Aims: To develop a rapid multiplex PCR method for simultaneous detection of five major foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella Enteritidis and Shigella flexneri, respectively). Methods and Results: Amplification by PCR was optimized to obtain high efficiency. Sensitivity and specificity assays were investigated by testing different strains. With a multipathogen enrichment, multiplex PCR assay was able to simultaneously detect all of the five organisms in artificially contaminated pork samples. The developed method was further applied to retail meat samples, of which 80% were found to be positive for one or more of these five organisms. All the samples were confirmed by traditional culture methods for each individual species. Conclusions: This study reported a rapid multiplex PCR assay using five primers sets for detection of multiple pathogens. Higher consistency was obtained between the results of multiplex PCR and traditional culture methods. Significance and Impact of the Study: This work has developed a reliable, useful and cost‐effective multiplex PCR method. The assay performed equally as well as the traditional cultural method and facilitated the sensitive detection both in artificially contaminated and naturally contaminated samples.

Thermostable nuclease is known to be an important pathogenic factor unique to Staphylococcus aureus and it is commonly presumed to have had the same

The aim of the present study was to investigate the antibiotic resistance profiles and the molecular epidemiology of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates from two production swine operations in Sichuan Province, China, between August 2002 and February 2007. The prevalence of ESBL-producing E. coli increased dramatically from 2.2% to 10.7% during this period. This increase appeared mostly related to dissemination of CTX-M–type ESBLs among E. coli isolates. Of 212 E. coli isolates studied, 14 harbored ESBL genes. Among them, 13 harbored bla_CTX-M-15/22 and one harbored bla_SHV-2. To our knowledge, this is the first study to identify bla_CTX-M-22 from production animals. One isolate in 2002 harbored bla_SHV-2, indicating that ESBL genes have been present in farm animals in China since at least 2002. Molecular characterization and pulsed-field gel electrophoresis of the ESBL-producing isolates suggested that different mechanisms may be involved in the dissemination of the CTX-M genes and revealed that additional resistance determinants for non–β-lactam antibiotics were carried by plasmids encoding certain ESBL genes. Results of this study provide an example of how ESBL genes, particularly those of CTX-M lineages, are rapidly spreading among E. coli isolates from commercial pig farms in Sichuan province of China.

Staphylococcus aureus is a pathogenic bacterium capable of developing biofilms, leading to nosocomial infection and cross-contamination of foods. The current study was focused on the detection of adhesin genes, staphylococcal nuclease and hemolytic activities, and biofilm formation among the isolates of S. aureus from different sources. Fifteen adhesin genes (bap, bbp, clfA, clfB, cna, ebpS, fib, fnbA, fnbB, eno, icaAD, icaBC, sasG, sasC, pls) involved in S. aureus cell aggregation and biofilm accumulation were detected by polymerase chain reaction using specific primer. The activities of staphylococcal nuclease and hemolysis were analyzed by using toluidine blue–DNA agar and sheep blood agar for each strain. The ability of biofilm formation among different S. aureus strains was tested by using the glass tube method and microtiter-plate method. Our results showed the diversity of biofilm formation from different sources. Some isolates were strong biofilm producers; some were weak biofilm producers; and some were nonbiofilm producers. Staphylococcal nuclease and hemolysis seem to play a certain inhibitory role in biofilm formation. The adhesin genes varied among different S. aureus strains. The bap gene was not present in any strains. The bbp gene was only detected in one strain. The detection rates of other adhesin genes were as follows: clfB and sasG (100%); cna, eno, fib, and ebpS (93.75%); fnbA, icaAD, and icaBC (87.50%); fnbB (68.75%); sasC (31.25%); clfA (25%); and pls (12.50%), respectively. The variation between phenotypic and genotypic characterization may be due to the heterogeneity in the genetic origins. There was no direct correlation in distribution of adhesin genes and biofilm formation, which indicates that a single gene or subset of genes cannot be utilized as a biofilm indicator for morphology. Our results also indicated that biofilm formation might be affected by many factors, which brings new challenges to the prevention of this serious pathogen due to biofilm-related infection and contamination.

Staphylococcus aureus is one of the major etiological pathogens of bovine mastitis. Its invasion into mammary epithelial cells has been proven to be a key event in the pathogenesis of mastitis. However, the specific pathogenic factors have not been clearly identified. Staphylococcus aureus often triggers infections by releasing virulence factor. Recent several studies reported that staphylococcal enterotoxin M was one of the most frequently found enterotoxin genes associated with bovine mastitis. Thus, the effect of staphylococcal enterotoxin M on inflammation and damage of the bovine mammary epithelial bovine mammary gland epithelial cell line (MAC-T) cells with 48 h treatment was explored in the present study. First, staphylococcal enterotoxin M protein was purified by a Ni-NTA spin column (GE Life Science, Westborough, MA). The levels of tumor necrosis factor-α, IL-6, and monocyte chemoattractant protein 1 (MCP-1) secretion were measured with the corresponding ELISA kits (R&D Systems, Abingdon, UK). Second, cell viability was assessed with a Cell Counting Kit-8 (Bioswamp, Wuhan, China) and the apoptotic percentage of cells was determined by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI; Beyotime, Nanjing, China) staining. Third, ATP concentration, reactive oxygen species (ROS) generation and lactate dehydrogenase (LDH) release were assayed with commercial kits, then mitochondrial membrane potential (ΔΨm) was estimated using fluorescent probe JC-1 (Beyotime). Finally, the production intercellular cell adhesion molecule-1 (ICAM-1), microtubule-associated protein 1A/1B-light chain 3 I/II (LC3 I/II), p62 (Proteintech, Rosemont, IL), and phosphorylation of IκBα, caspase 3, and mammalian target of rapamycin were detected by Western blot. The results showed that staphylococcal enterotoxin M induced inflammation of epithelial cells (upregulating tumor necrosis factor-α, IL-6, MCP-1, and ICAM-1 production) and activated NF-κB (promoting phosphorylation of IκBα). Furthermore, staphylococcal enterotoxin M impaired MAC-T cells via cell necrosis (enhancing LDH release), apoptosis (annexin V-FITC/PI stain, exacerbating oxidative stress, decreasing ΔΨm and intracellular ATP concentration, and activating caspase 3), but independent of autophagy (nonsignificantly increasing LC3-II, decreasing p62 expression, and activating mammalian target of rapamycin). Thereby, staphylococcal enterotoxin M induced the inflammatory property of bovine mammary epithelial cells by boosting cytokine, chemokine, and adhesion molecule production. Furthermore, it caused epithelial cell dysfunction via depressing cell viability and initiating cell necrosis and apoptosis. Because epithelial cells played important roles in orchestrating the inflammatory response and protecting bovine mammary tissue from mastitis, our results indicated that staphylococcal enterotoxin M may be associated with mastitis.