Chunlei Shi

Genetic diversity and virulence potential of Staphylococcus aureus isolates from raw and processed food commodities in Shanghai

The risk of zoonotic transmission to humans highlights the need to understand the molecular ecology of Staphylococcus aureus in foods. In this study, 142 S. aureus isolates obtained from various raw and processed foods from Shanghai, China were characterized to determine their genetic diversity and virulence gene content. A total of 16 clonal complexes (CCs), 34 staphylococcal protein A (spa) types, and 6 accessory gene regulator (agr) allelic groups were identified and analyzed among the 142 S. aureus isolates. Among these, the genotype CC188-t189-agr Ι was the most prevalent, constituting 28.2% of all isolates. The presence of virulence genes encoding 20 staphylococcal enterotoxins (se), toxic shock syndrome toxin (tsst1), exfoliative toxins (eta, etb, and etd), Panton-Valentine leukocidin (lukS-PV and lukF-PV), as well as methicillin resistance gene (mecA), was determined by PCR. Of these S. aureus isolates, 72.5% harbored toxin genes, in which the most frequent toxin gene was sep (43.7%), followed by sej (26.1%) and pvl (21.1%). In contrast, see, ses, set, tsst1, etb, and etd were not found in any of the isolates tested. Eight S. aureus isolates (5.6%, 8/142), seven from raw milk and one from frozen food, were mecA positive and resistant to oxacillin, thus were MRSA. The 142 S. aureus isolates displayed 52 different toxin gene profiles. Although no direct association was found between toxin gene profile and the S. aureus genotype, the isolates belonging to CC5, CC9, CC20, CC50, and CC72 clonal lineages in general carried more toxin genes (>5) compared with the isolates in other CCs. It was also revealed that raw milk and raw meat were the major sources of isolates containing multiple toxin genes. S. aureus isolates from food that were genetically highly related, displayed diverse toxin gene profiles, implying the significant role of horizontal gene transfer in the emergence of highly toxigenic S. aureus isolates.

Molecular typing of Vibrio parahaemolyticus isolates from the middle-east coastline of China

The occurrence of outbreaks of Vibrio parahaemolyticus gastroenteritis in China highlights the need for strain characterization and subtyping of this pathogenic species. A total of 56 epidemiologically-unrelated strains of V. parahaemolyticus were isolated from clinical samples, seafood and various environmental sites in the middle-east coastline of China from 2006 to 2008. The isolates were characterized using four molecular typing methods, including ribotyping, enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), pulsed-field gel electrophoresis (PFGE), and sequence analysis of the gyrB gene. Genetic profiles of cluster analysis from these molecular typing tests clearly showed that there were differences in potential pathogenicity among isolates from seafood and its environments. Genetic characterization of two isolates (F13 and QS2) that originated from seafood demonstrated that they were potentially pathogenic. Discriminatory indices of four typing methods for the 56 V. parahaemolyticus isolates were differentiated by Simpsons Index of Diversity. The discriminatory index of ERIC-PCR typing was maximal (D=0.942), while that of sequence analysis of the gyrB gene was minimal (D=0.702). The discriminatory ability was greatly enhanced (D=0.966) when ERIC-PCR was coupled with sequence analysis of the gyrB gene. These results suggest that ERIC-PCR combined with sequence analysis of gyrB gene may be a reliable, rapid typing strategy for V. parahaemolyticus strains.

PCR identification of Salmonella serogroups based on specific targets obtained by comparative genomics.

Comparative genomic approaches provide abundant information to reveal the diversity among Salmonella serogroups. In a local genomic sequence database, twenty-five Salmonella whole genomic sequences were divided into 6 (A, B, C1, C2, D and others) serogroups for mining the DNA fragments specific for serogroups A through D. For each serogroup, a reference sequence was selected and split into 1000-bp fragments in silico to align against all the other genomic sequences to obtain one or more serogroup-specific fragments. As a result, 2, 6, 7, 10, and 7 specific fragments were found for A, B, C1, C2 and D serogroups, respectively. Specific primer sets targeting these DNA fragments were designed for multiplex PCR assays identifying 21 Salmonella standard strains and 86 additional food isolates. The PCR results demonstrated good agreement with those from Salmonella serotyping. This means that the PCR assay may be able to identify 5 (A, B, C1, C2 and D) Salmonella serogroups and elucidate differences among them. Based on the gene annotations, the 32 serogroup-specific fragments were divided into 3 categories (membrane protein genes, rfb gene clusters, and fimbrial genes). Each gene from these three groups was conserved within the serogroup and was closely correlated with phenotypic characterization. This finding implies that these genes, which are associated with sugar synthesis and metabolism or glycosyl and O-actetyl transfer, impart the differences among Salmonella serogroups.

