Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

We have recently described a clinical isolate of Providencia rustigianii strain JH-1 carrying the genes for cytolethal distending toxin (CDT) in a conjugative plasmid. A cdtB mutant of strain JH-1, which lost CDT activity, was still found to retain invasiveness and diarrheagenicity. The strain was subjected to phenotypic and genetic analyses including whole genome sequencing (WGS) to explore the genetic determinants of the observed invasiveness and diarrheagenic properties. Analysis and annotation of WGS data revealed the presence of two distinct type III secretion systems (T3SS) in strain JH-1, one of which was located on the chromosome designated as cT3SS (3,992,833 bp) and the other on a mega-plasmid designated as pT3SS (168,819 bp). Comparative genomic analysis revealed that cT3SS is generally conserved in Providencia spp. but pT3SS was limited to a subset of Providencia spp., carrying cdt genes. Strain JH-1 was found to invade HeLa cells and induce fluid accumulation with characteristic pathological lesions in rabbit ileal loops. Remarkably, these phenomena were associated with the pT3SS but not cT3SS. The plasmid could be transferred by conjugation from strain JH-1 to other strains of P. rustigianii, Providencia rettgeri, and Escherichia coli with concomitant transfer of these virulence properties. This is the first report of a functional and mobile T3SS in P. rustigianii and its association with invasiveness and diarrheagenicity of this bacterium. These data suggest that P. rustigianii and other CDT-producing Providencia strains might carry T3SS and exert their diarrheagenic effect by exploiting the T3SS nano-machinery.IMPORTANCEThe precise mechanism of virulence of Providencia rustigianii is unclear, although some strains produce cytolethal distending toxin as a putative virulence factor. We have detected the presence of a type III secretion system (T3SS) for the first time on a plasmid in a P. rustigianii strain. Plasmid-mediated T3SS seems to be directly involved in virulence of P. rustigianii and may serve as a means of horizontal transfer of T3SS genes. Our results may have implication in understanding the mechanism of emergence of new pathogenic strains of P. rustigianii.

DOI： 10.1128/mbio.02297-24

PubMed

Publishing type：Research paper (scientific journal)  

Introduction

Campylobacter spp. are a public health concern, yet there is still no effective vaccine or medicine available.

Methods

Here, we developed a Campylobacter jejuni-specific antibody and found that it targeted a menaquinol cytochrome c reductase complex QcrC.

Results

The antibody was specifically reactive to multiple C. jejuni strains including clinical isolates from patients with acute enteritis and was found to inhibit the energy metabolism and growth of C. jejuni. Different culture conditions produced different expression levels of QcrC in C. jejuni, and these levels were closely related not only to the energy metabolism of C. jejuni but also its pathogenicity. Furthermore, immunization of mice with recombinant QcrC induced protective immunity against C. jejuni infection.

Discussion

Taken together, our present findings highlight a possible antibody- or vaccination-based strategy to prevent or control Campylobacter infection by targeting the QcrC-mediated metabolic pathway.

DOI： 10.3389/fmicb.2024.1415893

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Escherichia albertii is an emerging zoonotic foodborne pathogen. The clinical significance of this bacterium has increasingly been recognized worldwide. However, diagnostic method has not yet been established and its clinical manifestations are not fully understood. Here, we show that an Eacdt gene-based quantitative real-time PCR (qRT-PCR) developed in this study is 100% specific and sensitive when tested with 39 E. albertii and 36 non-E. albertii strains, respectively. Detection limit of the real-time PCR was 10 colony forming unit (CFU) and 1 pg of genomic DNA per PCR tube. When E. albertii was spiked with 4 × 100-106 CFU per mL to stool of healthy person, detection limit was 4.0 × 103 and 4.0 CFU per mL before and after enrichment culture, respectively. Moreover, the qRT-PCR was able to detect E. albertii in five children out of 246 (2%) but none from 142 adults suffering from gastroenteritis. All five E. albertii strains isolated carried eae and paa genes, however, only one strain harbored stx2f genes. Long-term shedding of stx2f gene-positive E. albertii in a child stool could be detected because of the qRT-PCR developed in this study which might have been missed if only conventional PCR and culture methods were employed. Furthermore, E. albertii isolated from siblings with diarrhea showed clonality by PFGE analysis. Taken together, these data suggest that the Eacdt gene-based qRT-PCR developed for the detection of E. albertii is useful and will assist in determining the real burden and clinical manifestation of E. albertii infections.

