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.

An outbreak of type B botulism in southern Viet Nam, 2020.

Objective: To investigate the cause of a botulism outbreak in several provinces in Viet Nam in 2020. Methods: An initial investigation was conducted to confirm the outbreak and to form hypotheses about the potential causes, followed by a case-control assessment of the plausible causative food item. Collected food samples were tested to identify the pathogen, and mouse bioassays were performed. Control measures were introduced to stop the outbreak and to prevent similar events in the future. Results: Twelve people in six southern provinces of Viet Nam were identified as having symptoms of botulism, of whom 11 were in critical condition requiring breathing support. A history of foods eaten in the 4 days before illness onset indicated that all the cases had eaten a tinned vegetarian pâté, and a case-control assessment showed that this was significantly associated with the outbreak, with an odds ratio of 35.2 (95% confidence interval: 3.4-¥). Clostridium botulinum type B was detected in three of eight pâté samples collected from the houses of cases. In the mouse bioassay for the toxicity of the pâté samples, all the mice died with clinical symptoms of botulism. Discussion: A tinned vegetarian pâté was the plausible cause of a botulism outbreak in Viet Nam in 2020. Revision of food safety regulations to improve quality control of tinned foods to prevent future outbreaks is recommended.

Untargeted Phylogenetic Group III of Multi-drug-Resistant Bacillus cereus Isolated Using Fraser Medium from Retail Chickens in Ho Chi Minh City.

The prevalence of food-borne bacteria in developing countries is less well understood than in developed countries. The ISO11290-1 isolation method is commonly used to study Listeria contamination in chicken; however, all isolates are identified as untargeted Bacillus cereus. This study aimed to determine the classification, antibiotic susceptibility, and virulence genes of B. cereus isolated from retail chickens in Vietnam. Bacterial isolation using the ISO11290-1 method yielded 12 strains of B. cereus from seven out of 60 chickens. For determining bacterial diversity, panC and multilocus sequence typing (MLST) analyses were performed. PanC analysis showed that all seven strains belong to the phylogenetic group III, to which the highest risk of foodborne illnesses was associated. MLST analysis showed that most strains contained a ST205 complex; further, all strains were found to be resistant to ampicillin, ciprofloxacin, and tetracycline. Virulence genes were also investigated. ces, a cereulide-related gene, was detected in 50% of the isolated strains, followed by cytK, nheA, and hblA enterotoxins in 41.7%, 16.7%, and 25% of the strains, respectively. In conclusion, B. cereus may be erroneously detected when attempting to detect Listeria in food using the ISO11290-1 method. Further study of the prevalence of B. cereus in Vietnamese food is needed to improve food safety.

Prevalence, Serovar, and Antimicrobial Resistance of Nontyphoidal Salmonella in Vegetable, Fruit, and Water Samples in Ho Chi Minh City, Vietnam.

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 ß-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.

Prevalence and characteristics of extended-spectrum ß-lactamase-producing Escherichia coli in domestic and imported chicken meats in Japan.

The purpose of this study was to investigate the prevalence of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (ESBL-Ec) in retail chicken meats in Japan. Fifty-six domestic and 50 imported (Brazil, n=36; United States, n=8; Thailand, n=6) chicken meat samples were analyzed. The 162 ESBL-Ec included 111 from 43 (77%) domestic samples and 51 from 26 (52%) Brazilian samples. Fifty-three and 30 of 111 and 51 ESBL-Ec from domestic and Brazilian chickens, respectively, were selected for ESBL genotyping. The blaCTX-M (91%), blaTEM (36%) and blaSHV (15%) genes were detected in ESBL-Ec isolated from domestic chickens, whereas blaCTX-M (100%) and blaTEM (20%) were detected in ESBL-Ec isolated from imported chickens. Among the blaCTX-M group, blaCTX-M-2 (45%) and blaCTX-M-1 (34%) were prevalent in domestic chicken isolates, whereas blaCTX-M-2 (53%) and blaCTX-M-8 (43%) were prevalent in imported chicken isolates. Domestic chicken isolates were mostly resistant to tetracycline (83%), followed by streptomycin (70%) and nalidixic acid (62%). Imported chicken isolates were resistant to streptomycin (77%), followed by nalidixic acid (63%) and tetracycline (57%). Notably, extensive multidrug resistance was detected in 60% (32/53) and 70% (21/30) ESBL-Ec from domestic and imported chickens, respectively. Virulence genes associated with diarrheagenic and extra-intestinal pathogenic E. coli were detected in ESBL-Ec isolated from domestic and imported chickens. These data suggest that ESBL-Ec in retail chicken meats could be a potential reservoir for antimicrobial resistance determinants and that some are potentially harmful to humans.

