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This study characterized the prevalence and antimicrobial resistance characteristics of foodborne Salmonella isolates from March 2016 to February 2017 in Shanghai, China. A total of 147 (14.2%) nonduplicate foodborne Salmonella isolates were obtained from 1035 food samples. The Salmonella isolates were most frequently identified in fresh meat samples (28.0%), followed by ready-to-eat foods (9.0%), frozen convenience foods (7.1%), and fresh produce (4.5%). The top 3 serovars were Salmonella Enteritidis (46.3%; 68/147), Salmonella Typhimurium (32.7%; 48/147), and Salmonella Derby (6.8%; 10/147). The majority of isolates were resistant to sulfisoxazole (93.9%; 138/147) and trimethoprim/sulfamethoxazole (61.2%; 90/147). Interestingly, frozen convenience food isolates exhibited an extremely high multidrug resistance rate (86.7%; resistant to ≥3 classes of antimicrobials). Among 81 quinolone-resistant isolates, aac(6')-Ib-cr (100%), oqx AB (84.0%), qnr S1 (23.5%), D87Y (49.4%), and D87N (33.3%) mutations in GyrA, and T57S in ParC (12.3%) were observed. The β-lactamase genes bla TEM-1 (100%) were present in 63 ampicillin-resistant isolates. Polymerase chain reaction-based plasmid replicon typing revealed that 147 isolates represented 6 plasmid incompatibility groups (IncFIIs, IncHI2, IncI1, IncP, IncFIC, and IncA/C), among which, IncFIIs (59.2%) and IncHI2 (26.5%) were predominant. The genetic relationship of isolates was elucidated using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). MLST results indicated that ST34 and ST11 were predominate types in Salmonella Typhimurium (56.3%; 27/48) and Salmonella Enteritidis (95.6%; 65/68), respectively. Importantly, 96.3% (26/27) of ST34 Salmonella Typhimurium isolates possessed the ACSSuT resistance pattern (ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline). PFGE analysis of ST34 isolates showed clonal dissemination across all four types of retail foods. Our findings highlight the high prevalence of antimicrobial-resistant Salmonella isolates in retail foods in Shanghai, especially the clonal expansion of ST34 isolates with MDR-ACSSuT resistance, which might pose a public health threat.

The enterotoxin gene cluster (egc) has been proposed to contribute to the Staphylococcus aureus colonization, which highlights the need to evaluate genetic diversity and virulence gene profiles of the egc-positive population. Here, a total of 43 egc-positive isolates (16.2%) were identified from 266 S. aureus isolates that were obtained from various food and clinical specimens in Shanghai. Seven different egc profiles were found based on the polymerase chain reaction (PCR) result for egc genes. Then, these 43 egc-positive isolates were further typed by multilocus sequence typing, pulsed-field gel electrophoresis (PFGE), multiple-locus variable-number tandem-repeat analysis (MLVA), and accessory gene regulatory (agr) typing. It showed that the 43 egc-positive isolates displayed 17 sequence types, 28 PFGE patterns, 29 MLVA types, and 4 agr types, respectively. Among them, the dominant clonal lineage was CC5-agr II (48.84%). Thirty toxin and 20 adhesion-associated genes were detected by PCR in egc-positive isolates. Notably, invasive toxin genes showed a high prevalence, such as 76.7% for Panton-Valentine leukocidin encoding genes, 27.9% for sec, and 23.3% for tsst-1. Most of the examined adhesion-associated genes were found to be conserved (76.7-100%), whereas the fnbB gene was only found in 8 (18.6%) isolates. In addition, 33 toxin gene profiles and 13 adhesion gene profiles were identified, respectively. Our results imply that isolates belonging to the same clonal lineage harbored similar adhesion gene profiles but diverse toxin gene profiles. Overall, the high prevalence of invasive virulence genes increases the potential risk of egc-positive isolates in S. aureus infection.

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.

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Staphylococcus aureus is a recognized pathogen in humans, which causes nosocomial infections and food poisoning. The transmission of antibiotic resistant S. aureus (ARSA), especially methicillin-resistant S. aureus, between food products and humans has become a serious problem. Hence, it is necessary to monitor S. aureus through the food supply chain. In this study, the disk diffusion method and epsilometer test were performed to determine the prevalence of ARSA in 78 foodborne isolates using 18 antibiotics. The highest resistance frequency was found for penicillin G (74.4%), followed by erythromycin (59.0%) and clindamycin (44.9%), whereas no vancomycin-resistant isolates were found. The 78 isolates could be subtyped into 31 resistance profiles and 11 clusters based on their antimicrobial susceptibility. Furthermore, Polymerase chain reaction (PCR) screening for the presence of 13 genes conferring antibiotic resistance was conducted. The presence of resistance genes was relatively high: blaTEM (80.8%), ermB (41.0%), grlA (38.5%), ermC (35.9%), and aac6'/aph2" (35.9%). The incidence of antibiotic resistance was significantly correlated to food types (p = 0.018), with isolates from meat and raw milk more resistant to antibiotics than those from frozen food and vegetables., (© 2014 Institute of Food Technologists®)

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 10(7) 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.