DOI： 10.1016/j.heliyon.2024.e30042

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：Domestic journal  

<p><i>Campylobacter fetus</i> is a zoonotic pathogen. Although the precise virulence mechanisms have not yet been fully elucidated, cytolethal distending toxin (CDT) is considered as one of the well-characterized virulence factors in <i>Campylobacter</i>. <i>In silico</i> analysis of the genome of <i>C. fetus</i> type strain ATCC27374^T indicates that there are three <i>cdt</i> gene clusters, <i>Cfcdt-I</i>, <i>Cfcdt-II</i> and <i>Cfcdt-III</i>. However, it is not clear whether these clusters are ubiquitously present in <i>C. fetus</i> and their association with diseases in humans and animals. In this study, we have analyzed the distribution and nucleotide sequences of these <i>cdt</i> gene clusters in 137 <i>C. fetus</i> strains isolated from human patients and healthy cattle. MLST and PFGE were also applied to determine clonal relationship between <i>C. fetus</i> strains isolated from patients and cattle. We found all <i>C. fetus</i> strains carry three <i>Cfcdt</i> gene clusters by colony hybridization assay and the strains belonged to 38 different pulsotypes. Whole genome sequencing of 38 <i>C. fetus</i> strains was carried out to determine the entire <i>cdt</i> gene cluster sequences and their sequence type (ST). Among 38 strains, six STs were identified, and each <i>cdt</i> gene cluster showed high similarity (>99%). Interestingly, some of these <i>Cfcdt</i> genes are more similar to the <i>cdt</i> genes of other <i>Campylobacter</i> species than other <i>Cfcdt</i> gene types. Altogether, the results suggest that three <i>Cfcdt</i> gene clusters are highly conserved in <i>C. fetus</i> and the strains belonging to ST-6 may be more pathogenic to human.</p>

DOI： 10.1292/jvms.24-0336

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

SARS-CoV-2 serological studies suggest that individual serum antibody repertoires can affect neutralisation breadth. Herein, we asked whether a BNT162b2 vaccine-induced epitope dominance pattern (i.e., predominant viral structural domain targeted by serum antibodies for virus neutralisation) affects cross-variant neutralisation. When a neutralisation assay against the ancestral strain was carried out using 16 vaccine sera preabsorbed with a recombinant receptor-binding domain (RBD) or an N-terminal domain (NTD) protein, three and 13 sera, respectively, showed lower neutralisation under NTD and RBD protein-preabsorbed conditions than under the other protein-preabsorbed conditions. This suggests that the NTD was responsible for virus neutralisation in three sera, whereas the other 13 sera elicited RBD-dominant neutralisation. The results also suggest the presence of infectivity-enhancing antibodies in four out of the 13 RBD-dominant sera. A neutralisation assay using SARS-CoV-2 variants revealed that NTD-dominant sera showed significantly reduced neutralising activity against the B.1.617.2 variant, whereas RBD-dominant sera retained neutralising activity even in the presence of infectivity-enhancing antibodies. Taken together, these results suggest the followings: (i) epitope dominance patterns are divided into at least two types: NTD-dominant and RBD-dominant; (ii) NTD-dominant sera have less potential to neutralise the B.1.617.2 variant than RBD-dominant sera; and (iii) infectivity-enhancing antibodies play a limited role in cross-variant neutralisation against the five variants tested.

DOI： 10.1016/j.vaccine.2023.08.076

PubMed

<p>　Total 101 fecal samples were collected from the wild birds which were protected at Kochi Prefecture. These were examined by sugar floatation method. Consequently, 29.7％ of feces were parasitologically positive; 20.8% of <i>Eimeria</i> and/or <i>Isospora</i> type oocysts, and 8.9% of Capillarid or Ascarid spp. Transmitted stages of these parasites could survive in the environments for a while after being shedded in feces. These results suggested that in the past, these infected birds directly or indirectly contacted with feces contaminated with the parasites.</p>

