A Comprehensive Epidemiological Research for Clinical Vibrio parahaemolyticus in Shanghai

Vibrio parahaemolyticus is one of the most important pathogen for seafood-borne gastroenteritis in Shanghai and the rest of the world. A total of 42 V. parahaemolyticus strains were isolated from 1900 fecal specimens collected from patients in Shanghai hospital presenting from January 2014 to December 2015. All isolates were evaluated for potential virulence factors [tdh, trh, and type three secretion system (T3SS) genes], typed using multilocus sequence typing (MLST) and screened for antimicrobial resistance phenotype and genotype. And for the first time, the relationship between virulence, genetic diversity and antimicrobial resistance of these isolates were identified. The results showed that 37 isolates carried the tdh gene (88.1%) and only seven isolates were positive for the trh gene. The T3SS1 and T3SS2 genes were detected in all strains and only trh-positive isolates are also containing the T3SS2β genes. MLST analysis of the 42 Shanghai isolates identified 20 sequence types (STs) with 16 novel STs and that these clinical V. parahaemolyticus strains showed high degrees of genetic diversity. All isolates expressed high levels of resistance against Ampicillin (100.0%), Streptomycin (100.0%), Cephazolin (92.9%), Kanamycin (92.8%) and Amikacin (90.5%), and eight out of 38 resistance genes (SHV, tet(B), strA, qnrA, gryA, qnrB, sulI, sulII) were detected in at least two isolates. This study confirms that antimicrobial resistance of clinical V. parahaemolyticus isolates is greater than those of environmental isolates. Furthermore, no clear correlation between antimicrobial resistance and virulence or genetic diversity was found in this study. These results add to epidemiological data of clinical V. parahaemolyticus isolates in Shanghai and highlight the need for additional mechanistic studies, especially antimicrobial resistance, to reduce the burden of disease caused by this pathogen in China

A mcr-1-Carrying Conjugative IncX4 Plasmid in Colistin-Resistant Escherichia coli ST278 Strain Isolated From Dairy Cow Feces in Shanghai, China

Enterobacteriaceae, including Escherichia coli, has been shown to acquire the colistin resistance gene mcr-1. A strain of E. coli, EC11, which is resistant to colistin, polymyxin B and trimethoprim-sulfamethoxazole, was isolated in 2016 from the feces of a dairy cow in Shanghai, China. Strain EC11 identifies with sequence type ST278 and is susceptible to 19 frequently used antibiotics. Whole genome sequencing of strain EC11 showed that this strain contains a 31-kb resistance plasmid, pEC11b, which belongs to the IncX4 group. The mcr-1 gene was shown to be inserted into a 2.6-kb mcr-1-pap2 cassette of pEC11b. Plasmid pEC11b also contained putative conjugal transfer components, including an oriT-like region, relaxase, type IV coupling protein, and type IV secretion system. We were successful in transferring pEC11b to E. coli C600 with an average transconjugation efficiency of 4.6 × 10-5. Additionally, a MLST-based analysis comparing EC11 and other reported mcr-positive E. coli populations showed high genotypic diversity. The discovery of the E. coli strain EC11 with resistance to colistin in Shanghai emphasizes the importance of vigilance in detecting new threats like mcr genes to public health. Detection of mcr genes helps in tracking, slowing, and responding to the emergence of antibiotic resistance in Chinese livestock farming

High Correlation Between Structure Development and Chemical Variation During Biofilm Formation by Vibrio parahaemolyticus

The complex three-dimensional structure of biofilms is supported by extracellular polymeric substances (EPSs) and additional insight on chemical variations in EPS and biofilm structure development will inform strategies for control of biofilms. Vibrio parahaemolyticus VPS36 biofilm development was studied using confocal laser scanning microscopy (CLSM) and Raman spectroscopy (RM). The structural parameters of the biofilm (biovolume, mean thickness, and porosity) were characterized by CLSM and the results showed that VPS36 biofilm formed dense structures after 48 h incubation. There were concurrent variations in carbohydrates and nucleic acids contents in the EPS as evidenced by RM. The Raman intensities of the chemical component in EPS, measured using Pearson’s correlation coefficient, were positively correlated with biovolume and mean thickness, and negatively correlated with porosity. The Raman intensity for carbohydrates correlated closely with mean thickness (p-value < 0.01) and the Raman intensity for nucleic acid correlated closely with porosity (p-value < 0.01). Additional evidence for these correlations were confirmed using scanning electron microscopic (SEM) and crystal violet staining

Removal of Foodborne Pathogen Biofilms by Acidic Electrolyzed Water

Biofilms, which are complex microbial communities embedded in the protective extracellular polymeric substances (EPS), are difficult to remove in food production facilities. In this study, the use of acidic electrolyzed water (AEW) to remove foodborne pathogen biofilms was evaluated. We used a green fluorescent protein-tagged Escherichia coli for monitoring the efficiency of AEW for removing biofilms, where under the optimal treatment conditions, the fluorescent signal of cells in the biofilm disappeared rapidly and the population of biofilm cells was reduced by more than 67%. Additionally, AEW triggered EPS disruption, as indicated by the deformation of the carbohydrate C-O-C bond and deformation of the aromatic rings in the amino acids tyrosine and phenylalanine. These deformations were identified by EPS chemical analysis and Raman spectroscopic analysis. Scanning electron microscopy (SEM) images confirmed that the breakup and detachment of biofilm were enhanced after AEW treatment. Further, AEW also eradicated biofilms formed by both Gram-negative bacteria (Vibrio parahaemolyticus) and Gram-positive bacteria (Listeria monocytogenes) and was observed to inactivate the detached cells which are a potential source of secondary pollution. This study demonstrates that AEW could be a reliable foodborne pathogen biofilm disrupter and an eco-friendly alternative to sanitizers traditionally used in the food industry

Safety assessment of Bifidobacterium longum JDM301 based on complete genome sequences

AIM: To assess the safety of Bifidobacterium longum (B. longum) JDM301 based on complete genome sequences

Detection limit of Clostridium botulinum spores in dried mushroom samples sourced from China

A survey of dried mushrooms (Lentinula edodes (Shiitake) and Auricularia auricula (Wood Ear)) sourced from China was carried out to determine the natural contamination of these mushrooms with spores of proteolytic Clostridium botulinum and non-proteolytic C. botulinum. The mushrooms were collected from supermarkets and retailers in 21 cities in China during October 2008. Spore loads of C. botulinum in mushrooms have a degree of uncertainty and variability and this study contributes valuable data for determining prevalence of spores of C. botulinum in mushrooms. An optimized detection protocol that combined selective enrichment culture with multiplex PCR was used to test for spores of proteolytic and non-proteolytic C. botulinum. Detection limits were calculated, using a maximum likelihood protocol, from mushroom samples inoculated with defined numbers of spores of proteolytic C. botulinum or non-proteolytic C. botulinum. Based on the maximum likelihood detection limit, it is estimated that dried mushroom A. auricula contained <550spores/kg of proteolytic C. botulinum, and <350spores/kg of non-proteolytic C. botulinum. Dried L. edodes contained <1500spores/kg of proteolytic C. botulinum and it was not possible to determine reliable detection limits for spores of non-proteolytic C. botulinum using the current detection protocol