In Silico Detection of Integrons and Their Relationship with Resistance Phenotype of Salmonella Isolates from a Brazilian Pork Production Chain.

The pork production chain is an important reservoir of antimicrobial resistant bacteria. This study identified and characterized integrons in Salmonella isolates from a Brazilian pork production chain and associate them with their antibiotic resistance pattern. A total of 41 whole-genome sequencing data of nontyphoidal Salmonella were analyzed using PlasmidSPAdes and IntegronFinder software. Nine isolates (21.9%) had some integrons identified (complete and/or incomplete). Six complete class 1 integrons were found, with streptomycin resistance genes (aadA1, aadA2) alone or downstream of a trimethoprim resistance gene (dfrA1, dfrA12), and some also containing resistance genes for sulfonamides (sul1, sul3) and chloramphenicol (cmlA1). Class 2 integron was detected in only one isolate, containing dfrA1-sat2-aadA1 gene cassettes. Five isolates harbored CALINs-clusters attC but lacking integrases-with antimicrobial resistance genes typically found in integron structures. In all, integrons were observed among four serotypes: Derby, Bredeney, Panama, and monophasic var. Typhimurium I 4,[5],12:i:-. The association of integrons with antibiotic resistance phenotype showed that these elements were predominantly identified in multidrug resistance isolates, and six of the seven gentamicin-resistant isolates had integrons. So, surveillance of integrons in Salmonella should be performed to identify the potential for the spread of antimicrobial resistance genes among bacteria.

Abundance of Colistin-Resistance Genes in Retail Meats in Vietnam.

The degree of contamination of retail meat with colistin-resistant bacteria and its potential contribution to dissemination within communities remains to be determined. Thus, we aimed to elucidate the contamination status of colistin-resistance genes, indicative of colistin-resistant bacteria, in retail meats in Vietnam. In total, 46 chicken and 49 pork meats from stores in Vietnam and Japan were examined. Multiplex real-time polymerase chain reaction with TaqMan probes was performed for detecting mcr-1, mcr-3, and Escherichia coli 16S rRNA. Colistin-resistant bacteria in meats were isolated using selective media. The minimum inhibitory concentrations of colistin were determined using the broth microdilution method. The results showed that 70.7% of chicken meats in Vietnam were contaminated with both mcr-1 and mcr-3. Meanwhile, mcr-1 and mcr-3 were detected in 15.9% and 40.9% of pork meat, respectively. Only mcr-3 was detected in 40% of chicken in Japan. In addition, mcr-1-harboring E. coli and mcr-3-harboring Aeromonas were isolated from chicken meats in Vietnam. Some of these isolates showed colistin resistance. These results showed that most retail meats were highly contaminated with colistin-resistance genes. Notably, our results suggest that mcr-3 is more prevalent in the contaminated samples compared with mcr-1.

History repeats itself: consumption of a local traditional roast pork meat recipe leads to Salmonella ser. Give cases in Greece.

INTRODUCTION: An outbreak of gastroenteritis due to Salmonella Give, a very rarely identified serotype in human isolates in Greece, occurred in participants of a religious festival in a rural area of southern Greece, in September 2022. The objectives of this study were to describe the outbreak in terms of epidemiology, identify the vehicle of transmission of the foodborne pathogen and recommend prevention measures. METHODS: The outbreak was linked to the consumption of a local traditional recipe of roasted pork meat served by a street food vendor. In 2018, the same food item, served in a restaurant in the same region, was implicated in another S. Give outbreak. RESULTS: Outbreak investigations revealed that outbreak-associated isolates, of food and human origin, belonged to the same S. Give strain. Significant deficiencies regarding food safety practices were identified. CONCLUSION: Technical knowledge about pathogen transmission paths is important in order for both food handlers and consumers to follow hygiene and sanitary measures, mainly in cases of mass gatherings, where large quantities of food are prepared, handled, cooked and served. Efficient official supervision, mainly during summer festivals, is required in order to avoid recurrence of foodborne infections by different combinations of pathogens/food commodities.

Sphingomonas aliaeris sp. nov., a new species isolated from pork steak packed under modified atmosphere.

Species belonging to the genus Sphingomonas have been isolated from environments such as soil, water and plant tissues. Many strains are known for their capability of degrading aromatic molecules and producing extracellular polymers. A Gram-stain-negative, strictly aerobic, motile, red-pigmented, oxidase-negative, catalase-positive, rod-shaped strain, designated DH-S5T, has been isolated from pork steak packed under CO2-enriched modified atmosphere. Cell diameters were 1.5×0.9 µm. Growth optima were at 30 °C and at pH 6.0. Phylogenetic analyses based on both complete 16S rRNA gene sequence and whole-genome sequence data revealed that strain DH-S5T belongs to the genus Sphingomonas, being closely related to Sphingomonas alpina DSM 22537T (97.4 % gene sequence similarity), followed by Sphingomonas qilianensis X1T (97.4 %) and Sphingomonas hylomeconis GZJT-2T (97.3 %). The DNA G+C content was 64.4 mol%. The digital DNA-DNA hybridization value between the isolate strain and S. alpina DSM 22537T was 21.0 % with an average nucleotide identity value of 77.03 %. Strain DH-S5T contained Q-10 as the ubiquinone and major fatty acids were C18 : 1 cis 11 (39.3 %) and C16 : 1 cis 9 (12.5 %), as well as C16 : 0 (12.1 %) and C14 : 0 2-OH (11.4 %). As for polar lipids, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, dimethylphosphatidylethanolamine and sphingoglycolipid could be detected, alongside traces of monomethylphosphatidylethanolamine. Based on its phenotypic, chemotaxonomic and phylogenetic characteristics, strain DH-S5T (=DSM 110829T=LMG 31606T) is classified as a representative of the genus Sphingomonas, for which the name Sphingomonas aliaeris sp. nov. is proposed.

