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Journal: Letters in Applied Microbiology
Title: Piperine, an active ingredient of white pepper suppresses multidrug reisistant growth of toxigenic Vibrio cholerae and other pathogenic bacteria.
Authors: Goutham B. Manjunath, Sharda P. Awasthi, M. Shamim Hasan Zahid, Noritoshi Hatanaka, Atsushi Hinenoya, Emiko Iwaoka, Shunji Aoki, Thandavarayan Ramamurthy, Shinji Yamasaki*.
Abstract: coming soon…
Journal: Japanese Journal of Infectious Diseases (JJID)
Authors: Atsushi Hinenoya, Sharda Prasad Awasthi, Noritomo Yasuda, Keigo Nagano, Jayedul Hassan, Keiji Takehira, Noritoshi Hatanaka, Shun Saito, Takashi Watabe, Miki Yoshizawa, Haruna Inoue, Shinji Yamasaki*.
Abstract: Escherichia albertii is an emerging zoonotic foodborne pathogen. Several outbreaks of E. albertii have occurred, particularly in Japan. Although birds have been considered as one of the most important reservoirs of this bacterium, information regarding its prevalence in birds is still scarce. We performed a survey of E. albertii in wild birds in Japan and examined the characteristics of these isolates. E. albertii-specific genes were detected in five cloacal swabs from 156 birds by PCR. Four E. albertii strains were isolated from a swallow with two different E. albertii strains and two pigeons in a flock using XRM-MacConkey agar. These isolates were assigned to biogroup 3, showed no resistance to any tested antimicrobials, and were classified into two EAO-genotypes (EAOg2 and EAOg33) and were untypable. Similar to clinical E. albertii strains, these isolates carried virulence genes, including eae (n = 4), paa (n = 4), Eccdt-I (n = 2), and stx2f (n = 1), as well as Eacdt. Furthermore, stx2f genes in a strain were located on an inducible bacteriophage, which can confer the ability to produce Stx2f in E. coli. In conclusion, Japanese wild birds carried E. albertii at levels similar to the reported prevalence in birds. These isolates may have the potential to cause gastroenteritis in humans.
A Happy News!
We did a collaborative research with Ms. Inoue, who graduated OPU in 2010 and now works for a zoo ‘Wanpark Kochi Animal Land’ in Kochi Japan, as a veterinary doctor.
The purpose is to know the prevalence of E. albertii, an emerging zoonotic pathogen, in Japanese wild birds. The research products were wrapped up in a paper and has just been accepted in the journal of JJID.
Journal: Frontier in Microbiology
Authors: Nobuo Arai, Tsuyoshi Sekizuka, Yukino Tamamura-Andoh, Lisa Barco, Atsushi Hinenoya, Shinji Yamasaki, Taketoshi Iwata, Ayako Watanabe-Yanai, Makoto Kuroda, Masato Akiba, Masahiro Kusumoto*.
Abstract: Salmonella enterica subsp. enterica serovar Typhimurium sequence type 34 (ST34) and its monophasic variant (Salmonella 4,[5],12:i:-) are among the most frequently isolated clones from both humans and animals worldwide. Our previous study demonstrated that Salmonella Typhimurium/4,[5],12:i:- strains isolated in Japan could be classified into nine clades and that clade 9 consisted of ST34 strains. In Japan, ST34/clade 9 was first found in the 1990s and has become predominant among food animals in recent years. In the present study, we analyzed the whole genome-based phylogenetic relationships and temporal information of 214 Salmonella Typhimurium/4,[5],12:i:- ST34/clade 9 strains isolated from 1998 to 2017 in Japan. The 214 strains were classified into two sublineages: the newly identified clade 9–2 diverged from clade 9 in the early 2000s and has predominated in recent years. Clonally expanding subclades in clades 9–1 or 9–2 lacked Gifsy-1 or HP1 prophages, respectively, and some strains in these subclades acquired plasmids encoding antimicrobial resistance genes. Additional genome reduction around the fljB gene encoding the phase 2-H antigen was generated by an IS26-mediated deletion adjacent to the transposon in clade 9–2. Although most of the clade 9 strains were isolated from cattle in Japan, the clonally expanding subclades in clade 9–2 (i.e., all and 24% strains of subclades 9–2a and 9–2b, respectively) were isolated from swine. The spread of clade 9 in recent years among food animals in Japan was responsible for the emergence of multiple host-adapted sublineages involving the clonally expanding subclades generated by mobile genetic element-mediated microevolution.
Dr. Noritoshi Hatanaka starts to work as the Assistant Professor of this laboratory.
Congratulations!
Wish you more and more success and prosperity!
