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As the cost of genome sequencing of foodborne pathogens decreases, it has become possible to sequence a large number of isolates and evaluate those using machine learning algorithms. This study aimed to utilize machine learning algorithms to predict the disease endpoints in untagged Salmonella genome sequences isolated from ground chicken. Our models recognized genetic patterns in the test dataset based on our training dataset obtained from an extensive literature review, using a semi-supervised approach. Using known genotypes as input features, the semi-supervised random forest model showed the highest overall accuracy of 0.94 (95% confidence interval: 0.85–0.99), and a Kappa value of 0.82, and predicted 87% of the disease endpoints. The model predicted genes associated with specific disease endpoints that were associated with virulence, which could be used as features in predictive modeling endeavors in the future. Our machine learning approach would be useful in different areas of food safety, including identifying pathogen sources, predicting antibiotic resistance, and risk assessment of foodborne pathogens.

Enteric pathogens, including Salmonella, are capable of long-term survival after desiccation and resist heat treatments that are lethal to hydrated cells. The mechanisms of dry-heat resistance differ from those of wet-heat resistance. To elucidate the mechanisms of dry-heat resistance in Salmonella, screening of the dry-heat resistance of 108 Salmonella strains, representing 39 serotypes, identified the 22 most resistant and the 8 most sensitive strains for comparative genome analysis. A total of 289 genes of the accessory genome were differently distributed between resistant and sensitive strains. Among these genes, 28 proteins with a putative relationship to stress resistance were selected for to quantify relative gene expression before and after desiccation and expression by solid-state cultures on agar plates relative to cultures growing in liquid culture media. Of these 28 genes, 15 genes were upregulated (P

The exposure of foodborne pathogens such as Salmonella enterica to a sub-lethal stress may protect bacterial cells against distinct stresses during the production of leafy greens, which can constitute potential health hazards to consumers. In this study, we evaluated how the prior exposure of S. enterica to sub-lethal food processing-related stresses influenced its subsequent persistence on baby spinach under cold (4 °C for 7 days) and temperature abuse (37 °C for 2 h + 4 °C for 7 days) conditions. We also compared the survival characteristics of pre-stressed S. enterica and Enterococcus faecium NRRL B-2354 as its surrogate on baby spinach. A cocktail of three S. enterica serovars, as well as S. Typhimurium ATCC 14028 wild type and its ΔrpoS mutant, and E. faecium NRRL B-2354, was first exposed to sub-lethal desiccation, oxidation, heat shock, and acid stresses. Afterward, baby spinach was inoculated with unstressed or pre-stressed cells at 7.0 log CFU/sample unit, followed by 7-day storage under cold and temperature abuse conditions. The unstressed S. enterica (fresh cells in sterile 0.85% saline) decreased rapidly within the first day and thereafter persisted around 5.5 log CFU/sample unit under both conditions. The desiccation-stressed S. enterica showed the highest bacterial counts (p &lt, 0.05) compared to other conditions. The unstressed S. enterica survived better (p &lt, 0.05) than the oxidation- and acid-stressed S. enterica, while there were no significant differences (p &gt, 0.05) between the unstressed and heat-shocked S. enterica. Unlike the wild type, temperature abuse did not lead to the enhanced survival of the ΔrpoS mutant after exposure to desiccation stress, indicating that the rpoS gene could play a critical role in the persistence of desiccation-stressed S. enterica subjected to temperature abuse. E. faecium NRRL B-2354 was more persistent (p &lt, 0.05) than the pre-stressed S. enterica under both conditions, suggesting its use as a suitable surrogate for pre-stressed S. enterica by providing a sufficient safety margin. Our results demonstrate the merit of considering the prior exposure of foodborne pathogens to sub-lethal stresses when validating the storage conditions for leafy greens.

In industrial-scale apple juice concentrate (AJC) processing, 5-hydroxymethylfurfural (5-HMF or 5-(hydroxymethyl)-2-furaldehyde) is a furanic compound that is formed as an intermediate under certain conditions during thermal treatments applied to foods, especially AJC. Understanding 5-HMF formation is significant, particularly because 5-HMF is undesirable in foods due to its harm to humans’ health, but its presence and content is also an indicator of the processing technique used to prepare the food. The aim of this study was to investigate the changes in 5-HMF and other physicochemical indicators during AJC processing, and to critically analyse the impacts of those physicochemical indicators on the generation of 5-HMF. In this study, 5-HMF content, conventional physicochemical parameters, two monosaccharides, one disaccharide, 18 amino acids, and 12 polyphenols were monitored during 8 crucial stages of AJC production. 5-HMF formed primarily during three thermal processing stages: preconcentration, enzymatic clarification, and quintuple effect concentration. Color value increased, and brown index decreased significantly during processing. Fructose and glucose were the most abundant saccharides present in apple juice. Asparagine and aspartic acid were the two highest contents of amino acids detected, and amino acids contents showed little variation after the resin decolorization stage. Phloridzin content was highest in the original apple fruit, but all polyphenols showed significant variability during processing, especially during adsorption after the resin decolorization stage. The formation of 5-HMF showed a positive correlation with soluble solids content, color value, total sugar, fructose, glucose, glutamic acid, asparagine, threonine, alanine, methionine, proline, gallic acid, and rutin content. The formation of 5-HMF is dependent upon the fructose, glucose and specific amino acids content under acidic and thermal conditions found in industrial-scale AJC processing. Understanding the factors influencing 5-HMF formation can help to control its formation in AJC processing.

Salmonella producing β-lactamases has spread rapidly worldwide and poses a serious threat to human and animal health. In this study, we characterized 220 ceftriaxone (CRO)-resistant isolates identified among 3153 Salmonella from humans, animals, food, and water collected in Shanghai, China. They were assessed for antimicrobial susceptibility, phenotypic identification of extended-spectrum β-lactamases (ESBLs), and β-lactamase genes and integrons. CRO resistance in Salmonella increased from 5.0% in 2011 to 8.4% in 2013. Salmonella Enteritidis (45.5%), Salmonella Typhimurium (20.9%) from humans, and Salmonella Indiana (14.5%) from poultry represented the majority of the CRO-resistant isolates. Many isolates were also resistant to other antimicrobials, including nalidixic acid (84.5%), sulfisoxazole (70.5%), and tetracycline (61.8%). Resistance to ciprofloxacin was also found in 33.6% of the isolates. Most isolates (98.2%) were confirmed as ESBL producers. Resistance genes such as blaCTX-M, blaTEM, and blaOXA were detected in 207 (94.1%), 99 (45%), and 53 (24.1%) isolates, respectively. Three types of integron I and one type of integron II were identified in 13 (5.9%) and 2 (0.9%) isolates, respectively. The integrons encompassed 10 different genes: dfrA1/12/17/25, aadA1/2/5, sat2, orfF, and ybeA. Our study underscores concern for increasing CRO resistance, and highlights the widespread ESBL genes in Salmonella enterica.

Listeria monocytogenes is a major foodborne pathogen that can contaminate food products and colonize food-producing facilities. Foodservice operations (FSOp) are frequently responsible for foodborne outbreaks due to food safety practices failures. We investigated the presence of and characterized L. monocytogenes from two FSOp (cafeterias) distributing ready-to-eat meals and verified FSOp’s compliance with good manufacturing practices (GMP). Two facilities (FSOp-A and FSOp-B) were visited three times each over 5 months. We sampled foods, ingredients, and surfaces for microbiological analysis, and L. monocytogenes isolates were characterized by phylogenetic analyses and phenotypic characteristics. GMP audits were performed in the first and third visits. A ready-to-eat salad (FSOp-A) and a frozen ingredient (FSOp-B) were contaminated with L. monocytogenes, which was also detected on Zone 3 surfaces (floor, drains, and a boot cover). The phylogenetic analysis demonstrated that FSOp-B had persistent L. monocytogenes strains, but environmental isolates were not closely related to food or ingredient isolates. GMP audits showed that both operations worked under “fair” conditions, and “facilities and equipment” was the section with the least compliances. The presence of L. monocytogenes in the environment and GMP failures could promote food contamination with this pathogen, presenting a risk to consumers.

