Zhenquan Yang

Isolation and molecular characterization of Vibrio parahaemolyticus from fresh, low-temperature preserved, dried, and salted seafood products in two coastal areas of eastern China

A total of 1293 seafood samples from fishing farm, retail markets, restaurants and cooking rooms of hotels in Jiangsu province and Shanghai city of China were collected and analyzed for the prevalence of Vibrio parahaemolyticus during July to October in 2007. Two hundred and fifty one isolates of V. parahaemolyticus were identified, of which 8 isolates were positive for tdh and 2 were positive for trh gene. Three tdh positive isolates were identified from low-temperature preserved seafood samples and 5 isolates from fresh seafood samples, of these tdh positive isolates, 3 were positive in ORF8-PCR test. The genetic diversity among V. parahaemolyticus isolates was assessed using random amplified polymorphic DNA (RAPD)-PCR and the results showed that there were 33 different genetic patterns that were clustered into nine groups (groups A to I) at 82% similarity level. About 31.9% of the isolates belong to type III9d that were widely distributed in fresh, iced, frozen, dried and salted seafood samples. Seven tdh positive isolates belonged to group A and one belonged to group C, 2 trh positive isolates were type I10d belonging to group F, which was identical to that of reference strains isolated from patients. This study demonstrated genetic variability within V. parahaemolyticus isolates from seafood in Chinese markets and confirmed the presence of toxigenic V. parahaemolyticus not only in fresh but also in iced and frozen seafood products indicating that low-temperature preserved seafood might be also a vehicle for transmitting pathogenic V. parahaemolyticus.

Predictive model of Vibrio parahaemolyticus growth and survival on salmon meat as a function of temperature

The growth and survival curves of a strain of pandemic Vibrio parahaemolyticus TGqx01 (serotype O3:K6) on salmon meat at different storage temperatures (range from 0 degrees C to 35 degrees C) were determined. In order to model the growth or inactivation kinetics of this pathogen during storage, the modified Gompertz and Weibull equations were chosen to regress growth and survival curves, respectively, and both equations produced good fit to the observed data (the average R2 value equals to 0.990 for modified Gompertz and 0.920 for Weibull equation). The effect of storage temperature on the specific growth rate (mu) was modeled by square root type equation, and the relationship between mu and lag time (lambda) was described by a rule of mu x lambda = constant. The shape factor (n) and scale factor (b) values of the Weibull equations versus the temperature (degrees C) were plotted and the temperature effects on these parameters were described by two linear empirical equations. The predicted growth and survival curves from the model were compared to real enumeration results, using the correlation coefficient (R2), bias factor (Bf) and accuracy factor (Af), to assess the performance of the established model. The results showed that the overall predictions for V. parahaemolyticus TGqx01 growth or inactivation on salmon at tested temperatures agreed well with observed plate counts, and the average R2, Bf and Af values were 0.958, 1.019 and 1.035, respectively.

Serodiversity, Pandemic O3:K6 Clone, Molecular Typing, and Antibiotic Susceptibility of Foodborne and Clinical Vibrio parahaemolyticus Isolates in Jiangsu, China

Vibrio parahaemolyticus is a major foodborne pathogen in China, Japan, and other Asian countries. In this study, we collected 437 strains of V. parahaemolyticus and investigated their serotypes, distribution of virulence genes, and presence of pandemic O3:K6 clone strains. A total of 327 strains were isolated from food and 110 strains were isolated from active surveillance hospitals or food outbreaks during 2005 to 2008. Presence of the tdh and trh genes is the key characteristic of virulent strains. Positive for both the tdh gene and group-specific polymerase chain reaction is the key characteristic of pandemic strains. A total of 9 O serogroups and 62 serovars were identified in all strains. Nine O serogroups and 56 serovars existed in 327 foodborne strains, and 6 O serogroups and 20 serovars existed in 110 clinical strains. Among the 327 food isolates, 6 isolates belonged to the pandemic clone with the orf8 gene (1 isolate was O1:KUT (untyped) and 5 isolates were O3:K6) and 4 isolates carried the trh gene (2 isolates belonged to O1:KUT and 2 isolates belonged to O5:KUT and O5:K17). Seventy-nine percent of the clinical isolates were pandemic strains, 9.4% of which lacked the orf8 gene. O3:K6 was the main serovar of the pandemic strains accounting for 83.5% of the clinical pandemic strains. Pandemic clonal serovars included O3:K6, O1:KUT, O1:K25, O1:K26, and O4:K68, and the newly emerging serovars O1:K36, O3:K25, and O3:K68 identified in the current study. O3:K6 was the dominant serovar in pandemic strains. All pandemic isolates had identical arbitrarily primed polymerase chain reaction fragment patterns, but did not share similar antibiotic sensitivity patterns. These results suggest that high serodiversity of V. parahaemolyticus was present in foodborne strains. Pathogenic isolates, especially pandemic isolates, were present in high-priced iced seafood and became the potential risk factor in food.