Journal: Microorganisms
Title: Critical role of 3’-downstream region of pmrB in polymyxin resistance in Escherichia coli BL21(DE3).
Authors: Fuzhou Xu#, Atsushi Hinenoya#, Ximin Zeng#, Xing-ping Li, Ziqiang Guan, Jun Lin* (#These authors contributed equally to this work)
Abstract: Polymyxins, such as colistin and polymyxin B, are the drugs used as a last resort to treat multidrug-resistant Gram-negative bacterial infections in humans. Increasing colistin resistance has posed a serious threat to human health, warranting in-depth mechanistic research. In this study, using a functional cloning approach, we examined the molecular basis of colistin resistance in Escherichia coli BL21(DE3). Five transformants with inserts ranging from 3.8 to 10.7 kb displayed significantly increased colistin resistance, three of which containing pmrB locus and two containing pmrD locus. Stepwise subcloning indicated that both the pmrB with a single G361A mutation and at least a 103 bp downstream region of pmrB are essential for conferring colistin resistance. Analysis of the mRNA level and stability showed that the length of the downstream region drastically affected the pmrB mRNA level but not its half-life. Lipid A analysis, by mass spectrometry, revealed that the constructs containing pmrB with a longer downstream region (103 or 126 bp) have charge-altering l-4-aminoarabinose (Ara4N) and phosphoethanolamine (pEtN) modifications in lipid A, which were not observed in both vector control and the construct containing pmrB with an 86 bp downstream region. Together, the findings from this study indicate that the 3′-downstream region of pmrB is critical for the PmrB-mediated lipid A modifications and colistin resistance in E. coli BL21(DE3), suggesting a novel regulatory mechanism of PmrB-mediated colistin resistance in E. coli.
Journal: International Journal of Food Microbiology
Authors: Phong Quoc Le, Sharda Prasad Awasthi, Noritoshi Hatanaka, Atsushi Hinenoya, Jayedul Hassan, Rabee Alhossiny Ombarak, Atsushi Iguchi, Nga Thuy Thi Tran, Khanh Van Thi Dao, Mai Quang Vien, Huy Xuan Le, Hung Thai Do, Yoshimasa Yamamoto, Shinji Yamasaki*.
Abstract: The aim of the study was to assess the presence of genes in ESBL-producing E. coli (ESBL-Ec) isolated from retail raw food in Nha Trang, Vietnam. A total of 452 food samples comprising chicken (n = 116), pork (n = 112), fish (n = 112) and shrimp (n = 112) collected between 2015 and 2017 were examined for the prevalence of ESBL-Ec. ESBL-Ec were detected in 46.0% (208/452) of retail food samples, particularly in 66.4% (77/116), 55.4% (62/112), 42.0% (47/112) 19.6% (22/112) of chicken, pork, fish and shrimp, respectively. Sixty-five out of the 208 (31.3%) ESBL-Ec isolates were positive for mcr genes including mcr-1, mcr-3 and both mcr-1 and mcr-3 genes in 56/208 (26.9%), 1/208 (0.5%) and 8/208 (3.9%) isolates, respectively. Particularly, there was higher prevalence of mcr-1 in ESBL-Ec isolates from chicken (53.2%, 41/77) in comparison to shrimp (22.7%, 5/22), pork (11.3%, 7/62) and fish (6.4%, 3/47). mcr-3 gene was detected in co-existence with mcr-1 in ESBL-Ec isolates from shrimp (9.1%, 2/22), pork (8.1%, 5/62) and fish (2.1%, 1/47) but not chicken. The 65 mcr-positive ESBL-Ec (mcr-ESBL-Ec) were colistin-resistant with the MICs of 4–8 μg/mL. All mcr-3 gene–positive isolates belonged to group A, whereas phylogenetic group distribution of isolates harboring only mcr-1 was B1 (44.6%), A (28.6%) and D (26.8%). PFGE analysis showed diverse genotypes, although some isolates demonstrated nearly clonal relationships. S1-PFGE and Southern hybridization illustrated that the mcr-1 and mcr-3 genes were located either on chromosomes or on plasmids. However, the types of mcr genes were harbored on different plasmids with varied sizes of 30–390 kb. Besides, the ESBL genes of CTX-M-1 or CTX-M-9 were also detected to be located on plasmids. Noteworthy, co-location of CTX-M-1 with mcr-1 or mcr-3 genes on the same plasmid was identified. The conjugation experiment indicated that the mcr-1 or mcr-3 was horizontally transferable. All mcr-ESBL-Ec isolates were multidrug resistance (resistance to ≥3 antimicrobial classes). Moreover, β-Lactamase-encoding genes of the CTX-M-1 (78.5%), CTX-M-9 (21.5%), TEM (61.5%) groups were found in mcr-ESBL-Ec. The astA gene was detected in 27 (41.5%) mcr-ESBL-Ec isolates demonstrating their potential virulence. In conclusion, mcr-1 and mcr-3 genes existed individually or concurrently in ESBL-Ec isolates recovered from retail raw food in Nha Trang city, which might further complicate the antimicrobial-resistant situation in Vietnam, and is a possible health risk for human.