The past few years have seen a significant increase in availability of whole genome sequencing information, allowing for its incorporation in predictive modeling for foodborne pathogens to account for inter- and intra-species differences in their virulence. However, this is hindered by the inability of traditional statistical methods to analyze such large amounts of data compared to the number of observations/isolates. In this study, we have explored the applicability of machine learning (ML) models to predict the disease outcome, while identifying features that exert a significant effect on the prediction. This study was conducted on Salmonella enterica, a major foodborne pathogen with considerable inter- and intra-serovar variation. WGS of isolates obtained from various sources (i.e., human, chicken, and swine) were used as input in four machine learning models (logistic regression with ridge, random forest, support vector machine, and AdaBoost) to classify isolates based on disease severity (extraintestinal vs. gastrointestinal) in the host. The predictive performances of all models were tested with and without Elastic Net regularization to combat dimensionality issues. Elastic Net-regularized logistic regression model showed the best area under the receiver operating characteristic curve (AUC-ROC; 0.86) and outcome prediction accuracy (0.76). Additionally, genes coding for transcriptional regulation, acidic, oxidative, and anaerobic stress response, and antibiotic resistance were found to be significant predictors of disease severity. These genes, which were significantly associated with each outcome, could possibly be input in amended, gene-expression-specific predictive models to estimate virulence pattern-specific effect of Salmonella and other foodborne pathogens on human health.

We tested a diverse set of 500 isolates of nontyphoidal Salmonella enterica subsp. enterica from various animal, food, and human clinical sources for susceptibility to antimicrobials currently lacking epidemiological cutoff values (ECOFFs) set by the European Committee on Antimicrobial Susceptibility Testing. A consortium of five different laboratories each tested 100 isolates, using broth microdilution panels containing twofold dilutions of ceftriaxone, cefepime, and colistin to determine the minimum inhibitory concentrations of each drug when tested against the Salmonella isolates. Based on the resulting data, new ECOFFs of 0.25 μg/mL for ceftriaxone, 0.12 μg/mL for cefepime, and 2 μg/mL for colistin have been proposed. These thresholds will aid in the identification of Salmonella that have phenotypically detectable resistance mechanisms to these important antimicrobials.

We characterized antibiotic resistance profiles, antibiotic resistance-associated genes, and pulsed-field gel electrophoresis (PFGE) patterns of 145 Salmonella enterica serotype Typhimurium isolates from human infections and retail foods that were possibly responsible for salmonellosis outbreaks from 2008 to 2012 in Shanghai, China. Resistance to at least three antibiotics was found in 66.7% of chicken isolates, 76.5% of duck isolates, 77.8% of pork isolates, and 80.5% of human isolates. Seven antibiotic resistance phenotypes were detected in chicken isolates, 16 in pork isolates, 17 in duck isolates, and 50 in human isolates. No significant difference (p 0.05) was found between Salmonella isolates derived from human salmonellosis and from retail foods in terms of the percent resistance of ampicillin, amoxicillin/clavulanic acid, ceftiofur, ceftriaxone, nalidixic acid, chloramphenicol, gentamicin, kanamycin, streptomycin, tetracycline, sulfisoxazole, and sulfamethoxazole/trimethoprim. PFGE using XbaI and BlnI showed that some Salmonella isolates recovered from human infections and retail foods had same or highly similar genetic profile. Same or similar antibiotic resistance profiles, antibiotic resistance associated genes (i.e., qnrA, qnrB, qnrS, aac(6')-Ib, and oqxAB), gene cassettes (i.e., aadA2, dfrA12-aadA2, and aadA1), and mutations were detected in those isolates that exhibited high genetic similarities. These findings highlighted the frequent presence of Salmonella Typhimurium in retail chicken, pork, duck, and humans.

The aim of this study was to investigate the toxin gene profile and antimicrobial resistance of Staphylococcus aureus isolates from raw chicken in the People's Republic of China. In total, 289 S. aureus isolates were characterized by antimicrobial susceptibility testing, and genes encoding enterotoxins, exfoliative toxins, Panton-Valentine leukocidin, and toxic shock syndrome toxin were revealed by PCR. Overall, 46.0% of the isolates were positive for one or more toxin genes. A high proportion of toxin genes were pvl (26.6%), followed by sej (12.5%), sea (9.0%), seh (8.3%), seb (6.9%), sec (6.9%), sed (4.8%), sei (3.1%), and see (2.4%). None of the isolates harbored seg, tsst-1, or exfoliative toxin genes. In total, 29 toxin gene profiles were obtained, and pvl (10.7%) was the most frequent genotype, followed by sea (5.9%), seb (4.8%), and sej (4.2%). Furthermore, 99.7% of the strains were resistant to at least one of the tested antimicrobial agents, and 87.2% of them displayed multidrug resistance. Resistance was most frequently observed to trimethoprim-sulfamethoxazole and erythromycin (86.2% for each), followed by tetracycline (69.9%), amoxicillin-clavulanic acid (45.0%), and ampicillin (42.6%). None of the strains were resistant to vancomycin. This study indicates that S. aureus isolates from raw chicken harbored multiple toxin genes and exhibited multiple antimicrobial resistance, which represents a potential health hazard for consumers.

Shiga toxin-producing E. coli (STEC) are major foodborne pathogens. While many studies have focused on the "top-7 STEC", little is known for minor serogroups. A total of 284 non-top-7 STEC strains isolated from cattle feces were subjected to whole-genome sequencing (WGS) to determine the serotypes, the presence of virulence genes and antimicrobial resistance (AMR) determinants. Nineteen typeable and three non-typeable serotypes with novel O-antigen loci were identified. Twenty-one AMR genes and point mutations in another six genes that conferred resistance to 10 antimicrobial classes were detected, as well as 46 virulence genes. The distribution of 33 virulence genes and 15 AMR determinants exhibited significant differences among serotypes (p 0.05). Among all strains, 81.7% (n = 232) and 14.1% (n = 40) carried stx2 and stx1 only, respectively; only 4.2% (n = 12) carried both. Subtypes stx1a, stx1c, stx2a, stx2c, stx2d, and stx2g were identified. Forty-six strains carried eae and stx2a and therefore had the potential cause severe diseases; 47 strains were genetically related to human clinical strains inferred from a pan-genome phylogenetic tree. We were able to demonstrate the utility of WGS as a surveillance tool to characterize the novel serotypes, as well as AMR and virulence profiles of uncommon STEC that could potentially cause human illness.

One thousand four hundred ninety-one Salmonella isolates recovered from retail foods including chicken, beef, fish, pork, dumplings, and cold dishes in China in 2007, 2008, 2010, 2011, and 2012 were analyzed for distribution of serotype and antimicrobial susceptibility. A total of 129 Salmonella serotypes were detected among 1491 isolates. Salmonella Enteritidis (21.5%), Typhimurium (11.0%), Indiana (10.8%), Thompson (5.4%), Derby (5.1%), Agona (3.8%), and Shubra (3.0%) were the seven most important serotypes in 1491 isolates. For antibiotic susceptibility, except 16 (1.1%) isolates were susceptible to all tested antibiotics, 131 (8.8%) resisted 1-2 and 1344 (90.1%) resisted three or more antibiotics. One thousand forty-six (70.2%) of 1491 Salmonella isolates were identified as multidrug-resistant (MDR) isolates, which could resist three or more categories of antibiotics. Resistance to sulfisoxazole (78.1%) was most common among the tested Salmonella, followed by tetracycline (70.6%), trimethoprim/sulfamethoxazole (68.0%), and nalidixic acid (63.4%). Resistances to amikacin (20.0%), levofloxacin (18.7%), gatifloxacin (17.9%), ceftriaxone (17.7%), and cefoxitin (13.2%) were less frequently detected. Resistance to fluoroquinolones was most common among Salmonella Shubra and Indiana isolates, while resistance to cephalosporins was frequently detected among Salmonella Thompson isolates. The results highlighted the diversity of Salmonella serotypes and the high prevalence of Salmonella MDR isolates in China. Compared with Salmonella Enteritidis and Typhimurium isolates, the higher fluoroquinolones and cephalosporins resistance rates of some individual serotypes (Salmonella Shubra, Indiana, and Thompson) also provided more information for further study related to fluoroquinolones or cephalosporin-resistant Salmonella.