Distribution of Genes Encoding Four Pathogenicity Islands (VPaIs), T6SS, Biofilm, and Type I Pilus in Food and Clinical Strains of Vibrio parahaemolyticus in China

Vibrio parahaemolyticus is a major cause of foodborne gastroenteritis in China, Japan, and other countries. The pandemic O3:K6 clone, which harbors thermostable direct hemolysin [tdh] gene and toxRS/new gene, is mainly responsible for the foodborne outbreaks after 1995. Previous studies showed that genes in the pathogenicity island-1 (VPaI-1) and VPaI-5 are harbored only by pandemic strains, whereas genes in VPaI-7 and type III secretion system 2 are closely associated with tdh-positive strains of V. parahaemolyticus. In this study, we examined the distribution of genes encoding VPaI-2, VPaI-3, VPaI-4, VPaI-6, type VI secretion systems (T6SS), biofilm, and type I pilus in 71 food and 116 clinical strains of V. parahaemolyticus. The results showed that most of the pandemic strains of V. parahaemolyticus harbored the complete genes of VPaI-2, T6SS, and type I pilus. In contrast, most of the pathogenic strains (harboring tdh gene or TDH-related hemolysin [trh] gene) and nonpathogenic strains (harboring neither tdh gene nor trh gene) contained partial genes of VPaI-2, T6SS, and type I pilus. Genes of VPaI-4 were exclusively present in the pandemic strains. Genes of VPaI-3 were present in most of the pandemic strains and a small percentage of nonpathogenic strains, mainly O3:K6 strains. VPaI-6 and biofilm-associated genes were harbored by almost all the strains, irrespective of their pandemic, pathogenic, or nonpathogenic traits.

Probiotic properties of lactic acid bacteria isolated from stool samples of longevous people in regions of Hotan, Xinjiang and Bama, Guangxi, China

A total of 567 lactic acid bacteria (LAB) strains were isolated from the stool samples of longevous people in regions of Hotan, Xinjiang and Bama, Guangxi, China. In order to reduce the number of strains for further examinations, 36 isolates were screened out for further examination whilst the other strains, which had lower probiotic properties, were not suitable for yogurt production due to the absence of growth in pH 3.5 MRS medium and no curding during fermentation, and so were excluded. The result of identification by API, sequence analysis of 16S rRNA and random amplified polymorphic DNA (RAPD) analysis showed that there were three strains of Lactobacillus acidophilus, 10 strains of Lactobacillus rhamnosus, three strains of Lactobacillus casein, three strains of Lactobacillus brevis, two strains of Enterococcus faecium, two strains of Enterococcus faecalis, four strains of Bifdibacterium infantis, three strains of Bifdibacterium brevise, three strains of Bifdibacterium bifidium, two strains of Bifdibacterium adolecentis and one strain of Bifdibacterium longam among the 36 isolates. These strains were evaluated by in vitro methods including survival upon exposure to pH 2.0, 3.0 and/or 0.3% oxgall and adhesion to the human colon adenocarcinoma cell line Caco-2 as well as antimicrobial activity against potential pathogens. The results presented here show that L. rhamnosus LV108, L. rhamnosus F, B. brevise R39 and B. infantis R42 are acid and bile tolerant, adhere to the cultured human intestinal Caco-2 cell line, antagonistic activity against potential pathogenic bacteria infection in vitro, and so are potential strains for probiotic use.

