Xiujuan Zhou

Development of a real time PCR assay for rapid detection of Vibrio parahaemolyticus from seafood.

A real time PCR assay for the detection of Vibrio parahaemolyticus in seafood samples was developed using a novel specific target and a competitive internal amplification control (IAC). The specificity of this assay was evaluated using 390 bacterial strains including V. parahaemolyticus, and other strains belonging to Vibrio and non-Vibrio species. The real time PCR assay unambiguously distinguished V. parahaemolyticus with a detection sensitivity of 4.8 fg per PCR with purified genomic DNA or 1 CFU per reaction by counting V. parahaemolyticus colonies. The assays of avoiding interference demonstrated that, even in the presence of 2.1 μg genomic DNA or 107 CFU background bacteria, V. parahaemolyticus could still be accurately detected. In addition, the IAC was used to indicate false-negative results, and lower than 94 copies of IAC per reaction had no influence on the detection limit. Ninety-six seafood samples were tested, of which 58 (60.4%) were positive, including 3 false negative results. Consequently, the real time PCR assay is effective for the rapid detection of V. parahaemotyticus contaminants in seafood.

Mutation of a Salmonella serogroup-C1-specific gene abrogates O7-antigen biosynthesis and triggers NaCl-dependent motility deficiency.

Several molecular detection marker genes specific for a number of individual Salmonella serogroups have been recently identified in our lab by comparative genomics for the genotyping of diverse serogroups. To further understand the correlation between serotype and genotype, the function of a Salmonella serogroup-C1-specific gene (SC_2092) was analyzed in this study. It was indicated from the topological prediction using the deduced amino acid sequence of SC_2092 that this putative protein was highly similar to the confirmed Wzx flippases. Furthermore, SDS-PAGE revealed that lipopolysaccharide (LPS) biosynthesis, specifically O-antigen synthesis, was incomplete in an SC_2092 in-frame deletion mutant, and no agglutination reaction with the O7 antibody was exhibited in this mutant. Therefore, it was revealed that this Salmonella serogroup-C1-specific gene SC_2092 encoded a putative flippase, which was required for O7-polysaccharide biosynthesis, and was designated here as wzxC1. Subsequently, the effects of the deletion of wzxC1 on bacterial motility and sodium chloride (NaCl) tolerance were evaluated. The wzxC1 mutant lacked swarming motility on solid surfaces and was impaired in swimming motility in soft agar. Moreover, microscopic examination and RT-qPCR exhibited that an increased auto-aggregation and a strong defect in flagella expression, respectively, were responsible for the reduced motility in this mutant. In addition, the wzxC1 mutant was more sensitive than the wild-type strain to NaCl, and auto-aggregation of mutant cells was observed immediately up on the addition of 1% NaCl to the medium. Interestingly, the motility deficiency of the mutant strain, as well as the cell agglomeration and the decrease in flagellar expression, were relieved in a NaCl-free medium. This is the first study to experimentally demonstrate a connection between a Salmonella serogroup specific gene identified by comparative genomics with the synthesis of a specific O-antigen biosynthesis. Also, our results show that the mutation of wzxC1 triggers a NaCl-dependent motility deficiency.

Impact of Sod on the Expression of Stress-Related Genes in Listeria monocytogenes 4b G with/without Paraquat Treatment

Listeria monocytogenes is a foodborne pathogen that causes listeriosis. Paraquat can generate reactive oxygen species (ROS) in cells, which results in oxidative stress. It was first shown that 1 mM of paraquat inhibited the growth rate of a superoxide dismutase (sod)-deletion mutant (∆sod) generated from L. monocytogenes 4b G but not in the wild-type, and induced the expression of other resistance genes (kat, fri, perR, sigB, and recA) as well as sod in the wild type. Interestingly, without paraquat treatment the expression of all the 5 genes were repressed in ∆sod compared to the wild type, while the expression of recA triggering SOS response, a global response to DNA damage, was increased in ∆sod in the presence of 1 mM paraquat. Taken together, these results suggest that SOD plays a central role in oxidant defense of L. monocytogenes 4b G, and SOS probably significantly impacts ∆sod survival under oxidative stress.

IncHI2 Plasmids Are Predominant in Antibiotic-Resistant Salmonella Isolates

The wide usage of antibiotics contributes to the increase in the prevalence of antibiotic-resistant Salmonella. Plasmids play a critical role in horizontal transfer of antibiotic resistance markers in Salmonella. This study aimed to screen and characterize plasmid profiles responsible for antibiotic resistance in Salmonella and ultimately to clarify the molecular mechanism of transferable plasmid-mediated antibiotic resistance. A total of 226 Salmonella isolates were examined for antimicrobial susceptibility by a disk diffusion method. Thirty-two isolates (14.2%) were resistant to at least one antibiotic. The presence of plasmid-mediated quinolone resistance (PMQR) genes and β-lactamase genes were established by PCR amplification. PCR-based replicon typing revealed that these 32 isolates represented seven plasmid incompatibility groups (IncP, HI2, A/C, FIIs, FIA, FIB, and I1), and the IncHI2 (59.4%) was predominant. Antibiotic resistance markers located on plasmids were identified through plasmid curing. Fifteen phenotypic variants were obtained with the curing efficiency of 46.9% (15/32). The cured plasmids mainly belong to the HI2 incompatibility group. The elimination of IncHI2 plasmids correlated with the loss of β-lactamase genes (blaOXA-1 and blaTEM-1) and PMQR genes (qnrA and aac(6′)-Ib-cr). Both IncHI2 and IncI1 plasmids in a S. enterica serovar Indiana isolate SJTUF 10584 were lost by curing. The blaCMY -2-carrying plasmid pS10584 from SJTUF 10584 was fully sequenced. Sequence analysis revealed that it possessed a plasmid scaffold typical for IncI1 plasmids with the unique genetic arrangement of IS1294-ΔISEcp1-blaCMY -2-blc-sugE-ΔecnR inserted into the colicin gene cia. These data suggested that IncHI2 was the major plasmid lineage contributing to the dissemination of antibiotic resistance in Salmonella and the activity of multiple mobile genetic elements may contribute to antibiotic resistance evolution and dissemination between different plasmid replicons.