Molecular methods for serovar determination of Salmonella

Abstract Salmonella is a diverse foodborne pathogen, which has more than 2600 recognized serovars. Classification of Salmonella isolates into serovars is essential for surveillance and epidemiological investigations; however, determination of Salmonella serovars, by traditional serotyping, has some important limitations (e.g. labor intensive, time consuming). To overcome these limitations, multiple methods have been investigated to develop molecular serotyping schemes. Currently, molecular methods to predict Salmonella serovars include (i) molecular subtyping methods (e.g. PFGE, MLST), (ii) classification using serovar-specific genomic markers and (iii) direct methods, which identify genes encoding antigens or biosynthesis of antigens used for serotyping. Here, we reviewed reported methodologies for Salmonella molecular serotyping and determined the “serovar-prediction accuracy”, as the percentage of isolates for which the serovar was correctly classified by a given method. Serovar-prediction accuracy ranged from 0 to 100%, 51 to 100% and 33 to 100% for molecular subtyping, serovar-specific genomic markers and direct methods, respectively. Major limitations of available schemes are errors in predicting closely related serovars (e.g. Typhimurium and 4,5,12:i:-), and polyphyletic serovars (e.g. Newport, Saintpaul). The high diversity of Salmonella serovars represents a considerable challenge for molecular serotyping approaches. With the recent improvement in sequencing technologies, full genome sequencing could be developed into a promising molecular approach to serotype Salmonella.

A rapid method for the detection of foodborne pathogens by extraction of a trace amount of DNA from raw milk based on amino-modified silica-coated magnetic nanoparticles and polymerase chain reaction

A method based on amino-modified silica-coated magnetic nanoparticles (ASMNPs) and polymerase chain reaction (PCR) was developed to rapidly and sensitively detect foodborne pathogens in raw milk. After optimizing parameters such as pH, temperature, and time, a trace amount of genomic DNA of pathogens could be extracted directly from complex matrices such as raw milk using ASMNPs. The magnetically separated complexes of genomic DNA and ASMNPs were directly subjected to single PCR (S-PCR) or multiplex PCR (M-PCR) to detect single or multiple pathogens from raw milk samples. Salmonella Enteritidis (Gram-negative) and Listeria monocytogenes (Gram-positive) were used as model organisms to artificially contaminate raw milk samples. After magnetic separation and S-PCR, the detection sensitivities were 8 CFU mL(-1) and 13 CFU mL(-1) respectively for these two types of pathogens. Furthermore, this method was successfully used to detect multiple pathogens (S. Enteritidis and L. monocytogenes) from artificially contaminated raw milk using M-PCR at sensitivities of 15 CFU mL(-1) and 25 CFU mL(-1), respectively. This method has great potential to rapidly and sensitively detect pathogens in raw milk or other complex food matrices.

Cloning and Characterization of the -Carotene Desaturase Gene fromChlorella protothecoidesCS-41

To elucidate the lutein biosynthesis pathway in the lutein-producing alga, Chlorella protothecoides CS-41, the ζ-carotene desaturase gene (zds) was isolated from Chlorella protothecoides using the approach of rapid amplification of cDNA ends. The full-length cDNA sequence was 2031 bp and contained 1755 bp putative open reading frame which encodes a 584 amino acid deduced polypeptide whose computed molecular weight was 63.7 kDa. Sequence homology research indicated that the nucleotide and putative protein had sequence identities of 72.5% and 69.5% with those of the green alga Chlamydomonas reinhardtii, respectively. Phylogenetic analysis demonstrated that the ZDS from C. protothecoides CS-41 had a closer relationship with those of chlorophyta and higher plants than with those of other species. In addition, we also found that the zds gene expression was upregulated in response to light.

Phenotypic, proteomic, and genomic characterization of a putative ABC-transporter permease involved in Listeria monocytogenes biofilm formation.

