Campylobacter fetus isolates from both human patients and healthy cattle carry three distinct cytolethal distending toxin (cdt) gene clusters.

Campylobacter fetus 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 Campylobacter. In silico analysis of the genome of C. fetus type strain of ATCC27374T indicates that there are three cdt gene clusters, Cfcdt-I, Cfcdt-II and Cfcdt-III. However, it is not clear whether these clusters are ubiquitously present in C. fetus and their association with diseases in humans and animals. In this study, we have analyzed the distribution and nucleotide sequences of these cdt gene clusters in 137 C. fetus strains isolated from human patients and healthy cattle. MLST and PFGE were also applied to determine clonal relationship between C. fetus strains isolated from patients and cattle. We found all C. fetus strains carry three Cfcdt gene clusters by colony hybridization assay and the strains belonged to 38 different pulsotypes. Whole genome sequencing of 38 C. fetus strains was carried out to determine the entire cdt gene cluster sequences and their sequence type (ST). Among 38 strains, six STs were identified, and each cdt gene cluster showed high similarity (>99%). Interestingly, some of these Cfcdt genes are more similar to the cdt genes of other Campylobacter species than other Cfcdt gene types. Altogether, the results suggest that three Cfcdt gene clusters are highly conserved in C. fetus and the strains belonging to ST-6 may be more pathogenic to human.

A plasmid-mediated type III secretion system associated with invasiveness and diarrheagenicity of Providencia rustigianii.

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.

Quorum regulated latent environmental cells of toxigenic Vibrio cholerae and their role in cholera outbreaks.

BACKGROUND: Diverse bacterial group behaviors are controlled by quorum sensing, a regulatory network of bacterial gene expression based on cell density, and involving communication through chemical signal molecules called autoinducers. Multidisciplinary research in toxigenic Vibrio cholerae the etiologic agent of cholera, appear to suggest group behavior in the ecology, epidemiology, pathogenesis and transmission of the pathogen. This review summarizes latest advances and known aspects of quorum regulated environmental survival form of V. cholerae, and their role in cholera outbreaks, as well as the significance of this knowledge in tracking the pathogen for prevention of cholera. MAIN BODY: Pathogenic V. cholerae naturally exists in aquatic reservoirs, and infects humans, often leading to epidemic outbreaks of cholera. Effective detection and monitoring of the pathogen in surface waters have been a research focus in preventing cholera outbreaks. However, in the aquatic reservoirs, V. cholerae persists mostly in a quiescent state referred to as viable but non-culturable (VBNC), or conditionally viable environmental cells (CVEC), which fail to grow in routine bacteriological culture. The presence of CVEC can, however, be observed by fluorescent antibody based microscopy, and they appear as clumps of cells embedded in an exopolysaccharide matrix. Current studies suggest that CVEC found in water are derived from in-vivo formed biofilms excreted by cholera patients. The transition to CVEC occurs when dilution of autoinducers in water blocks quorum-mediated regulatory responses that would normally disperse the cellular aggregates. Consequently, CVEC are resuscitated to actively growing cells if autoinducers are replenished, either in the laboratory, or naturally by other environmental bacteria or the intestinal microbiota when CVEC are ingested by humans or aquatic animals. CONCLUSION: Quorum sensing plays a crucial role in the environmental persistence of toxigenic V. cholerae in a latent state, and their periodic emergence to cause cholera outbreaks. Furthermore, the autoinducer driven resuscitation of these cells may be a basis for improving the detection of V. cholerae in water samples, and monitoring V. cholerae in their aquatic reservoirs in cholera endemic areas.

QcrC is a potential target for antibody therapy and vaccination to control Campylobacter jejuni infection by suppressing its energy metabolism.

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.

Detection of prolong excretion of Escherichia albertii in stool specimens of a 7-year-old child by a newly developed Eacdt gene-based quantitative real-time PCR method and molecular characterization of the isolates.

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.

Seasonality of detection rate of Escherichia albertii in wild raccoons (Procyon lotor) in Osaka, Japan.

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.

Characteristics of epitope dominance pattern and cross-variant neutralisation in 16 SARS-CoV-2 mRNA vaccine sera.

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.

Cefixime-tellurite-deoxycholate tryptic soy broth (CTD-TSB), a selective enrichment medium, for enhancing isolation of Escherichia albertii from wild raccoon fecal samples.

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.

Presence of Functionally Active Cytolethal Distending Toxin Genes on a Conjugative Plasmid in a Clinical Isolate of Providencia rustigianii.

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.

Endotoxin-Free Stx2B-C-CPE Vaccine and Its Optimized Adjuvant Regimen for Preventing Food Poisoning.

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.

Comparative evaluation of chlorous acid and sodium hypochlorite activity against SARS-CoV-2.