Ethanol adaptation induces direct protection and cross protection against freezing stress in Salmonella enterica serovar Enteritidis

Salmonella enterica serovar Enteritidis (Salm. Enteritidis) encounters mild ethanol stress during its life cycle. However, adaptation to a stressful condition may affect bacterial resistance to subsequent stresses. Hence, this work was undertaken to investigate the influences of ethanol adaptation on stress tolerance of Salm. Enteritidis.

Quantitative proteomics reveals the crucial role of YbgC for Salmonella enterica serovar Enteritidis survival in egg white

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a food-borne bacterial pathogen that can cause human salmonellosis predominately by contamination of eggs and egg products. However, its survival mechanisms in egg white are not fully understood, especially from a proteomic point of view. In this study, the proteomic profiles of S. Enteritidis in Luria-Bertani (LB) broth containing 50% and 80% egg white, and in whole egg white were compared with the profile in LB broth using iTRAQ technology to identify key proteins that were involved in S. Enteritidis survival in egg white. It was found that there were 303, 284 and 273 differentially expressed proteins in S. Enteritidis after 6 h exposure to whole, 80% and 50% egg white, respectively. Most of up-regulated proteins were primarily associated with iron acquisition, cofactor and amino acid biosynthesis, transporter, regulation and stress responses, whereas down-regulated proteins were mainly involved in energy metabolism, virulence as well as motility and chemotaxis. Three stress response-related proteins (YbgC, TolQ, TolA) of the tol-pal system responsible for maintaining cell membrane stability of Gram-negative bacteria were up-regulated in S. Enteritidis in response to whole egg white. Interestingly, deletion of ybgC resulted in a decreased resistance of S. Enteritidis to egg white. Compared with the wild type and complementary strains, a 3-log population reduction was observed in △ybgC mutant strain after incubation in whole egg white for 24 h. Cellular morphology of △ybgC mutant strain was altered from rods to spheres along with cell lysis in whole egg white. Furthermore, deletion of ybgC decreased the expression of tol-pal system-related genes (tolR, tolA). Collectively, these proteomic and mutagenic analysis reveal that YbgC is essential for S. Enteritidis survival in egg white.

Comprehensive Analysis Reveals Two Distinct Evolution Patterns of Salmonella Flagellin Gene Clusters

Salmonella is one of the primary causes of foodborne disease, especially Salmonella enterica subsp. enterica (I) which has caused ~99% of clinical salmonellosis cases for humans and domestic mammals. The flagella genes, fliC and fljB, which encode the Salmonella phase 1 and phase 2 antigens respectively, are considered as the Salmonella serotype determinant genes, and contribute to the virulence of Salmonella. However, the evolution of the two flagellin genes is still not well-understood. In this study, the fliC and fljB gene clusters were analyzed among 205 S. enterica subspecies I genomes. The dataset covered 87 different serovars of S. enterica subsp. enterica and included 9 genomes (six serovars) of four other Salmonella subspecies. Based on a pan-genome definition and flanked gene linkages, the fliC and fljB gene clusters were identified in 207 (91 serovars) and 138 (61 serovars) genomes, respectively. A phylogenetic tree constructed based on SNPs (Single Nucleotide Polymorphisms) of core genes were used to reflect the essential evolutionary relationships among various serovars. Congruence analysis was performed among the core genome and each gene of fliC and fljB gene clusters, with only fliA and fliS showing congruence to Salmonella core genome. Congruence was also observed among fliB, fliC/fljB, and fliD genes, and their phylogeny revealed a division into two major groups, which strongly corresponded to monophasic and biphasic serovars. Besides, homologous recombination events referring fliB, fliC, and fliD were found to have mainly occurred within each group. These results suggested two distinct evolutionary patterns of Salmonella flagellin gene clusters. Further insight on the evolutionary implication of the two patterns and a framework for phase variation mechanism are needed to be further processed.

Genome-Scale Screening and Validation of Targets for Identification of Salmonella enterica and Serovar Prediction

Salmonella enterica is the most common foodborne pathogen worldwide, with 2,500 recognized serovars. Detection of S. enterica and its classification into serovars are essential for food safety surveillance and clinical diagnosis. The PCR method is useful for these applications because of its rapidity and high accuracy. We obtained 412 candidate detection targets for S. enterica using a comparative genomics mining approach. Gene ontology (GO) functional enrichment analysis of these candidate targets revealed that the GO term with the largest number of unigenes with known function (38 of 177, 21.5%) was significantly involved in pathogenesis (P < 10(-24)). All the candidate targets were then evaluated by PCR assays. Fifteen targets showed high specificity for the detection of S. enterica by verification with 151 S. enterica strains and 34 non-Salmonella strains. The phylogenetic trees of verified targets were highly comparable with those of housekeeping genes, especially for differentiating S. enterica strains into serovars. The serovar prediction ability was validated by sequencing one target (S9) for 39 S. enterica strains belonging to six serovars. Identical mutation sites existed in the same serovar, and different mutation sites were found in diverse serovars. Our findings revealed that 15 verified targets can be potentially used for molecular detection, and some of them can be used for serotyping of S. enterica strains.