A total of 120 Listeria monocytog enes isolates (107 from foods and 13 from humans) in Shanghai, China, were characterized by serogroup typing and virulence genes detection with PCR, antimicrobial susceptibility testing, and molecular subtyping using pulsed-field Gel Electrophoresis (PFGE). Isolates belonged to three L. monocytogenes serogroups, 1/2c, 3c (n = 47, 39.1%) was the most prevalent, followed by 1/2a, 3a (n = 44, 36.7%) and 1/2b, 3b, 7 (n = 29, 24.2%). Although the isolates were all susceptible to most antibiotics tested, 13 isolates were resistant to ceftriaxone and seven to tetracycline. None of the isolates were resistant to multiple antibiotics. The tet (M) genes were detected among tetracycline-resistant isolates. The isolates all harbored virulence genes hly , prfA , plcA , plcB , inlA , inlB , inlI ; 116 isolates (96.7%) were positive for inlC ; 78 (65.0%) for inlG ; 119 (99.2%) for inlJ ; 8 (6.67%) for llsX . A total of 74 distinct patterns were generated for the 119 isolates tested using Apa I and Asc I with Pulsed Field Gel Electrophoresis (PFGE). Three clinical isolates shared the same PFGE patterns with retail meet (chicken, duck, and beef) isolates. These findings indicated that food of animal origin likely serve as a major vehicle in transmitting human listeriosis in Shanghai. Active surveillance for L. monocytogenes is needed for better understanding the epidemiology of the pathogen, and for effective control and prevention of human listeriosis.

An enzyme-linked immunosorbent assay (ELISA) was developed to screen for the presence of botulinal toxin types A, B, and E in inoculated food studies. A commercially available trivalent antitoxin (Connaught Laboratories, Ontario) was used as a capture antibody and biotinylated for use as a secondary antibody. An avidin-alkaline phosphatase conjugate coupled with an enzymebased amplification system resulted in a high degree of sensitivity. Detection levels of purified neurotoxins in gelatin phosphate buffer were 9 LD50 for type A and

Aims The purpose of this experiment was to study the bacterial diversity and predominance of spoilage bacteria in chicken skin at different thermal treatment temperatures (60, 70, 80, 90, 100, 110, 120°C). Method and results Bacteria in chicken skin was collected, then propidium monoazide treatment to remove the DNA of dead cell, total DNA was extracted by Tiandz Bacterial DNA Kit, and investigated by high-throughput sequencing of the v3/v4 regions of the 16S rDNA gene. A total of 796 008 high-quality bacterial sequences were obtained for assessing the microbial diversity of chicken skin from seven thermal treatment group and control group. The results showed that the bacterial diversity in chicken skin at 90°C was lowest. And Acinetobacter (25·88%), Clostridium (20·70%), Bacteroides (13·93%) and Myroides (13·13%) were the main flora at 25°C; The Clostridium was dominant genus of the samples heat-treated by 60, 70, 80 and 90°C, the proportion of this genus were up to 64·86, 77·42, 52·22 and 87·30% respectively. The Bacillus was the main flora of the samples heat-treated by 100, 110 and 120°C, and the relative percentages were 39·44, 79·61 and 45·96% respectively. In addition, high-temperature-resistant Serratia was found in chicken skin. Conclusions The study revealed that the relationship between thermal treatment temperature and bacterial diversity and dominant spoilage bacteria in chicken skin, which had a strong guiding significance for the control and prediction of micro-organisms in foods. Significance and impact of the study The results of this paper could provide a theoretical basis for meat products containing chicken skin, including the safe use of chicken skin, determination of sterilization process parameters and selection of preservatives for compounding, which has strong practicality in China.

Salmonella enterica serovar Stanley is an important serovar that has been increasingly identified in human salmonellosis. The present study aimed to investigate the antimicrobial resistance and molecular typing of 88 Salmonella Stanley strains isolated from humans (diarrhea patients, n = 64; and healthy carrier, n = 1), foods (aquatic products, n = 16; vegetable, n = 1; and pork, n = 1), and environment (waste water, n = 2; and river water, n = 3) in Shanghai, China from 2006 to 2012. Nearly half of the strains were resistant to sulfafurazole (43/88, 48.9%), and many were resistant to streptomycin (35/88, 39.8%), tetracycline (22/88, 25%), and nalidixic acid (19/88, 21.6%). Approximately a quarter of the strains (24/88, 27.3%) were resistant to more than three antimicrobials, and five had ACSSuT resistance type. Six clusters (A-F) were identified by pulsed-field gel electrophoresis (PFGE) with 80% similarity. Interestingly, strains in the same cluster identified by PFGE possessed similar antibiotic resistance patterns. PFGE typing also indicated that aquatic products might serve as a transmission reservoir for Salmonella Stanley infections in humans.

Multidrug-resistant (MDR) plasmids play an important role in disseminating antimicrobial resistance genes. To elucidate the antimicrobial resistance gene compositions in A/C incompatibility complex (IncA/C) plasmids carried by animal-derived MDR Salmonella Newport, and to investigate the spread mechanism of IncA/C plasmids, this study characterizes the complete nucleotide sequences of IncA/C plasmids by comparative analysis. Complete nucleotide sequencing of plasmids and chromosomes of six MDR Salmonella Newport strains was performed using PacBio RSII. Open reading frames were assigned using prokaryotic genome annotation pipeline (PGAP). To understand genomic diversity and evolutionary relationships among Salmonella Newport IncA/C plasmids, we included three complete IncA/C plasmid sequences with similar backbones from Salmonella Newport and Escherichia coli: pSN254, pAM04528, and peH4H, and additional 200 draft chromosomes. With the exception of canine isolate CVM22462, which contained an additional IncI1 plasmid, each of the six MDR Salmonella Newport strains contained only the IncA/C plasmid. These IncA/C plasmids (including references) ranged in size from 80.1 (pCVM21538) to 176.5 kb (pSN254) and carried various resistance genes. Resistance genes floR, tetA, tetR, strA, strB, sul, and mer were identified in all IncA/C plasmids. Additionally, bla

Rapid, reliable, and robust detection of Salmonella in produce remains a challenge. In this study, loop-mediated isothermal amplification (LAMP) was comprehensively evaluated against real-time quantitative PCR (qPCR) for detecting diverse Salmonella serovars in various produce items (cantaloupe, pepper, and several varieties of lettuce, sprouts, and tomato). To mimic real-world contamination events, produce samples were surface-inoculated with low concentrations (1.1-2.9 CFU/25 g) of individual Salmonella strains representing ten serovars and tested after aging at 4 °C for 48 h. Four DNA extraction methods were also compared using produce enrichment broths. False-positive or false-negative results were not observed among 178 strains (151 Salmonella and 27 non-Salmonella) used to evaluate assay specificity. The detection limits for LAMP were 1.8-4 CFU per reaction in pure culture and 10(4)-10(6) CFU per 25 g (i.e., 10(2)-10(4) CFU per g) in produce without enrichment, comparable to those obtained by qPCR. After 6-8 h of enrichment, both LAMP and qPCR consistently detected these low concentrations of Salmonella of diverse serovars in all produce items except sprouts. The PrepMan Ultra sample preparation reagent yielded the best results among the four DNA extraction methods. Upon further validation, LAMP may be a valuable tool for routine Salmonella testing in produce. The difficulty of detecting Salmonella in sprouts, whether using LAMP or qPCR, warrants further study.