The effect of Lactobacillus rhamnosus hsryfm 1301 on the intestinal microbiota of a hyperlipidemic rat model.

BackgroundGrowing evidence indicates that intestinal microbiota regulate our metabolism. Probiotics confer health benefits that may depend on their ability to affect the gut microbiota. The objective of this study was to examine the effect of supplementation with the probiotic strain, Lactobacillus rhamnosus hsryfm 1301, on the gut microbiota in a hyperlipidemic rat model, and to explore the associations between the gut microbiota and the serum lipids.MethodsThe hyperlipidemic rat model was established by feeding rats a high-fat diet for 28 d. The rats’ gut microbiota were analyzed using high-throughput sequencing before and after L. rhamnosus hsryfm 1301 supplementation or its fermented milk for 28 d. The serum lipids level was also tested.ResultsThe rats’ primary gut microbiota were composed of Bacteroidetes, Firmicutes, Proteobacteria, Spirochaetes and Verrucomicrobia. The abundance and diversity of the gut microbiota generally decreased after feeding with a high-fat diet, with a significant decrease in the relative abundance of Bacteroidetes, but with an increase in that of Firmicutes (P < 0.05). Administration of L. rhamnosus hsryfm 1301 or its fermented milk for 28 d, could recover the Bacteroidetes and Verrucomicrobia abundance and could decrease the Firmicutes abundance, which was associated with a significant reduction in the serum lipids’ level in the hyperlipidemic rats with high-fat diet induced. The abundance of 22 genera of gut bacteria was changed significantly after probiotic intervention for 28 d (P < 0.05). A positive correlation was observed between Ruminococcus spp. and serum triglycerides, Dorea spp. and serum cholesterol (TC) and low-density lipoprotein (LDL-C), and Enterococcus spp. and high-density lipoprotein. The Butyrivibrio spp. negatively correlated with TC and LDL-C.ConclusionsOur results suggest that the lipid metabolism of hyperlipidemic rats was improved by regulating the gut microbiota with supplementation of L.rhamnosus hsryfm 1301 or its fermented milk for 28 d.

Systematic functional pandemic strain-specific genes, three genomic islands, two T3SSs in foodborne, and clinical Vibrio parahaemolyticus isolates in China.

Vibrio parahaemolyticus is one of the most important pathogens capable of causing foodborne gastroenteritis in China, Japan, and other countries. Pathogenic V. parahaemolyticus has been known to produce either thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), or both. The emergence of a new clone in 1995, V. parahaemolyticus O3:K6, has resulted in the first documented pandemic spread of V. parahaemolyticus. In this study, 235 isolates from clinical and food sources were characterized by determining the presence of known virulence factors (tdh, trh), systematic genetic markers (toxRS/new, pandemic group-specific sequence [PGS], orf8) specific for V. parahaemolyticus O3:K6 clone and its clonal derivatives, three important genomic islands (GIs) (VPaI-1, VPaI-5, and VPaI-7), and two type III secretion systems (T3SS1 and T3SS2). Our results showed that all 235 isolates harbored all or part of the T3SS1 genes. All the 103 tdh-positive strains harbored all or part of the VPaI-7 and T3SS2 genes. A total of 91 isolates including six foodborne isolates belonged to a pandemic clone in which eight isolates lacked orf8. All pandemic strains harbored VPaI-1 and VPaI-5 except one O4:K68 strain that lacked VPaI-5 altogether. Twelve clinical pathogenic strains had VPaI-7 and T3SS2 but lacked VPaI-1 and VPaI-5. Thirteen nonpathogenic clinical strains and 119 foodborne strains, including six foodborne pathogenic trh-positive strains, only harbored T3SS1 genes. These results indicated that O3:K6 and its serovariants were the main pandemic clone in China. VPaI-1 and VPaI-5 genes were specifically correlated with pandemic strains while VPaI-7 and T3SS2 were closely associated with tdh-positive strains.