A 5'-nuclease real-time PCR assay using a minor groove binding probe was developed for the detection of Salmonella enterica from artificially contaminated foods. S. enterica-specific sequences were identified by a comparative genomic approach. Several species-specific target sequences were evaluated for specificity. A real-time PCR assay was developed targeting a nucleotide sequence within the putative type III secretion ATP synthase gene (ssaN). An internal amplification control (IAC) probe was designed by randomly shuffling the target probe sequence and a single-stranded oligonucleotide was synthesized to serve as an IAC. The assay demonstrated 100% inclusivity for the 40 Salmonella strains tested and 100% exclusivity for 24 non-Salmonella strains. The detection limit of the real-time PCR assay was 41.2 fg/PCR with Salmonella Typhimurium genomic DNA and 18.6 fg/PCR using Salmonella Enteritidis genomic DNA; 8 and 4 genome equivalents, respectively. In the presence of a natural background flora derived from chicken meat enrichment cultures, the sample preparation and PCR method were capable of detecting as few as 130 Salmonella cfu/mL. Using the developed real-time PCR method to detect Salmonella in artificially contaminated chicken, liquid egg and peanut butter samples, as few as 1 cfu/10 g of sample was detectable after a brief (6h) non-selective culture enrichment., ((c) 2009. Published by Elsevier B.V. All rights reserved.)

We developed single base extension-tags (SBE-tags) microarray to detect eight common food-borne pathogens, including Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, Salmonella, Enterobacter sakazaki, Shigella, Escherichia coli O157:H7 and Campylobacter jejuni. With specific PCR primers identified and integrated for eight food-borne pathogens, target sequences were amplified and purified as template DNA of single base extension-tags reaction. The products were hybridized to microarrays and scanned for fluorescence intensity. The experiment showed a specific and simultaneous detection of eight food-borne pathogens. The system limits is 0.1 pg for a genomic DNA and 5x10(2) CFU/mL for Salmonella typhimurium cultures. The single base extension-tags assay can be used to detect food-borne pathogens rapidly and accurately with a high sensitivity, and provide an efficient way for diagnosis and control of disease caused by food-borne pathogens.

Cross-protection to environmental stresses by ethanol adaptation in Salmonella poses a great threat to food safety because it can undermine food processing interventions. The ability of Salmonella enterica serovar Enteritidis ( S. Enteritidis) to develop acid resistance following ethanol adaptation (5% ethanol for 1 h) was evaluated in this study. Ethanol-adapted S . Enteritidis mounted cross-tolerance to malic acid (a two-fold increase in minimum bactericidal concentration), but not to acetic, ascorbic, lactic, citric and hydrochloric acids. The population of S . Enteritidis in orange juice (pH 3.77) over a 48-h period was not significantly ( p > 0.05) influenced by ethanol adaptation. However, an increased survival by 0.09–1.02 log CFU/ml was noted with ethanol-adapted cells of S . Enteritidis compared to non-adapted cells in apple juice (pH 3.57) stored at 25 °C ( p < 0.05), but not at 4 °C. RT-qPCR revealed upregulation of two acid tolerance-related genes, rpoS (encoding σ S ) and SEN1564A (encoding an acid shock protein), following ethanol adaptation. The relative expression level of the acid resistance gene hdeB did not change. The resistance phenotypes and transcriptional profiles of S . Enteritidis suggest some involvement of rpoS and SEN1564A in the ethanol-induced acid tolerance mechanism. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a major foodborne pathogen that is well known for its high mortality rate in humans upon infection. This study aimed to investigate the prevalence and serotype diversity of L. monocytogenes isolates in various foods. From July 2013 to June 2014, a total of 648 food samples, originating from raw milk, quick-frozen food, vegetables, raw meats, cooked meats, aquatic products, dairy products and bean products, were collected from open-air markets, large supermarkets, delis and dairy farms, in Shanghai, China. Forty samples (6.2%) tested positive for L. monocytogenes , confirmed by both bacteriological and molecular methods. Among the food categories surveyed, the highest prevalence was observed in raw meat samples at 16.0% (15/94), followed by 9.9% (10/101) in quick-frozen food samples and 7.3% (4/55) in cooked meat samples. Serotyping showed that majority of the isolates (67.5%) belonged to serotype 1/2a, followed by 1/2b (22.5%), 1/2c (7.5%) and 3c (2.5%). Isolates were assessed for their ability to form biofilms. Significant differences in biofilm forming abilities were observed between storage temperatures and serotypes. Anti-biofilm efficacy of industrial disinfectants, chlorine dioxide (ClO 2 ) and hydrogen peroxide (H 2 O 2 ), was examined by measuring minimal effective concentration (MEC) on L. monocytogenes in biofilm. A positive correlation was observed among the biofilm’s resistance to H 2 O 2 and the biomass of biofilms. ClO 2 was found to be more effective against biofilm and inactivated all the listerial cells in the biofilm state, whereas H 2 O 2 was not able to kill all the cells in biofilms at the recommended concentrations according to National Food Safety Standard of China. Our study suggests that there is potential public health risk of L. monocytogenes infection in Shanghai. Efficacy against L. monocytogenes biofilm should be considered when evaluating disinfectants. [ABSTRACT FROM AUTHOR]