β-Lactamases and extended-spectrum β-lactamases (ESBLs) producing pathogenic bacteria were widely studied previously in China, but were seldom focused on foodborne Salmonella. In this study, an investigation concerning β-lactamases and ESBLs producing Salmonella recovered from retail raw chickens was performed. Sixty of 699 foodborne Salmonella isolates were detected as β-lactamases and ESBLs-producing ones that covered 12 Salmonella serotypes and exhibited different pulsed-field gel electrophoresis genotypes. Forty-four of 60 β-lactamases and ESBLs-producing strains were simultaneously resistant to ampicillin, amoxicillin/clavulanic acid, ceftiofur, ceftriaxone, and cefoxitin. The most commonly detected β-lactamases and ESBLs-encoding gene was bla(TEM-1) (n = 44), followed by bla(OXA-1) (n = 38), bla(CMY-2) (n = 29), bla(PSE-1-like) (n = 1), bla(CTX-M-3) (n = 16), and bla(CTX-M-15) (n = 1), respectively. Fourteen, 24, 21, and 1 isolates were detected simultaneously positive for 1, 2, 3, and 4 of the detected β-lactamases and ESBLs-encoding genes, respectively. A Salmonella strain simultaneously co-carrying bla(TEM-1), bla(OXA-1), bla(CMY-2), and bla(CTX-M-3) was first reported in the present study. Amino acid substitution of Trp244Cys/His247Leu was detected in PSE-1, Val218Asp in CMY-2, and Asp242Gly in CTX-M-15 enzymes, respectively. A difference was found among the amino acid sequences of the detected OXA-1, CMY-2, CTX-M, PSE-1, and TEM-1. The results demonstrated that β-lactamases and ESBLs were emerging and prevalent in foodborne Salmonella.

Escherichia coli O157:H7 is, to date, the major E. coli serotype causing food-borne human disease worldwide. Strains of O157 with other H antigens also have been recovered. We analyzed a collection of historic O157 strains ( n = 400) isolated in the late 1980s to early 1990s in the United States. Strains were predominantly serotype O157:H7 (55%), and various O157:non-H7 (41%) serotypes were not previously reported regarding their pathogenic potential. Although lacking Shiga toxin ( stx ) and eae genes, serotypes O157:H1, O157:H2, O157:H11, O157:H42, and O157:H43 carried several virulence factors ( iha , terD , and hlyA ) also found in virulent serotype E. coli O157:H7. Pulsed-field gel electrophoresis (PFGE) showed the O157 serogroup was diverse, with strains with the same H type clustering together closely. Among non-H7 isolates, serotype O157:H43 was highly prevalent (65%) and carried important enterohemorrhagic E. coli (EHEC) virulence markers ( iha , terD , hlyA , and espP ). Isolates from two particular H types, H2 and H11, among the most commonly found non-O157 EHEC serotypes (O26:H11, O111:H11, O103:H2/H11, and O45:H2), unexpectedly clustered more closely with O157:H7 than other H types and carried several virulence genes. This suggests an early divergence of the O157 serogroup to clades with different pathogenic potentials. The appearance of important EHEC virulence markers in closely related H types suggests their virulence potential and suggests further monitoring of those serotypes not implicated in severe illness thus far.

The aim of this study was to investigate the prevalence of Staphylococcus aureus in quick-frozen dumplings and to characterize these strains. A total of 120 dumpling samples, including lamb (n = 13), vegetarian (n = 14), seafood (n = 12), and pork (n = 81) stuffing, were collected in Shaanxi province in China and screened for S. aureus. All S. aureus isolates were characterized by antimicrobial susceptibility testing, and detection of genes encoding staphylococcal enterotoxins, exfoliative toxins A and B (eta and etb), toxic shock syndrome toxin 1 (tsst-1), and resistance to methicillin-oxacillin (mecA). In all, 60.0% of all samples were positive for S. aureus, and 117 S. aureus isolates, including seven mecA-positive strains, were recovered from these positive samples. In addition, all mecA-positive S. aureus isolates were recovered from products of animal origin. In these S. aureus isolates, resistance was observed most frequently to ampicillin (92.3%) and penicillin (86.3%), followed by clarithromycin, erythromycin, midecamycin, tetracycline, and kanahemycin (from 53.8 to 28.2%). All isolates were sensitive to cefoperazone, minocycline, vancomycin, and ofloxacin. The predominant toxin gene was sec (38.5%), followed by seg (19.7%), sej (16.2%), see (12.8%), sea (11.1%), and seb (10.3%), whereas eta, etb, and tsst-1 genes were not detected. These findings indicate that S. aureus was present commonly in quick-frozen dumplings, accompanied by multiple antimicrobial resistance and toxin genes. Our findings highlight the urgency for stricter hygiene strategies in food production and the prudent use of antibiotics in the breeding industry.

Salmonellosis is one of the most common bacterial foodborne diseases worldwide. Poultry and poultry products are considered significant vehicles for foodborne salmonellosis transmission. We subtyped 725 Salmonella isolates recovered from 1152 retail raw chickens using pulsed-field gel electrophoresis (PFGE), and 448 ampicillin-resistant ones among 725 isolates using plasmid analysis in this study. Based on 80% similarity, 126 PFGE clusters were identified among 725 Salmonella isolates. Sixty one (8.4%) of 725 isolates belonged to PFGE cluster C96, followed by cluster C5 (38/725, 5.2%), and cluster C18 (38/725, 5.2%). Fifty two distinct PFGE patterns were detected among the isolates in Shaanxi Province, and 67, 64, 82, 67, 55, 88, and 47 patterns were identified in isolates in Henan, Sichuan, Beijing, Guangxi, Shanghai, Guangdong, and Fujian, respectively. Plasmids were detected in 331 (73.9%) of the 448 ampicillin-resistant Salmonella isolates. One hundred and twenty three plasmid profiles were identified, and P3, P11, P30, P12, were defined as predominant ones. Combination of PFGE and plasmid analysis was an effective way to enhance the genotypic discrimination ability to Salmonella , especially for S . Typhimurium isolates. Our results indicated that multiple Salmonella subtypes were prevalent in retail raw chicken in China, and they were potential hazard for food safety.

Escherichia coli serotype O157:H7 is a zoonotic food- and waterborne bacterial pathogen that causes a high hospitalization rate and can cause life-threatening complications. Increasingly, E. coli O157:H7 infections appear to originate from fresh produce. Ruminants, such as cattle, are a prominent reservoir of E. coli O157:H7 in the United States. California is one of the most agriculturally productive regions in the world for fresh produce, beef, and milk. The close proximity of fresh produce and cattle presents food safety challenges on a uniquely large scale. We performed a survey of E. coli O157:H7 on 20 farms in California to observe the regional diversity and prevalence of E. coli O157:H7. Isolates were obtained from enrichment cultures of cow feces. Some farms were sampled on two dates. Genomes from isolates were sequenced to determine their relatedness and pathogenic potential. E. coli O157:H7 was isolated from approximately half of the farms. The point prevalence of E. coli O157:H7 on farms was highly variable, ranging from zero to nearly 90%. Within farms, generally one or a few lineages were found, even when the rate of isolation was high. On farms with high isolation rates, a single clonal lineage accounted for most of the isolates. Farms that were visited months after the first visit might have had the same lineages of E. coli O157:H7. Strains of E. coli O157:H7 may be persistent for months on farms. IMPORTANCE This survey of 20 cow-calf operations from different regions of California provides an in depth look at resident Escherichia coli O157:H7 populations at the molecular level. E. coli O157:H7 is found to have a highly variable prevalence, and with whole-genome sequencing, high prevalences in herds were found to be due to a single lineage shed from multiple cows. Few repeat lineages were found between farms in this area; therefore, we predict that E. coli O157:H7 has significant diversity in this area beyond what is detected in this survey. All isolates from this study were found to have pathogenic potential based on the presence of key virulence gene sequences. This represents a novel insight into pathogen diversity within a single subtype and will inform future attempts to survey regional pathogen populations.

