Sharda Prasad Awasthi's research while affiliated with Osaka Prefecture University and other places

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.

Escherichia albertii has increasingly been recognized as an important emerging zoonotic enteropathogen. Raccoon is shown to be one of the most vital reservoirs of this pathogen. E. albertii has been detected in 993 (62%) out of 1,606 wild raccoons in Osaka, Japan from 2017 to 2020 by Eacdt-PCR. The detection rate of E. albertii was increased from May to December (winter) and gradually decreased from January to April (spring). Furthermore, we could isolate E. albertii from 30% (196/664) of Eacdt-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 E. albertii in wild raccoon being higher in winter and lower in spring.

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 to 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.

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.

Escherichia albertii has recently been recognized as a zoonotic enteropathogen associated with food-poisoning. The reservoirs and transmission routes of this bacterium to humans are still unclear. In this study, we performed a survey of E. albertii in fecal specimens of wild and safeguarded animals in Okayama Prefecture and its prefectural borders, Japan to understand its reservoir in the environment. Forty-two E. albertii were isolated from 10 and 31 droppings of 59 crows and 125 starlings, respectively. Fifty-two E. albertii were isolated from 906 mammal droppings, and out of 52 isolates, origin of 33, 6 and 1 isolates were from martens, foxes, and rabbit, respectively, however, origin of 12 isolates remained unknown. Three E. albertii were isolated from two and one feces of 159 dogs and 76 cats, respectively. Pulsed-filed gel electrophoresis analysis grouped 97 E. albertii strains into 66 pulsotypes including 36 and 30 pulsotypes of isolates from mammals and birds, respectively. E. albertii strains isolated in this study were genetically diverse. Although clonal relationship was not observed between mammal and bird isolates, there were intra- and inter-species relationship in mammalian isolates. All E. albertii strains were positive for eae and Eacdt virulencegenes. Furthermore, 20 and 7 strains also carried Eccdt-I and stx2f genes, respectively. Taken together, the results indicate that genetically diverse and potentially virulent E. albertii are distributed among various wild and safeguarded animals in Okayama Prefecture, and the animals could also be reservoirs of E. albertii.

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 (HClO 2 ) has been shown to be an effective antimicrobial agent. However, at present there is no experimental evidence showing that HClO 2 can inactivate SARS-CoV-2. Therefore, in this study, we examined the potential of HClO 2 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 HClO 2 for 10 s, viral titre was decreased by 5 log of 50% tissue culture infective dose per mL (TCID 50 ml ⁻¹ ). However, the same concentration of NaClO could not inactivate SARS-CoV-2 as effectively as HClO 2 did even after incubation for 3 min. Furthermore, 10 ppm HClO 2 also inactivated more than 4.0 log of TCID 50 within 10 s in the presence of 5 % fetal bovine serum used as mixed organic matters. Our results obtained with HClO 2 are more effective against SARS-CoV-2 as compared to NaClO that can be used for disinfectant against SARS-CoV-2 .

Emergence and rapid spread of multi-drug resistant (MDR) bacteria including Vibrio cholerae are a global public health issue. Much attention has been paid to natural compounds, such as spices and herbs to find novel antimicrobial compounds as they are considered to be cheaper alternatives to develop as a drug. Here, we show that methanol extract of white pepper could inhibit growth of V. cholerae O1 El Tor variant, responsible for the recent outbreaks/epidemics. Furthermore, we demonstrate for the first time that piperine, the major component of white pepper showed a dose-dependent bactericidal effect on V. cholerae growth irrespective of their biotypes and serogroups in presence of 200 and 300 µg ml-1 of piperine, respectively. Piperine also inhibited growth of MDR strains of Pseudomonas aeruginosa, Escherichia coli isolated from poultry and enterohemorrhagic/enteroaggregative E. coli O104 in presence of 200 µg ml-1 . Interestingly, we did not observe any significant inhibitory effect of piperine on E. coli strains isolated from healthy person even up to 200 µg ml-1 . Our data suggest that piperine could be a novel antimicrobial agent in therapeutic and preventive applications against infections caused by pathogenic bacteria including MDR strains.

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 the prevalence in birds is still scanty. We performed a survey of E. albertii in wild birds in Japan, and examined characteristics of the isolates. E. albertii specific gene was detected in 5 cloacal swabs out of 156 birds by PCR. Four E. albertii were isolated from a swallow with 2 different E. albertii strains and 2 pigeons in a flock by XRM-MacConkey agar. These isolates were assigned to biogroup 3, shown no resistance to any antimicrobials tested, and classified into 2 EAO-genotypes (EAOg2 and EAOg33) and 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) in addition to Eacdt. Interestingly, stx2f genes in a strain were located on an inducible bacteriophage, which can confer the ability to produce Stx2f to E. coli. In conclusion, Japanese wild birds carried E. albertii at the similar levels to the reported prevalence in birds. These isolates may have a potential to cause gastroenteritis in humans.

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.