Vibrio parahaemolyticus Strains of Pandemic Serotypes Identified from Clinical and Environmental Samples from Jiangsu, China

Vibrio parahaemolyticus has emerged as a major foodborne pathogen in China, Japan, Thailand, and other Asian countries. In this study, 72 strains of V. parahaemolyticus were isolated from clinical and environmental samples between 2006 and 2014 in Jiangsu, China. The serotypes and six virulence genes including thermostable direct hemolysin (TDR) and TDR-related hemolysin (TRH) genes were assessed among the isolates. Twenty five serotypes were identified and O3:K6 was one of the dominant serotypes. The genetic diversity was assessed by multilocus sequence typing (MLST) analysis, and 48 sequence types (STs) were found, suggesting this V. parahaemolyticus group is widely dispersed and undergoing rapid evolution. A total of 25 strains of pandemic serotypes such as O3:K6, O5:K17, and O1:KUT were identified. It is worth noting that the pandemic serotypes were not exclusively identified from clinical samples, rather, nine strains were also isolated from environmental samples; and some of these strains harbored several virulence genes, which may render those strains pathogenicity potential. Therefore, the emergence of these “environmental” pandemic V. parahaemolyticus strains may poses a new threat to the public health in China. Furthermore, six novel serotypes and 34 novel STs were identified among the 72 isolates, indicating that V. parahaemolyticus were widely distributed and fast evolving in the environment in Jiangsu, China. The findings of this study provide new insight into the phylogenic relationship between V. parahaemolyticus strains of pandemic serotypes from clinical and environmental sources and enhance the MLST database; and our proposed possible O- and K- antigen evolving paths of V. parahaemolyticus may help understand how the serotypes of this dispersed bacterial population evolve.

In vitro and in vivo Studies on the Antioxidant Effects of Soymilk Fermented with Streptococcus thermophilus grx02

Soymilk fermented with Streptococcus thermophilus grx02 (FSM) was evaluated for its antioxidant properties. Antioxidant effects of FSM in vitro were measured with 2, 2-diphenyl-1-picrylhydrazyl (DPPH) based assay and by determining the inhibitory effect on the formation for malonyldialdehyde (MDA), and on the ability to chelate ferrous ions; the respective average IC50 values were 49.5, 24.2 and 8.7 mg/mL. Antioxidant enzymatic activities in liver of alcohol-fed mice, biochemical markers in serum and an index of lipid peroxidation were determined, and additionally the changes in hepatocytes of ICR mice were compared. The results showed that, compared with the control group, alanine transaminase (ALT) activity, aspartate transaminase (AST) activity and triglycerides (TG) of negative group increased (p < 0.05). Moreover, the serum ALT and AST activities of FSM group was lower than negative group (p < 0.05). However, there was no significant difference (p > 0.05) in the albumin (ALB) and total protein (TP) values (in positive, negative, control, and FSM groups) in the serum. Compared with the negative group, alcohol dehydrogenase (ADH), superoxide dismutase (SOD) activities and glutathione (GSH) content increased in liver (p < 0.05) in FSM group. The MDA of FSM group in liver was lower than the negative group (p < 0.05).

iTRAQ-based quantitative proteomic analysis of dark-germinated soybeans in response to salt stress

Soybean germination under stressful conditions, especially salt stress, has been verified to be an effective way of accumulating gamma-aminobutyric acid (GABA) in dark-germinated soybeans. In this study, a combination of physiological characteristics and isobaric tags for relative and absolute quantitation (iTRAQ) in a proteomic-based approach was used to investigate the protein changes in dark-germinated soybeans under salt stress. A total of 201 differentially abundant proteins (DAPs) were identified and divided into 13 functional groups. Under salt stress, 20 metabolic pathways were significantly enriched in dark-germinated soybeans. GABA content and antioxidase activity were increased while the growth and development of soybeans were inhibited by the salt stress. Promoting the synthesis of ROS-scavenging enzymes, maintaining the protein metabolic balance and re-establishing cellular homeostasis were very important strategies for growth stimulation in response to salt stress. In summary, these results showed comprehensive proteome coverage of dark-germinated soybeans in response to salt treatment, and increased our understanding of the molecular processes involved in plant networks responding to stresses.