DOI： 10.5686/jjzwm.28.103

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

AIM: The aim of this study was to develop a selective enrichment broth for efficient isolation of Escherichia albertii. METHODS AND RESULTS: A total of 412 raccoon rectal swabs suspended in PBS (phosphate-buffered saline) were tested by a real-time PCR to quantify the number of E. albertii followed by its isolation. The number of E. albertii in the PBS suspension strongly affected the isolation rate (1.2%-89%), which notably dropped (≤33%) when the number was <4 log10 CFU ml-1. However, enrichment of PBS suspension containing raccoon feces in tryptic soy broth containing cefixime, tellurite, and deoxycholate (CTD-TSB), the selective medium developed in this study, remarkably improved the isolation efficiency (up to 48%) of E. albertii. CONCLUSIONS: CTD-TSB is a useful enrichment culture medium for E. albertii and contributes to increase its isolation rate.

DOI： 10.1093/jambio/lxad123

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Providencia rustigianii is potentially enteropathogenic in humans. Recently, we identified a P. rustigianii strain carrying a part of the cdtB gene homologous to that of Providencia alcalifacines that produces an exotoxin called cytolethal distending toxin (CDT), encoded by three subunit genes (cdtA, cdtB, and cdtC). In this study, we analyzed the P. rustigianii strain for possible presence of the entire cdt gene cluster and its organization, location, and mobility, as well as expression of the toxin as a putative virulence factor of P. rustigianii. Nucleotide sequence analysis revealed the presence of the three cdt subunit genes in tandem, and over 94% homology to the corresponding genes carried by P. alcalifaciens both at nucleotide and amino acid sequence levels. The P. rustigianii strain produced biologically active CDT, which caused distension of eukaryotic cell lines with characteristic tropism of CHO and Caco-2 cells but not of Vero cells. S1-nuclease digested pulsed-field gel electrophoresis followed by Southern hybridization analysis demonstrated that the cdt genes in both P. rustigianii and P. alcalifaciens strains are located on large plasmids (140 to 170 kb). Subsequently, conjugation assays using a genetically marked derivative of the P. rustigianii strain showed that the plasmid carrying cdt genes in the P. rustigianii was transferable to cdt gene-negative recipient strains of P. rustigianii, Providencia rettgeri, and Escherichia coli. Our results demonstrated the presence of cdt genes in P. rustigianii for the first time, and further showed that the genes are located on a transferable plasmid, which can potentially spread to other bacterial species.

DOI： 10.1128/iai.00121-22

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

BACKGROUND: Clostridium perfringens and Shiga toxin (Stx)-producing Escherichia coli (STEC) are common causes of food poisoning. We previously demonstrated the efficacy of Stx2B-C-CPE, a fusion protein of the C-terminal region of C. perfringens enterotoxin (C-CPE) and Shiga toxin 2 B subunit (Stx2B), as a bivalent vaccine against C. perfringens and STEC infections. METHODS: Here, we applied an E. coli expression system and Triton X-114 phase separation to prepare tag- and endotoxin-free Stx2B-C-CPE for use in vaccine formulations. RESULTS: As we anticipated, endotoxin removal from the purified antigen reduced both Stx2B- and C-CPE-specific IgG antibody responses in subcutaneously immunized mice, suggesting that endotoxin contamination influences the immunological assessment of Stx2B-C-CPE. However, the combined use of aluminum and Alcaligenes lipid A adjuvants improved IgG antibody responses to the injected antigen, thus indicating the suitability of purified Stx2B-C-CPE for vaccine formulation. CONCLUSIONS: Our current findings provide important knowledge regarding the design of an effective commercial Stx2B-C-CPE vaccine.