Salmonella Senftenberg is an important nontyphoidal Salmonella serovar that causes gastrointestinal disease worldwide. In total, 130 Salmonella Senftenberg strains obtained from humans, food, and the environment in Shanghai, People's Republic of China, were characterized for antimicrobial susceptibility and subjected to molecular typing. Our findings indicated that most (96 of 130, 73.8%) of the strains were susceptible to all 13 antimicrobial compounds tested, whereas only two strains (1.5%) were resistant to two antimicrobial compounds. In total, 56 pulsed-field gel electrophoresis profiles were identified, including four main pulsed-field gel electrophoresis profiles (X2, X3, X4, and X5) that showed 95.7% genetic similarity. Our study revealed that the strains of Salmonella Senftenberg from food and the environment shared a high correlation of genetic similarity with those from humans, highlighting the potential links that exist among the strains recovered from different sources in Shanghai.

The aim of this study was to investigate the existence and characteristics of Salmonella enterica in dried milk-related infant foods. Twenty-four (3.4%) of 705 samples, including 5 (2.0%) of 246 powdered infant formula, 18 (4.0%) of 445 infant rice cereal, and 1 (7.1%) of 14 other infant foods, were positive for Salmonella. Fifteen serotypes were identified in 40 Salmonella isolates; Salmonella Duesseldorf (15.0%) and Salmonella Indiana (15.0%) were more frequently detected than other serotypes. Resistance to chloramphenicol (82.5%) was most common, followed by tetracycline (57.5%), ceftiofur (52.5%), kanamycin (52.5%), streptomycin (50.0%), gentamycin (45.0%), nalidixic acid (35.0%), ceftriaxone (32.5%), ciprofloxacin (25.0%), amikacin (20.0%), and cefoxitin (15.0%). Twenty-eight (70.0%) isolates were resistant to ≥ 8 antimicrobials, with 5 (12.5%) being resistant to 14 antimicrobials. Amino acid substitutions in gyrase A (GyrA) were most frequently detected as Ser83Arg/Asp87Glu and in p53-associated Parkin-like cytoplasmic protein (ParC), they were all Ser80Arg; the quinolone resistance gene qnrS (47.5%) was commonly detected as well as aminoglycoside acetyltransferase [aac(6')-Ib; 25.0%], qnrA (17.5%), and qnrB (15.0%) genes. Thirty distinct pulsed-field gel electrophoresis patterns were identified among 40 isolates; no identical pulsed-field gel electrophoresis pattern was detected among Salmonella isolates with the same serovar that was recovered in 2010 and 2012. Our results suggest that dried milk-related infant foods could be contaminated with Salmonella and highlight that the dangers to infant health should not be neglected.

Shiga toxin-producing Escherichia coli (STEC) strains ( n = 194) representing 43 serotypes and E. coli K-12 were examined for clustered regularly interspaced short palindromic repeat (CRISPR) arrays to study genetic relatedness among STEC serotypes. A subset of the strains ( n = 81) was further analyzed for subtype I-E cas and virulence genes to determine a possible association of CRISPR elements with potential virulence. Four types of CRISPR arrays were identified. CRISPR1 and CRISPR2 were present in all strains tested; 1 strain also had both CRISPR3 and CRISPR4, whereas 193 strains displayed a short, combined array, CRISPR3-4. A total of 3,353 spacers were identified, representing 528 distinct spacers. The average length of a spacer was 32 bp. Approximately one-half of the spacers (54%) were unique and found mostly in strains of less common serotypes. Overall, CRISPR spacer contents correlated well with STEC serotypes, and identical arrays were shared between strains with the same H type (O26:H11, O103:H11, and O111:H11). There was no association identified between the presence of subtype I-E cas and virulence genes, but the total number of spacers had a negative correlation with potential pathogenicity ( P < 0.05). Fewer spacers were found in strains that had a greater probability of causing outbreaks and disease than in those with lower virulence potential ( P < 0.05). The relationship between the CRISPR- cas system and potential virulence needs to be determined on a broader scale, and the biological link will need to be established.

Various ready-to-eat (RTE) foods are becoming increasingly popular in the world and could be easily contaminated with various microorganisms including certain pathogens. A total of 342 RTE food samples, including 32 cooked meats, 123 vegetable salads, 26 boiled peanuts, 109 cold noodles, and 52 dried tofu samples, were collected in Shaanxi Province, People's Republic of China, during the period of July to October 2012 and screened for Staphylococcus aureus. All S. aureus isolates were characterized by antimicrobial susceptibility and PCR for detecting nine enterotoxin genes (sea to sej). Overall, 25.4% of samples were positive for S. aureus, including 10 (31.3%) cooked meats, 34 (27.6%) salad vegetables, 6 (23.1%) boiled peanuts, 20 (18.3%) cold noodles, and 17 (32.7%) dried tofu samples. Of the isolated S. aureus organisms, 98.4% were resistant to at least one antimicrobial agent and 58.6% to three or more antimicrobials. Resistance to erythromycin (78.1%) and tetracycline (40.6%) was most frequently detected, while all isolates were sensitive to vancomycin and amikacin. Moreover, 55.5% of isolates were positive for one or more enterotoxin genes. The genes sed (25.8%) and sea (19.5%) were commonly detected among the isolates; seg, sei, and sej were not found. Our findings indicate that RTE foods in Shaanxi were contaminated with S. aureus isolates that harbored multiple toxin genes and exhibited multiple-drug resistance. Appropriated hygienic measures should be taken by producers, retailers, and consumers to reduce the risk posed by S. aureus in RTE foods.

Listeria monocytogenes is an important foodborne pathogen of public health concern. A total of 902 retail food samples, including 342 ready-to-eat (RTE) foods, 366 infant foods, and 194 raw chickens were collected randomly in supermarkets and farmers' markets in 12 geographic areas in Shaanxi Province, China and screened for L. monocytogenes. All L. monocytogenes isolates were characterized by antimicrobial susceptibility testing, serotyping, and pulsed-field gel electrophoresis (PFGE). Twenty-seven (3.0%) samples were positive for L. monocytogenes, and 39 L. monocytogenes isolates were recovered from positive samples. Of these L. monocytogenes isolates, 21 isolates (53.8%) showed resistance to at least one antimicrobial. The isolates displayed resistance most frequently to oxacillin (18 isolates, 46.2%), followed by tetracycline (five isolates, 12.8%), erythromycin (four isolates, 10.3%), trimethoprim/sulfamethoxazole (three isolates, 7.7%), chloramphenicol (two isolates, 5.1%), and vancomycin (one isolate, 2.6%). All isolates were sensitive or displayed intermediate resistance to gentamicin, ampicillin, ciprofloxacin, and amikacin. Four serotypes including serotype 1/2b, 4b, 4e, and 1/2a were identified in those foodborne isolates. PFGE analysis demonstrated that some isolates with the same PFGE patterns came from different food sources, and isolates from the same food source tend to cluster closely. Presence of L. monocytogenes of clinically important serotypes in retail foods and their antimicrobial resistance constitute a potential risk for the public. Appropriate measures should be taken by government, industry, and consumers to reduce the risk posed by this ubiquitous pathogen.