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 .

Isolation and characterization of Escherichia albertii from wild and safeguarded animals in Okayama Prefecture and its prefectural borders, Japan.

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 virulence genes. 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.

Longitudinal surveillance and comparative characterization of Escherichia albertii in wild raccoons in the United States.

Escherichia albertii is an emerging enteric bacterial pathogen causing watery diarrhea, abdominal distension, vomiting and fever in humans. E. albertii has caused many foodborne outbreaks in Japan and was also reported in other countries worldwide. However, the important animal reservoirs of this pathogen are still largely unknown, impeding us to combat this emerging pathogen. Recently, we reported that wild raccoons (Procyon lotor) and broiler chickens are significant reservoirs of E. albertii in Japan and the U.S., respectively. Here, we performed a longitudinal surveillance to monitor prevalence of E. albertii in wild raccoons in the U.S. and conducted comprehensive comparative analyses of the E. albertii of different origins. A total of 289 fecal swab samples were collected from wild raccoons in Tennessee and Kentucky in the U.S. (2018-2020). Approximately 26% (74/289) of the raccoons examined were PCR-positive for E. albertii and eventually 22 E. albertii isolates were obtained. PFGE analysis showed the U.S. raccoon E. albertii were phylogenetically distant even though the corresponding raccoons were captured from a small area. Unlike the high prevalence of multidrug resistance (83%) observed in previous chicken E. albertii survey, antibiotic resistance was rarely observed in all the U.S. raccoon and 22 Japan raccoon strains with only one Japan strain displaying multidrug resistance (2%). Whole genome sequencing of 54 diverse E. albertii strains and subsequent comparative genomics analysis revealed unique clusters that displayed close evolutionary relationships and similar virulence gene profiles among the strains of different origins in terms of geographical locations (e.g., U.S. and Japan) and hosts (raccoon, chicken, swine, and human). Challenge experiment demonstrated raccoon E. albertii strains could successfully colonize in the chicken intestine at 3 and 8 days postinfection. A pilot environmental survey further showed all the four tested water samples from Tennessee river were E. albertii-positive; two different E. albertii strains, isolated from a single water sample, showed close relationships to those of human origin. Together, the findings from this study provide new insights into the ecology, evolution, and pathobiology of E. albertii, and underscore the need to control the emerging E. albertii in a complex ecosystem using One Health approach.

ShellCoat, a Calcinated Calcium Solution, Effectively Inactivates SARS-CoV-2.

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.

Pufferfish (Tetraodon cutcutia) Sampled from a Freshwater River Serves as an Intermediate Reservoir of a Sucrose Nonfermenting Variant of Vibrio cholerae PS-4.

We describe the genomic characteristics of Vibrio cholerae strain PS-4 that is unable to ferment sucrose on a thiosulfate citrate bile salt sucrose (TCBS) agar medium. This bacterium was isolated from the skin mucus of a freshwater pufferfish. The genome of strain PS-4 was sequenced to understand the sucrose nonfermenting phenotype. The gene encoding the sucrose-specific phosphotransferase system IIB (sucR) was absent, resulting in the defective sucrose fermenting phenotype. In contrast, genes encoding the glucose-specific transport system IIB (ptsG) and fructose-specific transport system IIB (fruA) showed acid production while growing with respective sugars. The overall genome relatedness indices (OGRI), such as in silico DNA-DNA hybridization (isDDH), average nucleotide identity (ANI), and average amino acid identity (AAI), were above the threshold value, that is, 70% and 95 to 96%, respectively. Phylogenomic analysis based on genome-wide core genes and the nonrecombinant core genes showed that strain PS-4 clustered with Vibrio cholerae ATCC 14035T. Further, genes encoding cholera toxin (ctx), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), toxin-coregulated pilus (tcp), and lipopolysaccharide biosynthesis (rfb) were absent. PS-4 showed hemolytic activity and reacted strongly to the R antibody. Therefore, the Vibrio cholerae from the pufferfish adds a new ecological niche of this bacterium. IMPORTANCE Vibrio cholerae is native of aquatic environments. In general, V. cholerae ferments sucrose on thiosulfate citrate bile salt sucrose (TCBS) agar and produces yellow colonies. V. cholerae strain PS-4 described in this study is a sucrose nonfermenting variant associated with pufferfish skin and does not produce yellow colonies on TCBS agar. Genes encoding sucrose-specific phosphotransferase system IIB (sucR) were absent. The observed phenotype in the distinct metabolic pathway indicates niche-specific adaptive evolution for this bacterium. Our study suggests that the nonfermenting phenotype of V. cholerae strains on TCBS agar may not always be considered for species delineation.

Hypochlorous acid solution is a potent antiviral agent against SARS-CoV-2.