DOI： 10.31083/j.fbl2801015

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：Domestic journal  

<p><i>Escherichia albertii </i>has increasingly been recognized as an important emerging zoonotic enteropathogen. Raccoon is shown to be one of the most vital reservoirs of this pathogen. <i>E. albertii </i>has been detected in 993 (62%) out of 1,606 wild raccoons in Osaka, Japan from 2017 to 2020 by <i>Eacdt</i>-PCR. The detection rate of <i>E. albertii </i>was increased from May to December (winter) and gradually decreased from January to April (spring). Furthermore, we could isolate <i>E. albertii</i> from 30% (196/664) of <i>Eacdt</i>-PCR positive samples and the monthly isolation rate seems to correlate with its detection rate. These data indicate that there is a seasonality regarding the prevalence of <i>E. albertii </i>in wild raccoon being higher in winter and lower in spring. </p>

DOI： 10.1292/jvms.23-0372

PubMed

Authorship：Lead author   Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

A novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), suddenly emerged in China in 2019, spread globally and caused the present COVID-19 pandemic. Therefore, to mitigate SARS-CoV-2 infection effective measures are essential. Chlorous acid (HClO2) has been shown to be an effective antimicrobial agent. However, at present there is no experimental evidence showing that HClO2 can inactivate SARS-CoV-2. Therefore, in this study, we examined the potential of HClO2 to inactivate SARS-CoV-2 in presence or absence of organic matter and the results were compared with that of sodium hypochlorite (NaClO), another potent antimicrobial agent. When concentrated SARS-CoV-2 was incubated with 10 ppm HClO2 for 10 s, viral titre was decreased by 5 log of 50% tissue culture infective dose per mL (TCID50 ml-1). However, the same concentration of NaClO could not inactivate SARS-CoV-2 as effectively as HClO2 did even after incubation for 3 min. Furthermore, 10 ppm HClO2 also inactivated more than 4.0 log of TCID50 within 10 s in the presence of 5 % fetal bovine serum used as mixed organic matters. Our results obtained with HClO2 are more effective against SARS-CoV-2 as compared to NaClO that can be used for disinfectant against SARS-CoV-2 .

DOI： 10.1099/acmi.0.000354

PubMed

Kind of work：Joint Work   International / domestic magazine：International journal  

Publishing type：Research paper (scientific journal)   International / domestic magazine：Domestic journal  

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.

DOI： 10.7883/yoken.JJID.2021.355

PubMed

大阪市と高知市の野鳥を対象にEscherichia albertii保有状況を調べた。ハトのクロアカスワブ検体129件と13種類の野鳥のクロアカスワブ検体27件を使用した。増菌培養後、Eacdt遺伝子を標的としたPCRでスクリーニングし、陽性検体をXRM-マッコンキー寒天培地またはマッコンキー寒天培地で培養した。その結果、ツバメ2羽(7.4%)、ハト3羽(2.3%)がPCR陽性となり、全体の検出率は3.2%(5/156)であった。培養検査ではツバメ1羽とハト2羽からE.albertiiが分離された。PFGE解析により、ハト由来株は同一クローン株、ツバメ由来株から2つのクローン株が確認された。分離4株は生物グループ3に割り当てられ、供試16種抗菌薬に感受性を示した。O抗原遺伝子型はツバメ由来2株が型別不能とEAOg2、ハト由来2株はEAOg33であった。分離4株はEacdtの他にeaeとpaaを保有していた。さらにツバメ由来1株はstx2f、ハト由来2株はEccdt-I遺伝子を保有していた。4株のインチミンサブタイプは多様であったが、LEE病原性アイランドの統合部位はphe Uのみであった。マイトマイシンC存在下、プラークアッセイによる溶原化ファージ誘発の結果、stx2fは誘発性ファージ上に位置し、ファージはレシピエント大腸菌にStx2f産生能を付与した。ドナー株のベロ毒素力価が増強された。

日本で食品添加物として認可されているShellCoatが、急性呼吸器症候群の原因ウイルスであるSARS-CoV-2を不活性化できるか検討した。有機化合物ウシ胎仔血清(FBS)の存在下で、SARS-CoV-2に対するShellCoatの抗ウイルス効果も評価した。濃縮SARS-CoV-2をFBSの存在下または不在下で、10秒間ShellCoatで処理すると、ウイルス価は50%組織培養感染力/mL(TCID50/mL)のlog値が4以上低下した。同条件で20秒以上処理すると、ウイルス価は検出限界(logTCID50/m=2.1)以下となった。以上より、ShellCoatは有機物質の存在下でも、SARS-CoV-2に対する強力な抗ウイルス物質であると考えられた。

Publishing type：Research paper (scientific journal)  

DOI： 10.1111/lam.13646

Other URL： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/lam.13646

Authorship：Lead author   Kind of work：Joint Work   International / domestic magazine：International journal  