A total of 67 Listeria monocytogenes isolates from 698 raw meat samples were characterized for molecular serogroup identification and antimicrobial susceptibility. Approximately one third (32.8%) of the isolates belonged to molecular serogroup 1/2a, 3a, followed by 1/2c, 3c (26.9%), 1/2b, 3b, 7 (22.4%), 4b, 4d, 4e (16.4%) and 4a, 4c (1.5%). Most of the L. monocytogenes isolates were susceptible to 14 antimicrobials tested but several were resistant to tetracycline, ciprofloxacin and nitrofurantoin. An additional 30 L. monocytogenes isolates from chicken and produce in our collection were also included to determine the presence of significant virulence markers. All 97 isolates carried inlC and inlJ except for a lineage III isolate 110-1. Most Listeriolysin S (LLS)-carrying isolates (11/12) belonged to lineage I, whereas the remaining one isolate belonged to lineage III. Five 4b, 4d, 4e isolates including two from turkey and three from produce belonged to Epidemic Clone I (ECI). Four molecular serogroup associated mutation types that lead to premature stop codons (PMSCs) in inlA were identified. PFGE and inlA sequence analysis results were concordant, and different virulence potential within 1/2a, 3a and 4b, 4d, 4e isolates were observed. The study revealed that a subset of isolates from meat and produce belonged to ECI, harbored inlC, inlJ and LLS, and produced full length InlA, suggesting that they be capable of causing human illness.

The aim of this study was to determine the prevalence of Staphylococcus aureus ( S. aureus ) and methicillin-resistant S. aureus (MRSA) in whole raw chickens in China. A total of 1152 whole chicken samples were collected from three types of retail markets (large supermarket, small supermarket, and farmers market) in six provinces and two national cities in China and screened for the presence of. S. aureus , then the S. aureus isolates were further examined for the presence of MRSA. The overall positive rate for S. aureus was 24.2%. S. aureus prevalence was highest in Shaanxi province (39.6%), followed by Fujian province (31.3%), then Sichuan and Guangdong provinces (26.4% in each province), Shanghai (23.6%), Beijing (19.4%), Henan province (13.9%), the lowest prevalence was found in Guangxi province (13.2%). S. aureus prevalence was significantly different among the six provinces and two national cities ( P S. aureus prevalence in chickens collected from large supermarket, small supermarket, and farmers market were 26.3%, 25%, and 21.4%, respectively, but differences were not significant ( P > 0.05). These findings indicate the need for identifying the factors contributing to the contamination at the farm, processing and retail level, and for the implementation of good manufacturing practice to better control the pathogen throughout poultry industry.

The prevalence and characteristics of non-O157 Shiga toxin-producing Escherichia coli (STEC) in retail ground meat from the Washington D.C. area were investigated in this study. STEC from 480 ground beef and pork samples were identified using PCR screening followed by colony hybridization. The STEC isolates were serogrouped and examined for the presence of virulence genes (stx1, stx2, eae and hlyA), and antimicrobial susceptibility. PFGE was used to identify the clonal relationships of STEC isolates, and PCR-RFLP was employed to determine stx subtypes. In addition, the cytotoxicity of STEC isolates was determined using a Vero cell assay. STEC were identified in 12 (5.2%) of 231 ground pork and 13 (5.2%) of 249 ground beef samples. Among 32 STEC isolates recovered from the 25 samples, 12 (37.5%) carried stx2dact and 7 (21.9%) carried hlyA, but none carried eae. Nine isolates were identified as O91, and 17 (53.1%) isolates were resistant to two or more antimicrobials. Verotoxicity was detected in 26 (81.3%) of the STEC isolates. Thus, the retail ground meat was contaminated with a heterogeneous population of non-O157 STEC, some of which were potential human pathogens.

Terrapins and turtles are known to transmit Salmonella to humans. However, little was known about the occurrence of this pathogen in soft-shelled terrapin that is a popular delicacy in Chinese and other East Asian cuisines. We isolated and characterized 82 (24.4%) isolates of Salmonella from 336 fecal samples of soft-shelled terrapins (51 of 172; 29.7%) and pet turtles (31 of 164; 18.9%) in Shanghai. Salmonella Thompson was the most common serotype (17.1%) among others. Many isolates (84.1%) were resistant to multiple antimicrobials (≥3). Molecular analysis of Salmonella Thompson and Salmonella Typhimurium using pulsed-field gel electrophoresis unveiled a close genetic relationship between several human and terrapin isolates. Our results highlight the risk associated with the handling and consumption of turtles and their role in the spread of Salmonella in the human salmonellosis.

Mutations in gyrase and topoisomerase genes (gyrA, gyrB, parC and parE) were examined among 30 multidrug-resistant (MDR) Salmonella isolates recovered from retail meats using PCR and DNA sequencing analysis. Six isolates had a single gyrA mutation (Ser83 → Phe or Ser83 → Tyr or Asp87 → Asn) with ciprofloxacin (Cip) minimum inhibitory concentrations (MICs) ranging from 0.125 to 0.5 μg/ml except 1 with 16 μg/ml. Three isolates had both a gyrA mutation (Ser83 → Tyr or Ser83 → Phe) and a parC mutation (Ser80 → Arg) (Cip MICs, 8 to 16 μg/ml). Fifteen isolates had both double mutations in gyrA (Ser83 → Phe and Asp87 → Gly or Asp87 → Asn) and a single mutation (Ser80 → Arg) in parC (Cip MICs, 8 to ≥ 16 μg/ml). Three had double gyrA mutations (Ser83 → Phe, Asp87 → Gly or Asp87 → Asn), one parC mutation (Ser80 → Arg), and 1 parE mutation (Lys428 → Gln or Gly442 → Ser) (Cip MICs, 8 to ≥ 16 μg/ml). One had double gyrA mutations (Ser83 → Phe, Asp87 → Gly or Asp87 → Asn), one parC mutation (Ser80 → Arg), and 2 parE mutations (Lys441 → Ile and Asp494 → Asn) (Cip MICs, 8 to ≥ 16 μg/ml). Isolates with single gyrA mutations were less resistant to quinolone and fluoroquinolones than those together with additional parC and/or parE mutations in Salmonella. Our findings confirmed the importance of point mutations in gyrase and topoisomerase in reduced susceptibility to quinolone and fluoroquinolones. Mutations in gyrA confer low-level quinolone and fluoroquinolone resistance, while additional gyrA mutation(s) together with parC and/or parE mutations increase the resistance to a high level.

Dry infant foods are not sterile and could be contaminated with various bacteria including certain pathogens. The aim of this study was to investigate the prevalence of Staphylococcus aureus in infant foods and to characterize these strains. A total of 367 infant food samples, including 143 samples of powdered infant formula milk (PIF) and 224 samples of infant rice cereal (IRC), were collected in the Shaanxi Province of China during the period of July to August 2010 and screened for S. aureus. All S. aureus isolates were characterized by antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and detection of genes encoding enterotoxins, exfoliative toxins, Panton-Valentine leukocidin (PVL), and toxic shock syndrome toxin 1. Among all the samples examined, sixteen of 143 PIF samples (11.2%) and 14 of 224 IRC samples (6.3%) were positive for S. aureus. From these positive samples, 29 S. aureus strains were isolated from PIF and 25 from IRC. Of these S. aureus isolates, 83.3% were resistant to at least one antimicrobial, 35.2% to three or more antimicrobials. Resistance was most frequently observed to erythromycin (75.9%), followed by ciprofloxacin (51.9%) and trimethoprim/sulfamethoxazole (27.8%), while significantly fewer isolates were resistant to gentamicin (22.2%), tetracycline (18.5%), or cefoxitin (3.7%). In addition, 63.0% of isolates were positive for one or more toxin genes tested. The three most predominant toxin genes were pvl (40.7%), seg (38.9%), and sec (18.5%), followed by sea (7.4%), seb (7.4%), sed (5.6%), and see (5.6%). The ets, tsst-1, seh, sei, and sej genes were not detected. A total of 39 PFGE patterns were generated among 51 selected food isolates. Our findings indicate that PIF and IRC in the Shaanxi province were contaminated with S. aureus, and many S. aureus isolates harbored multiple toxin genes and exhibited multiple antimicrobial resistance. In addition, these S. aureus isolates were genetically diverse. The presence of S. aureus strains in these infant foods poses a potential threat to infant health.