Highlights
1. High prevalence of mcr-1 positive ESBL-E. coli isolated from raw food in Vietnam.
2. mcr-ESBL-E. coli isolates could be potentially virulent due to presence of astA gene.
3. First report of mcr-3 gene in E. coli from fish and shrimp in Vietnam.
4. Concurrence of mcr-1 and mcr-3 genes on chromosomes of a single E. coli isolate.
5. Co-existence of CTX-M-1 with mcr-1 or mcr-3 on the same plasmid in ESBL-E. coli.
Journal: Foodborne Pathogens and Diseases
Authors: Dao Thi Anh Nguyen, Sharda Prasad Awasthi, Phuong Hoai Hoang, Phuc Do Nguyen, Jayedul Hassan, Noritoshi Hatanaka, Atsushi Hinenoya, Chinh Van Dang, Shah M. Faruque, Shinji Yamasaki*.
Abstract:
In this study, we investigated the prevalence, serovar distribution, and antimicrobial resistance pattern of Salmonella isolates from vegetable, fruit, and water samples in Ho Chi Minh City, Vietnam. Salmonella was detected in 75% (30/40), 57.1% (12/21), 17.5% (28/160), and 2.5% (1/40) of river water, irrigation water, vegetable, and ice water samples, respectively. However, no Salmonella was isolated from 160 fruit and 40 tap water samples examined. A total of 102 isolates obtained from 71 samples belonged to 34 different serovars, of which Salmonella Rissen was the most prevalent, followed by Salmonella London, Salmonella Hvittingfoss, and Salmonella Weltevreden. Certain Salmonella serovars such as Newport, Rissen, and Weltevreden were isolated from both vegetable and water samples. Antimicrobial resistance was most commonly observed against tetracycline (35.3%), followed by chloramphenicol (34.3%), ampicillin (31.4%), trimethoprim/sulfamethoxazole (23.5%), and nalidixic acid (10.8%). Of 102 isolates analyzed, 52 (51%) showed resistance to at least 1 antimicrobial class whereas 27 (26.5%) showed multidrug resistant (MDR) phenotype, being resistant to at least three different classes of antimicrobials. Determination of the presence and type of β-lactamase genes showed the cooccurrence of blaTEM-1 and blaCMY-2 in one Salmonella Agona isolate from a river water sample. Taken together, these data indicated that both environmental water and vegetables were contaminated with Salmonella, including MDR strains, and that environmental water used in irrigation might have been the source of Salmonella contamination in the vegetables.
Keywords: Salmonella; Vietnam; blaCMY-2, vegetable; environmental water; extended-spectrum β-lactamase.
Journal: Journal of Veterinary Medical Science
Title: Prevalence, O-genotype and Shiga toxin (Stx) 2 subtype of Stx-producing Escherichia coli strains isolated from Argentinean beef cattle.
Authors: Kentaro Okuno, Sharda P. Awasthi, German A. Kopprio, Atsushi Iguchi, Noritoshi Hatanaka, Atsushi Hinenoya, Ruben J. Lara, Shinji Yamasaki*.
Abstract: The aims of this study were to investigate prevalence, O-genotype, and virulence gene profile including Shiga toxin (Stx) 2 gene-subtype of Stx-producing Escherichia coli (STEC) in beef cattle from the Bahía Blanca in Argentina. Rectal swabs were collected from 283 beef cattle in 2012. stx genes were detected in 90 (32%) out of the 283 rectal swabs by stx gene-specific PCR assay. The positive cases were 13 with stx1, 58 with stx2, and 19 with both stx1 and stx2. Among 90 stx gene-positive samples, 45 STEC strains were isolated, which included 3 stx1, 34 stx2, and eight stx1 and stx2 genes positive isolates. O-genotyping grouped 45 STEC strains into 19 different O-genotypes such as Og8, Og145, Og171, Og185 (4 from each), Og22, Og153, Og157 (3 from each) and others. Various stx2 gene-subtypes were identified in 42 STEC strains: 13 positive cases for stx2a, 11 for stx2c, 3 for stx2g, 10 for stx2a and stx2d, 4 for stx2a and stx2c, and 1 for stx2b, stx2c and stx2g. efaI gene, generally prevalent in clinical strains, was detected in relatively high in the STEC strains. These data suggest that stx2a and stx2c were distributed not only in O145 and O157 but also in minor O-genotypes of STEC in Argentina.