Publishing type：Research paper (scientific journal)   Kind of work：Joint Work  

Authorship：Lead author   Publishing type：Research paper (scientific journal)  

It is well known that black and green tea extracts, particularly polyphenols, have antimicrobial activity against various pathogenic microbes including viruses. However, there is limited data on the antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged rapidly in China in late 2019 and which has been responsible for coronavirus disease 2019 (COVID-19) pandemic globally. In this study, 20 compounds and three extracts were obtained from black and green tea and found that three tea extracts showed significant antiviral activity against SARS-CoV-2, whereby the viral titre decreased about 5 logs TCID50 per ml by 1·375 mg ml−1 black tea extract and two-fold diluted tea bag infusion obtained from black tea when incubated at 25°C for 10 s. However, when concentrations of black and green tea extracts were equally adjusted to 344 µg ml−1, green tea extracts showed more antiviral activity against SARS-CoV-2. This simple and highly respected beverage may be a cheap and widely acceptable means to reduce SARS-CoV-2 viral burden in the mouth and upper gastrointestinal and respiratory tracts in developed as well as developing countries.

DOI： 10.1111/lam.13591

Other URL： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/lam.13591

Authorship：Lead author   International / domestic magazine：International journal  

Authorship：Lead author   Publishing type：Research paper (scientific journal)   International / domestic magazine：Domestic journal  

SARS-CoV-2, an acute respiratory syndrome-causing virus, suddenly emerged at the end of 2019 in China, and rapidly spread all over the world. In this study, we examined whether a calcinated calcium solution (ShellCoat) , which has been approved as a food additive in Japan can inactivate SARS-CoV-2. Furthermore, antiviral activity of ShellCoat against SARS-CoV-2 was also evaluated in the presence of organic matter, namely, fetal bovine serum (FBS) . When concentrated SARS-CoV-2 were treated with ShellCoat for 10 sec in presence or absence of FBS as organic matters, the viral titer was decreased more than 4 logs 50% tissue culture infective dose per mL (TCID50/mL) but use of ShellCoat for 20 sec or more under similar experimental conditions the viral titer was below the detection limit (≦2.1 logs TCID50/mL) . These results clearly indicate that the ShellCoat is a powerful antiviral agent against SARS-CoV-2 even in the presence of organic matters.

DOI： 10.4265/bio.27.53

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.ijfoodmicro.2022.109540

Kind of work：Joint Work   International / domestic magazine：International journal  

Authorship：Lead author   Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

A new coronavirus (SARS-CoV-2) abruptly emerged in Wuhan, China in 2019 and rapidly spread globally to cause the COVID-19 pandemic. In this study, we examined the anti-SARS-CoV-2 activity of the potent disinfectant Cleverin, the major disinfecting component of which is chlorine dioxide (ClO2) and the results were compared with that of sodium hypochlorite in the presence or absence of 0.5% or 1.0% fetal bovine serum (FBS). When SARS-CoV-2 viruses were treated with 0.8 ppm ClO2 or sodium hypochlorite, viral titre was decreased only by 1 log TCID50/mL in 3 min. However, the viral titre was decreased by more than 4 logs TCID50/mL when treated with 80 ppm of each chemical for 10 sec regardless of presence or absence of FBS. It should be emphasized that treatment with 24 ppm of ClO2 inactivated more than 99.99% SARS-CoV-2 within 10 sec or 99.99% SARS-CoV-2 in 1 min in the presence of 0.5% or 1.0% FBS, respectively. In contrast, 24 ppm of sodium hypochlorite was able to inactivate only 99% or 90% SARS-CoV-2 in 3 min under similar conditions. Notably, except ClO2 the other components of Cleverin such as sodium chlorite, decaglycerol monolaurate and silicone showed no significant antiviral activity. Altogether, the results strongly suggest that although ClO2 and sodium hypochlorite are strong antiviral agents in absence of organic matters but in presence of organic matters ClO2 is a more potent antiviral agent against SARS-CoV-2 than sodium hypochlorite.

