kits were >85.0% and >90.0%, respectively. These results indicate that the GeneFields® EHEC/SS kit can be used for genetic screening of S. enterica, Shigella spp., and EHEC in human stool specimens with sensitivities and specificities similar to those of the commercially available real-time PCR kits.

Journal: Japanese Journal of Infectious Diseases (NIID)

Title: Serotypes, pathogenic potential and antimicrobial resistance of Escherichia coli isolated from subclinical bovine mastitis milk samples in Egypt.

Authors: Rabee Alhossiny Ombarak, Mahmoud Gamaleldin Zayda, Atsushi Hinenoya, Shinji
Yamasaki*

Abstract:  Subclinical mastitis (SCM) is regarded as not only a problem to dairy producers worldwide but also a threat to human health due to the potential bacterial contamination of milk and dairy products, particularly those made from raw milk. In this study, E. coli were isolated from fourteen (9.3%) SCM milk samples. The isolated E. coli (n=14) were serotyped, their potential pathogenicity and antimicrobial resistances (AMRs) were investigated. Serotyping results showed that the E. coli isolates belonged to the O55:H7 (n=2), O111:H4 (n=2), O127:H6 (n=2), O128:HUN (n=2), O26:HUN (n=1), O44:H18 (n=1), O114:H21 (n=1), O86:HUN (n=1), O124:HUN (n=1) and O127:H7 (n=1) serogroups. Potential pathogenicity was detected in 93% (13/14) of the isolates. In particular, thirteen isolates possessed at least one of the examined virulence genes. Ten isolates (71%) exhibited AMR to at least one of the tested antimicrobials, 4 (40%) of them were- multidrug-resistant and one isolates showed ESBL production. The obtained results indicate that SCM acts as a source for the spread of potentially pathogenic E. coli strains that are resistant to many groups of antibiotics which may constitute a hazard for both public and animal health.