Data regarding Salmonella on raw poultry are very limited in China. The objective of this study was to determine the prevalence of Salmonella on raw poultry at the retail level in six provinces and two national cities in China. Whole chicken carcasses (n = 1,152) were collected from three types of retail markets (large, small, and wet). All samples were analyzed for the presence of Salmonella by using the U.S. Department of Agriculture, Food Safety Inspection Service method. Of 1,152 chicken samples, overall Salmonella prevalence was 52.2%. The highest prevalence was observed in Guangxi Province (65.3%), next in Guangdong Province (64.6%), and then in Beijing (63.9%), Shaanxi Province (50.7%), Henan Province (47.9%), Shanghai (44.4%), and Fujian Province (42.4%), and lowest prevalence was observed in Sichuan Province (38.9%). Salmonella prevalence was significantly different among the six provinces and two national cities. Salmonella prevalence was highest in the wet markets (54.4%) compared with the large markets (50.3%) and the small markets (52.1%), but differences were not significant (P > 0.05). Good manufacturing practices, good agricultural practices, and hazard analysis critical control point systems for Salmonella control in poultry production at the farm, processing, and retail level should be implemented.

Campylobacter isolates (n = 297; 202 C. jejuni and 95 C. coli isolates) recovered from 2,513 retail meat samples (chicken breasts, ground turkey, ground beef, and pork chops) were examined for antimicrobial susceptibility. The isolates were further analyzed for genetic relatedness by pulsed-field gel electrophoresis (PFGE) using SmaI and KpnI restriction enzymes, and a subset of isolates (n = 174) were subtyped by multilocus sequence typing (MLST). The resistance most frequently observed was that to doxycycline (27.6%), followed by ciprofloxacin (13.8%) and erythromycin (6.4%). All isolates were susceptible to gentamicin and meropenem. C. coli showed higher resistance to doxycycline than did C. jejuni (42.1 versus 20.8%) and lower resistance to ciprofloxacin than did C. jejuni (10.5 versus 15.3%). Erythromycin resistance was only observed in C. coli. PFGE using SmaI plus KpnI digestion generated 168 clusters from 297 isolates: 115 from C. jejuni and 53 from C. coli. MLST revealed 44 sequence types (STs) under 10 clonal complexes from 120 C. jejuni and 27 STs under two clonal complexes from 54 C. coli. There was a positive association between PFGE and STs; however, PFGE showed greater discriminatory power than MLST. Subtyping data did not correlate with antimicrobial resistance phenotypes.

In order to improve the safety of refrigerated ready-to-eat food products prepared at retail deli departments, a better understanding of current practices in these establishments is needed. Food employees in deli departments at six chain and three independent retail establishments in Maryland and Virginia were observed, using notational analysis, as they prepared deli products for sale. The frequency of contact with objects and deli products before sale, hand washing and glove changing during preparation, and equipment, utensil, and surface cleaning and sanitizing was determined. Compliance with the U.S. Food and Drug Administration's 2005 model Food Code recommendations, which must be adopted by the individual state and local jurisdictions that are responsible for directly regulating retail establishments, was also assessed. Observations indicated there were a large number of actions for which hand washing was recommended at independent and chain stores (273 recommended of 1,098 total actions and 439 recommended of 3,073 total actions, respectively). Moreover, 67% (295 of 439) of the actions for which hand washing was recommended at the chain stores and 86% (235 of 273) of those at the independent stores resulted from employees touching non-food contact surfaces prior to handling ready-to-eat food. Compliance with hand washing recommendations was generally low and varied depending on store type with independent stores exhibiting lower compliance than chain stores (5 instances of compliance for 273 recommended actions and 73 instances of compliance for 439 recommended actions, respectively). Potential risk mitigation measures that may reduce the frequency of hand washing actions needed during ready-to-eat food preparation in retail deli departments are discussed. More research is needed to determine the impact of such measures on food safety.

A total of 764 retail meat including 515 chicken, 91 pork, 78 beef and 80 lamb samples were collected in Shaanxi Province of China in 2007-2008 to determine the prevalence of Salmonella. The isolates were characterized using serotyping, antimicrobial susceptibility testing, and the presence of bla(CMY-2) and bla(TEM) and class I integrons. Selective serovars were further subtyped using PFGE. Approximately 54% (276) of chicken, 31% (28) of pork, 17% (13) of beef and 20% (16) of lamb samples were positive of Salmonella. Among 24 serovars identified, Enteritidis (31.5%) was most common, followed by Typhimurium (13.4%), Shubra (10.0%), Indiana (9.7%), Derby (9.5%) and Djugu (7.0%). Nearly 80% of the isolates (283) were resistant to at least one antimicrobial, and 53% (191) to more than three antimicrobials. Resistance was most frequently observed to sulfamethoxazole (67%), to trimethoprim/sulfamethoxazole (58%) and to tetracycline (56%). Furthermore, many isolates were resistant to nalidixic acid (35%), ciprofloxacin (21%) and ceftriaxone (16%). Most isolates of Shubra (89%) and Indiana (88%) were resistant toor = 9 antimicrobials, compared to only 11% of Enteritidis and 9% of Infantis that showed similar resistance. Class I integrons were detected in 10% of the isolates, and contained aadA, tetR, dhfr, bla(PSE-1), bla(DHA-1) and bla(VEB-1) gene cassettes alone or various combinations. Ceftriaxone- and/or cefoperazone-resistant isolates (n=62) carried bla(TEM) (51.6%) and/or bla(CMY-2) (56.5%). A total of 116 PFGE patterns were generated among 210 selected isolates. Our findings indicated that Salmonella contamination was common in retail meats, and that the Salmonella isolates were phenotypically and genetically diverse. Additionally, many Salmonella isolates were resistant to multiple antimicrobials.

Listeria monocytogenes isolates recovered from retail ready-to-eat (RTE) meats, raw chickens and fresh produce were characterized by serogroup identification using PCR, genotyping using pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. Five L. monocytogenes serogroups were identified. Of the 167 isolates 68 (41%) belonged to serogroup 1/2b, 3b; 53 (32%) belonged to serogroup 4b, 4d, 4e; 43 (26%) belonged to serogroup 1/2a, 3a; 2 (1.2%) belonged to serogroup 1/2c, 3c; and 1 (0.6%) belonged to serogroup 4a, 4c. PFGE generated 120 patterns which correlated well with PCR serogrouping. Most L. monocytogenes isolates were resistant to sulfonamide (73%) and some were resistant to tetracycline (8.4%) and ciprofloxacin (1.8%). Tetracycline resistance was conjugatively transferable and the tet(M) gene was identified in 14 tetracycline-resistant isolates as well as their transconjugants. These findings indicate that L. monocytogenes present in food were diverse, and that resistance to one or more antibiotics among these isolates was common. In addition, the presence of potential serotype 4b in all food categories is of public health concern, as serotype 4b has been the serotype most frequently associated with human listeriosis.

Ethanol extracts of Chardonnay grape and black raspberry seed flours were evaluated for their capacity to suppress lipid oxidation, preserve important fatty acids, and inhibit microbial growth. They were also tested for radical scavenging activity against DPPH and peroxyl radicals as reflected in oxygen radical absorbance capacity (ORAC), and total phenolic content (TPC). Both tested seed flour extracts suppressed lipid oxidation and rancidity development in fish oil. Black raspberry seed flour extract significantly reduced the degradation of biologically important n − 3 PUFA under accelerated oxidative conditions. Black raspberry and Chardonnay seed flour extracts at 165 and 160 μg seed flour equivalents/mL, respectively exhibited bacteriocidal activity against Escherichia coli and growth inhibition of Listeria monocytogenes under experimental conditions. Both seed flour extracts exhibited DPPH radical quenching activity and Chardonnay had the stronger ORAC of 663 μmol Trolox equivalents per gram seed flour and the higher TPC of 99 mg gallic acid equivalents/g flour. The data from this study suggest the potential for developing natural food preservatives from these seed flours for improving food stability, quality, safety, and consumer acceptance.