Development of a multiplex PCR targeting eae, stx and cdt genes in genus Escherichia and detection of a novel cdtB gene in Providencia rustigianii.

This study was aimed to develop a multiplex PCR (m-PCR) for the detection of Escherichia coli attaching and effacing (eae), Shiga toxin (stx) and cytolethal distending toxin (cdt) genes encoding important virulence factors of diarrheagenic E. coli such as EPEC, STEC, and Escherichia albertii. For this purpose, the m-PCR was designed to detect eae, all the subtypes of stx (stx1, stx2a-g except stx2f) and cdt (I-V) genes. The m-PCR was validated with 58 and 55 target gene-positive and negative strains of different sources, respectively. Sensitivity and specificity of the m-PCR were 100%. The m-PCR could also detect the eae, stx and cdt genes in bacteria spiked into stool specimens with or without enrichment culture. Clinical specimens collected from children with diarrhea were tested by the m-PCR, and 27 eae and 32 cdt genes were detected. Among them, three cdt-II and one untypable cdt gene-positive bacteria were isolated and identified as E. albertii and Providencia rustigianii, respectively. This is the first report demonstrating the presence of cdtB gene in P. rustigianii. These results indicate that the m-PCR is useful for surveillance of eae, stx and cdt gene-positive bacteria, not only EPEC, STEC and E. albertii but also P. rustigianii.

Antimicrobial resistance profiles and molecular characterization of Escherichia coli strains isolated from healthy adults in Ho Chi Minh City, Vietnam.

In this study, we attempted to isolate Escherichia coli from healthy adults in Ho Chi Minh City, Vietnam, and characterized its antimicrobial resistance profile, extended-spectrum ß-lactamase (ESBL) genotype, phylogenetic grouping and virulence gene profile. A total of 103 E. coli isolates were obtained, and most of them were antimicrobial resistant such to streptomycin (80.6%), tetracycline (67.0%), ampicillin (65.0%), sulfamethoxsazole/trimethoprim (48.5%), nalidixic acid (43.7%), chloramphenicol (34.0%), cefotaxime (15.5%), ciprofloxacin (15.5%), kanamycin (12.6%), ceftazidime (10.7%), fosfomycin (4.9%) and gentamicin (2.9%). However, all these E. coli strains were susceptible to imipenem. Surprisingly, of 103 strains, 74 (71.8%) and 43 (41.7%) strains showed resistance to more than 3 and 5 classes of antimicrobials, respectively. Furthermore, 10 E. coli strains were ESBL-producers and positive for blaCTX-M genes (7 for blaCTX-M-9 and 3 for blaCTX-M-1), while five were additionally positive for blaTEM genes. S1-nuclease pulsed-field gel electrophoresis analysis revealed that 7 and 3 strains of E. coli carry blaCTX-M genes on their large plasmid and chromosome, respectively. Phylogenetic analysis exhibited that majority of the E. coli strains was grouped into A (44.7%), followed by B1 (23.3%), B2 (18.4%) and D (13.6%). Virulence genes associated with diarrheagenic E. coli, such as astA, EAF, eaeA, elt and eagg were also detected in ESBL-producing E. coli as well as antimicrobial resistant strains. These data suggest that commensal E. coli of healthy human could be a reservoir for antimicrobial resistance determinants and some of them might be harmful to human.