Journal: Zoonoses and Public Health
Title: Isolation and characterization of Escherichia albertii in poultry at the pre-harvest level.
Authors: Atsushi Hinenoya, Xing-ping Li, Ximin Zeng, Orhan Sahin, Rodney A. Moxley, Catherine M. Logue, Barbara Gillespie, Shinji Yamasaki*, Jun Lin*.
Abstract: Escherichia albertii, often misidentified as Escherichia coli, has become an emerging foodborne human enteric pathogen. However, the prevalence and major animal reservoirs of this significant pathogen are still not clear. Here, we performed comprehensive microbiological, molecular, comparative genomics and animal studies to understand the status and features of E. albertii in the US domestic and food animals. Although no E. albertii was identified in a total of 1,022 diverse E. coli strains isolated from pets and food animals in a retrospective screening, in a pilot study, E. albertii was successfully isolated from a broiler farm (6 out of 20 chickens). The chicken E. albertii isolates showed clonal relationship as indicated by both pulsed‐field gel electrophoresis (PFGE) and whole‐genome sequence analysis. The isolated chicken E. albertii displayed multidrug resistance; all the resistance determinants including the extended‐spectrum beta‐lactamase gene, carried by plasmids, could be conjugatively transferred to E. coli, which was further confirmed by S1‐PFGE and Southern hybridization. Whole‐genome sequence‐based phylogenetic analysis showed the chicken E. albertii strains were phylogenetically close to those of human origins. Challenge experiment demonstrated that the E. albertii strains isolated from human and wild bird could successfully colonize in the chicken intestine. Together, this study, for the first time, reported the isolation of E. albertii in poultry at the pre‐hrvest level. The findings from multi‐tier characterization of the chicken E. albertii strains indicated the importance of chickens as a reservoir for E. albertii. A large scale of E. albertii survey in poultry production at the pre‐harvest level is highly warranted in the future.
Journal: Journal of Medical Microbiology
Authors: Sharda Prasad Awasthi, Nityananda Chowdhury, Noritoshi Hatanaka, Atsushi Hinenoya, Thandavarayan Ramamurthy, Masahiro Asakura and Shinji Yamasaki*
Abstract:
Introduction. Cholix toxin (ChxA) is an ADP-ribosylating exotoxin produced by Vibrio cholerae. However, to date, there is no quantitative assay available for ChxA, which makes it difficult to detect and estimate the level of ChxA produced by V. cholerae.
Hypothesis/Gap Statement. It is important to develop a reliable and specific quantitative assay to measure the production level of ChxA, which will help us to understand the role of ChxA in V. cholerae pathogenesis.
Aim. The aim of this study was to develop a bead-based sandwich ELISA (bead-ELISA) for the quantification of ChxA and to evaluate the importance of ChxA in the pathogenesis of V. cholerae infection.
Methodology. Anti-rChxA was raised in New Zealand white rabbits, and Fab-horse radish peroxidase conjugate was prepared by the maleimide method to use in the bead-ELISA. This anti-ChxA bead-ELISA was applied to quantify the ChxA produced by various V. cholerae strains. The production of ChxA was examined in different growth media such as alkaline peptone water (APW), Luria-Bertani broth and AKI. Finally, the assay was evaluated using a mouse lethality assay with representative V. cholerae strains categorized as low to high ChxA-producers based on anti-ChxA bead-ELISA.
Results. A sensitive bead-ELISA assay, which can quantify from 0.6 to 60 ng/ml of ChxA, was developed. ChxA was mostly detected in the extracellular cell-free supernatant and its production level varied from 1.2 ng/ml to 1.6 µg/ml. The highest ChxA production was observed when V. cholerae strains were cultured in LB broth, but not in APW or AKI medium. The ChxA-producer V. cholerae strains showed 20–80 % lethality and only the high ChxA II-producer was statistically more lethal than a non-ChxA-producer, in the mice model assay. ChxA I and II production levels were not well correlated with mice lethality, and this could be due to the heterogeneity of the strains tested.
Conclusion. ChxA I to III was produced mostly extracellularly at various levels depending on strains and culture conditions. The bead-ELISA developed in this study is useful for the detection and quantification of ChxA in V. cholerae strains.
This study, for the first time, developed a cholix toxin-specific sandwich bead-ELISA by which the toxin production of V. cholerae can be quantitatively measured.