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) 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 with the reduction of 104 of 50% tissue culture infectious dose per ml (TCID50 per ml) within 10 s. When its concentration increased to 59.4 ppm, the virus titre decreased to below the detection limit (reduction of 5 logs TCID50 ) within 10 s even in the presence of 0.1% foetal bovine serum. 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 per ml or below the detection limit. Because the titre 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 s. CONCLUSIONS: In a carrier test, 125 ppm HClO solution for 10 min incubation is adequate to inactivate 4 logs TCID50 per ml of SARS-CoV-2 or more while in a suspension test 56.3 ppm HClO is adequate to inactivate 5 logs TCID50 per ml of SARS-CoV-2 when incubated for only 10 s regardless of presence or absence of organic matter. SIGNIFICANCE AND IMPACT OF THE 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 s. 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.

Detection, Isolation, and Molecular Characterization of Escherichia albertii from Wild Birds in West Japan.

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.

Last glacial temperature reconstructions using coupled isotopic analyses of fossil snails and stalagmites from archaeological caves in Okinawa, Japan.

We applied a new geoarchaeological method with two carbonate archives, which are fossil snails from Sakitari Cave and stalagmites from Gyokusen Cave, on Okinawa Island, Japan, to reconstruct surface air temperature changes over the northwestern Pacific since the last glacial period. Oxygen isotope ratios (δ18O) of modern and fossil freshwater snail shells were determined to infer seasonal temperature variations. The observational and analytical data confirm that δ18O values of fluid inclusion waters in the stalagmite can be regarded as those of spring waters at the sites where snails lived. Our results indicate that the annual mean, summer, and winter air temperatures were lower by 6-7 °C at ca. 23 thousand years ago (ka) and 4-5 °C at ca. 16-13 ka than those of the present day. Our reconstruction implies that surface air cooling was possibly two times greater than that of seawater around the Ryukyu Islands during the Last Glacial Maximum, which potentially enhanced the development of the East Asian summer monsoon during the last deglaciation. Considering the potential uncertainties in the temperature estimations, the climatic interpretations of this study are not necessarily definitive due to the limited number of samples. Nevertheless, our new geoarchaeological approach using coupled δ18O determinations of fossil snails and stalagmite fluid inclusion waters will be useful for reconstructing snapshots of seasonally resolved time series of air temperatures during the Quaternary.

Occurrence and multidrug resistance of Campylobacter spp. at duck farms and associated environmental and anthropogenic risk factors in Bangladesh.

BACKGROUND: The alarming rise in multi-drug resistant (MDR) zoonotic pathogens, including Campylobacter spp., has been threatening the health sector globally. In Bangladesh, despite rapid growth in poultry sector little is known about the potential risks of zoonotic pathogens in homestead duck flocks. The aim of this study was to understand the occurrence, species diversity, and multi-drug resistance in Campylobacter spp., and identify the associated risk factors in duck farms in Bangladesh. METHODS: The study involved 20 duck farms at 6 sub-districts of Mymensingh, Bangladesh. Monthly occurrence of Campylobacter spp. in potential sources at the farms during February-September, 2018, was detected by culture and PCR-based methods. Campylobacter isolates were examined for resistance to different antimicrobials. Risk factors, concerning climatic and environmental disposition, farm management, and anthropogenic practices, of Campylobacter infection were estimated by participatory epidemiological tools. RESULTS: Occurrence of Campylobacter spp. was detected in overall 36.90% (155/420) samples, more frequently in drinking water (60%, 30/50), followed by cloacal swab (37.50%, 75/200), egg surface swab (35%, 35/100) and soil of the duck resting places (30%, 15/50) but was not detected in feed samples (n = 20). PCR assays distinguished the majority (61.30%, 95/155) of the isolates as C. coli, while the rest (38.70%, 60/155) were C. jejuni. Notably, 41.7% (25/60) and 31.6% (30/95) strains of C. jejuni and C. coli, respectively, were observed to be MDR. The dynamics of Campylobacter spp., distinctly showing higher abundance during summer and late-monsoon, correlated significantly with temperature, humidity, and rainfall, while sunshine hours had a negative influence. Anthropogenic management-related factors, including, inadequate hygiene practices, use of untreated river water, wet duck shed, flock age (1-6 months), and unscrupulous use of antimicrobials were identified to enhance the risk of MDR Campylobacter infection. CONCLUSION: The present study clearly demonstrates that duck farms contribute to the enhanced occurrence and spread of potentially pathogenic and MDR C. coli and C. jejuni strains and the bacterial dynamics are governed by a combined interaction of environmental and anthropogenic factors. A long-term holistic research at the environment-animal-human interface would be integral to divulge health risk reduction approaches tackling the spread of Campylobacter spp. from duck farms.