The abilities of 34 Campylobacter jejuni and 9 Campylobacter coli isolates recovered from retail meats to adhere to and invade human intestinal epithelial T84 cells were examined and compared with those of a well-characterized human clinical strain, C. jejuni 81-176, to better assess the pathogenic potential of these meat isolates. The meat isolates exhibited a wide range of adherence and invasion abilities; a few of the isolates adhered to and invaded T84 cells almost as well as did C. jejuni 81-176. There was a significant correlation between the adherence ability and the invasion ability of the Campylobacter isolates. The presence of eight putative virulence genes in these Campylobacter isolates that are potentially responsible for adherence and invasion or that encode cytolethal distending toxin was determined using PCR. All Campylobacter isolates possessed flaA, cadF, pldA, cdtA, cdtB, and cdtC, and most (91%) also contained the ciaB gene. However, the virB11 gene, carried by virulence plasmid pVir, was absent in almost all the Campylobacter isolates. Our findings indicated that C. jejuni and C. coli present in retail meat were diverse in their ability to adhere to and invade human intestinal epithelial cells and that the putative virulence genes were widespread among the Campylobacter isolates. Thus, despite of the presence of the putative virulence genes, only some but not all Campylobacter strains isolated from retail meat can effectively invade human intestinal epithelial cells in vitro.

An improved method was developed for the rapid isolation and identification of Salmonella in chicken carcasses. Considering the heavy presence of Proteus mirabilis in the poultry-production environment, we investigated combinations of selective bacterial growth media that promote the growth of Salmonella but inhibit that of P. mirabilis. The improved method was evaluated using standard methods described in the Bacteriological Analytical Manual (BAM) by the U.S. Food and Drug Administration (FDA), and the Microbiology Laboratory Guidebook published by the U.S. Department of Agriculture Food Safety Inspection Service (USDA/FSIS). A total of 44 chicken carcasses were analyzed for Salmonella using the three methods. The FDA/BAM method showed a poor sensitivity and specificity, especially when the tetrathionate (TT) broth was applied. The improved method offered an excellent specificity (>95%) and shortened the time of isolation and identification from 4 to 5 days to less than 3 days, consequently reducing the costs.

A suspension array assay was developed for molecular serotyping of the seven most prevalent Shiga toxin–producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, O145, and O157). Fluorescence values of 59 STEC were 30 to >270 times greater than the signals of negative controls, demonstrating the method's effectiveness for the molecular serotyping of STEC.

The capability of an assay to detect Listeria monocytogenes from artificially inoculated fresh-cut produce such as cantaloupe and mixed salad was demonstrated. An oligonucleotide probe that becomes fluorescent upon hybridization to the target DNA (Molecular Beacon, MB) was used in a real-time polymerase chain reaction (PCR) assay. As few as 4 to 7 colony-forming units (CFU) of L. monocytogenes per 25 g of artificially contaminated produce could be detected. A comparison of 2 commercially available kits using MB-PCR (iQ-Check, Bio-Rad Laboratories) and conventional PCR (BAX, Dupont Qualicon) was performed on artificially inoculated produce. The time required to detect L. monocytogenes (from produce to PCR) was considerably shorter for the iQ-Check protocol (approximately 26 h) compared with the BAX-PCR (approximately 52 h). The iQ-Check protocol was also used to confirm the identity of the L. monocytogenes isolates obtained during a microbiological screen of conventional and organic leaf lettuce and alfalfa sprout samples from local supermarkets. The iQ check protocol was successful in differentiating L. monocytogenes isolates from other Listeria spp. such as L. welshimeri, L. innocua, and L. ivanovii. This is the 1st report of the application of the MB probe being used for real-time detection of L. monocytogenes in whole and fresh-cut produce.

The purpose of this review article is to summarize steadily accruing evidence from around the world which indicate retail meat and poultry are a reservoir of antimicrobial-resistant Escherichia coli. The majority of E. coli recovered from retail meat and poultry have been found resistant to at least one antimicrobial, and an increasing proportion have been found resistant to clinically important, frontline antimicrobials, including trimethoprim-sulphamethoxazole, fluoroquinolones, and third-generation cephalosporins. Continued surveillance throughout the food production continuum is needed to detect emerging resistance phenotypes.

Escherichia coli O157:H7, a Shiga toxin-producing E. coli, has been the causative agent of many cases of severe, often life-threatening foodborne illness. Because of the importance of E. coli O157:H7 to public health, many molecular typing methods have been developed to determine its transmission routes and source of infection during epidemiological investigations. Pulsed-field gel electrophoresis (PFGE) is currently used by public health organizations to track infections of E. coli O157:H7 and other foodborne pathogens. In this study, we compared the ability of PFGE, multilocus sequence typing (MLST), and repetitive-element PCR (Rep-PCR) to distinguish among 92 E. coli O157:H7 isolates from cattle, food, and infected humans. Several virulence genes, including the intimin gene (eaeA), the hemolysin gene (hlyA), and the H7 fimbrial gene (fliC), and a housekeeping gene for beta-glucuronidase (uidA) were included in MLST. Rep-PCR reactions were performed using a commercially available typing kit (Bacterial Barcodes Inc., Houston, Tex.) with the provided Uprime-RI primer set. Results of the study indicated that PFGE provided the most discrimination among the techniques, identifying 72 distinct PFGE profiles for the isolates; Rep-PCR elucidated 14 different profiles, whereas MLST generated five profiles. Additionally, there did not appear to be any correlation among the typing methods examined in this study. Therefore, to date, PFGE remains the technique of choice for molecular subtyping of E. coli O157:H7.

Four hundred and seventy-two generic Escherichia coli isolates were recovered from ground and whole retail beef, chicken, pork, and turkey obtained from Greater Washington, DC, USA during the years 1998 to 2000. Many of the isolates displayed resistance to tetracycline (59%), sulfamethoxazole (45%), streptomycin (44%), cephalothin (38%) and ampicillin (35%). Resistance was also observed, but to a lesser extent, to gentamicin (12%), nalidixic acid (8%), chloramphenicol (6%), ceftiofur (4%) and ceftriaxone (1%). Sixteen percent of the isolates displayed resistance to one antimicrobial, followed by 23% to two, 23% to three, 12% to four, 7% to five, 3% to six, 2% to seven and 2% to eight. Three E. coli isolates were shown to possess Shiga toxin genes (stx2) via PCR; all were O non-typeable and were recovered from ground beef samples purchased on the same day at the same supermarket. One of the Shiga toxin-producing E. coli (STEC) isolates was susceptible to each of the antimicrobials tested, whereas one displayed resistance to cephalothin and sulfamethoxazole, and one displayed resistance to ampicillin, cephalothin, gentamicin, streptomycin, sulfamethoxazole and tetracycline. Findings from this study indicate that retail raw meats may often be contaminated with antimicrobial-resistant E. coli.

Salmonella enterica serotype Agona (Salmonella Agona) has been among the top 10 serotypes that cause human diarrheal diseases in China. A total of 95 Salmonella Agona (67 from humans, and 28 from animals, food of animal origins, and environmental sources) recovered in Shanghai, China from 2005 to 2011 were subjected to antimicrobial susceptibility testing and molecular subtyping using pulsed-field gel electrophoresis (PFGE). Approximately 68.4% of the Salmonella Agona isolates were pansusceptible to 15 antimicrobial agents tested, and 4 isolates (4.21%) were resistant to at least 3 antimicrobials. PFGE analysis resulted in 41 unique patterns, of which 4 major PFGE patterns (X3, X4, X5, and X6) were grouped together at 96.1% similarity. Isolates of the four patterns included those from food (pork, beef, and chicken) and humans. Our findings showed that the same clones of Salmonella Agona were recovered from human patients and food, and that food of animal origin was potentially a major vehicle of Salmonella Agona in human salmonellosis in Shanghai.