DOI： 10.1016/j.jhin.2021.09.006

PubMed

Authorship：Lead author   Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

AIM: A novel coronavirus, termed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suddenly appeared in Wuhan, China and has caused pandemic. In this study, we evaluated antiviral activity of purified hypochlorous acid (HClO) solution against coronaviruses such as SARS-CoV-2 and transmissible gastroenteritis virus (TGEV) responsible for pig diseases. MATERIALS AND RESULTS: In a suspension test, 28.1 ppm HClO solution inactivated SARS-CoV-2 in phosphate buffered saline (PBS) with the reduction of 104 of 50% tissue culture infectious dose/mL (TCID50 /mL) within 10 sec. When its concentration increased to 59.4 ppm, the virus titer decreased to below the detection limit (reduction of 5 logs TCID50 ) within 10 sec even in the presence of 0.1% fetal bovine serum (FBS). In a carrier test, incubation with 125 ppm HClO solution for 10 min or 250 ppm for 5 min inactivated SARS-CoV-2 by more than 4 logs TCID50 /mL or below the detection limit. Since the titer of TGEV was 10 fold higher, TGEV was used for SARS-CoV-2 in a suspension test. As expected, 56.3 ppm HClO solution inactivated TGEV by 6 logs TCID50 within 30 sec. CONCLUSIONS: In a carrier test 125 ppm HClO solution for 10 min incubation is adequate to inactivate 4 logs TCID50 /mL of SARS-CoV-2 or more while in a suspension test 56.3 ppm HClO is adequate to inactivate 5 logs TCID50 /mL of SARS-CoV-2 when incubated for only 10 sec regardless of presence or absence of organic matter. SIGNIFICANCE AND IMPACT OF STUDY: Effectiveness of HClO solution against SARS-CoV-2 was demonstrated by both suspension and carrier tests. HClO solution inactivated SARS-CoV-2 by 5 logs TCID50 within 10 sec. HClO solution has several advantages such as none toxicity, none irritation to skin and none flammable. Thus, HClO solution can be used as a disinfectant for SARS-CoV-2.

DOI： 10.1111/jam.15284

PubMed

Publishing type：Research paper (scientific journal)  

The mcr-1 gene in 125 ESBL-producing Gram-negative bacteria obtained from animal products was screened and the location and transferability of the mcr-1 gene were examined. The mcr-1 gene was detected by PCR in one Escherichia coli isolate (RC263) obtained from hard cheese made from raw milk. Antimicrobial susceptibility test showed that the strain RC263 was phenotypically multidrug-resistant with colistin and nine other antimicrobials. Further genetic analysis indicated that this isolate also harboured the blaTEM and blaCTX-M-9 genes. S1-PFGE followed by Southern blotting verified that the mcr-1 gene was located on an approximately 153 kb plasmid. The plasmid was transferred to a recipient E. coli strain by conjugation, suggesting that the mcr-1 gene may spread to other Gram-negative bacteria. In conclusion, ESBL-producing E. coli carrying a transferable plasmid with mcr-1 gene was present in hard cheese in Egypt.

DOI： 10.1016/j.idairyj.2021.104986

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

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.

DOI： 10.1016/j.ijfoodmicro.2021.109164

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

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.

DOI： 10.1089/fpd.2020.2891

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：Domestic journal  

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.

DOI： 10.1292/jvms.21-0002

PubMed

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

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-1 of ChxA, was developed. ChxA was mostly detected in the extracellular cell-free supernatant and its production level varied from 1.2 ng ml-1 to 1.6 µg ml-1. 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.

DOI： 10.1099/jmm.0.001311

PubMed

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

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.

DOI： 10.3201/eid2606.191436

PubMed

Kind of work：Joint Work  

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

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.

DOI： 10.1016/j.diagmicrobio.2020.115006

PubMed

Authorship：Lead author   Publishing type：Research paper (scientific journal)  

© 2019 Elsevier Ltd 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.

DOI： 10.1016/j.foodcont.2019.107046

Authorship：Lead author   Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

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.

DOI： 10.1016/j.mimet.2020.105916

PubMed

Kind of work：Joint Work  

International / domestic magazine：International journal  

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.

DOI： 10.1016/j.diagmicrobio.2019.04.018

PubMed

Kind of work：Joint Work  

Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.mimet.2018.12.012

PubMed

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Kind of work：Joint Work  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation  

Presentation type：Poster presentation