The foodborne pathogen Listeria monocytogenes is able to form biofilms in food processing environments. Previously, we have reported that an lm.G_1771 gene (encoding a putative ABC-transporter permease) was involved in negative regulation of L. monocytogenes biofilm formation using LM-49, a biofilm-enhanced mutant isolated on Tn917 mutagenesis (AEM 2008 p.7675-7683). Here, the possible action of this ABC-transporter permease in L. monocytogenes biofilm formation was characterized by phenotypic, proteomic, and genomic analyses using an lm.G_1771 gene deletant (Δ1771). The Δ1771 mutant exhibited the same enhanced ability for biofilm formation as the LM-49 strain using a crystal violet staining assay. DNA microarrays and two-dimensional gel electrophoresis revealed 49 and 11 differentially expressed (twofold or more) genes or proteins in Δ1771, respectively. The transcriptomics study indicated that lm.G_1771 could play a vital role in regulating candidate genes involved in biofilm formation such as genes encoding cell surface proteins (Dlt), cell surface anchor proteins (SrtA), and transcriptional regulators (GntR) contributing to negative regulation of biofilm formation by L. monocytogenes. The mutant Δ1771 was more sensitive to Triton X-100 and less resistant to cationic antibiotics, which might be explained by the down-regulation of dlt operon in this deletant and the fact that dlt involves the incorporation of D-alanine residues into lipoteichoic acids, resulting in a positive net charge on the teichoic acids. Therefore, lm.G_1771 is considered to be involved in negative regulation of biofilm formation, and the results from this work provide a possible molecular mechanism of biofilm formation regulated by lm.G_1771 in L. monocytogenes.

The Expression of Superoxide Dismutase (SOD) and a Putative ABC Transporter Permease Is Inversely Correlated during Biofilm Formation in Listeria monocytogenes 4b G

Little is known about the molecular basis of biofilm formation in Listeria monocytogenes. The superoxide dismutase (SOD) of the deletion mutant of lm.G_1771 gene, which encodes for a putative ABC transporter permease, is highly expressed in biofilm. In this study, the sod gene deletion mutant Δsod, and double deletion mutant of the sod and lm. G_1771 genes Δ1771Δsod were used to investigate the role of SOD and its relationship to the expression of the putative ABC transporter permease in biofilm formation. Our results showed that the ability to form a biofilm was significantly reduced in the Δsod mutant and the Δ1771Δsod double mutant. Both Δsod and Δ1771Δsod mutants exhibited slow growth phenotypes and produced more reactive oxygen species (ROS). The growth was inhibited in the mutants by methyl viologen (MV, internal oxygen radical generator) treatment. In addition, the expression of one oxidation resistance gene (kat), two stress regulators encoding genes (perR and sigB), and one DNA repair gene (recA) were analyzed in both the wild-type L. monocytogenes 4b G and the deletion mutants by RT-qPCR. The expression levels of the four genes were increased in the deletion mutants when biofilms were formed. Taken together, our data indicated that SOD played an important role in biofilm formation through coping with the oxidant burden in deficient antioxidant defenses.

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.

Identification of Staphylococcus argenteus in Eastern China based on a nonribosomal peptide synthetase (NRPS) gene.

AIM To investigate whether the Staphylococcus argenteus is present in Eastern China and to verify the utility of a new screening process. MATERIALS & METHODS Phenotype observation, PCR assay targeting a hypothetical nonribosomal peptide synthetase (NRPS) gene, phylogenetic analysis of rpoB and multilocus sequence typing were used to screen and identify strains of S. argenteus from 839 presumptive S. aureus isolates. RESULTS Eighty-nine (89/839, 10.6%) of the presumptive S. aureus isolates produced white colonies on tryptone soya agar plates. Of the white-colony isolates, six (6/89, 7%) were S. argenteus, 75 (75/89, 84%) were S. aureus and eight (8/89, 9%) were other bacteria. CONCLUSION The PCR-based method targeting the NRPS gene can simultaneously identify and distinguish S. argenteus and S. aureus. All representative sequences of rpoB generated in this study were deposited in GenBank under accession numbers SJTU F20002, KT767581; SJTU F20269, KT767582; SJTU F20419, KT767583; SJTU F20420, KT767584; SJTU F20124, KT767585; SJTU F21164, KT767586; SJTU F21285, KT767587; SJTU F21224, KT767588; SJTU F21155, KT767589; SJTU F21294, KT767590; SJTU F20030, KT767591; SJTU F20044, KT767592; SJTU F20135, KT767593; SJTU F20123, KT767594; SJTU F21319, KT767595, respectively. All the new sequence types (STs) were submitted to a multilocus sequence typing database and the assigned ST numbers are ST3261 (151-469-20-101-145-150-131), ST3262 (12-3-1-1-4-4-410) and ST3267 (2-471-2-2-6-3-2).