Biological control of mites by xerophile Eurotium species isolated from the surface of dry cured ham and dry beef cecina.

Some meat dry products, including dry cured ham and dry beef cecina, are cured in cellars at moderately cold temperature allowing the growth of a lawn of fungi on their surface. During the curing process, frequently these products became contaminated with fungivore mites of the Acaridae family that feed on fungal mycelium and spores. AIMS: The aim of this article is to study the possible biological control of mites by fungi that form part of the normal microbiota of these meat products. METHODS AND RESULTS: Some yellow/orange pigmented fungi growing on the ham surface decreased the proliferation of mites; therefore, we isolated from ham and cecina xerophilic yellow/orange coloured fungal strains that were identified as members of the genus Eurotium (recently reclassified as Aspergillus section Aspergillus). Using molecular genetic tools, we have identified 158 strains as Eurotium rubrum (Aspergillus ruber), Eurotium repens (Aspergillus pseudoglaucus) and Eurotium chevalieri (Aspergillus chevalieri). Two strains, E. rubrum C47 and E. rubrum C49, showed strong miticidal activity. The toxic compound(s) are associated with the formation of cleistothecia. In synchronized mite development experiments, we observed that all stages of the mite lifecycle were inhibited by the E. rubrum C47 strain. In addition, we searched for miticidal activity in 13 culture collection Eurotium strains isolated from different habitats, and found that only one, Eurotium cristatum NRRL 4222 (Aspergillus cristatus) has a strong miticidal activity. CONCLUSIONS: These fungal strains have proliferated on the surface of ham and cecina for decades, and possibly have acquired miticidal activity as a resistance mechanism against fungivores. SIGNIFICANCE AND IMPACT OF THE STUDY: Biological control of infecting mites by favouring growth of E. rubrun C47, in place of the normal mixed population of Aspergillus and Penicillium, is an attractive approach to control mite infestations.

Presence and quantification of pathogenic Arcobacter and Campylobacter species in retail meats available in Japan.

We present estimations for the amounts of Arcobacter (A. butzleri, A. cryaerophilus and A. skirrowii) and Campylobacter (C. jejuni, C. coli and C. fetus) species in retail chicken, pork and beef meat using PCR-MPN. Arcobacter butzleri, A. cryaerophilus and C. jejuni were found in 100, 60 and 55% of chicken samples, respectively. No other Arcobacter or Campylobacter species were found in chicken. The MPNs of A. butzleri, A. cryaerophilus and C. jejuni were greater than 103 per 100 g in 50, 0 and 5% of samples, respectively. The MPN of A. butzleri was higher than that of C. jejuni in 95% of samples. In pork, A. butzleri and A. cryaerophilus were detected in 10 and 11 (50 and 55%) of 20 samples, respectively. No other Arcobacter or Campylobacter species were found in pork. Only one pork sample had more than 103 MPN per 100 g of A. cryaerophilus. For beef, only two samples tested positive for A. cryaerophilus, at 4600 and 92 MPN per 100 g. Overall, we found that the presence and MPNs of Arcobacter species are very high in chicken. In contrast, the positive ratios of Arcobacter in pork were high as chicken samples, but MPNs were lower than in chicken.

Persistence of Yersinia enterocolitica bio-serotype 4/O:3 in a pork production chain in Minas Gerais, Brazil.

Yersinia enterocolitica bio-serotype 4/O:3 was previously identified in a pork production chain in Brazil and the obtained isolates presented high identity by pulsed-field gel electrophoresis (PFGE, XbaI). For the current study, an additional 147 porcine samples (tonsils = 100, palate = 30, head meat = 17) were collected from the same pork production chain 2-years later and 14 (9.5%) tested positive for Y. enterocolitica. Isolates (n = 24, 1 to 2 per positive sample) were bio-serotype 4/O:3 and harbored virulence genes ail, inv, wbbU, virF, myfA, ystA, ymoA, hreP and sat, and the multidrug resistance related genes emrD, marC and yfhD. PFGE (XbaI) demonstrated no differences among isolates (100% similarity) and were identical to some Y. enterocolitica isolates (n = 13) obtained previously from the same pork chain. A second PFGE analysis (NotI) confirmed the high degree of similarity among isolates obtained over time, demonstrating the persistence of an apparent clonal Y. enterocolitica bio-serotype 4/O:3 in this particular pork production chain in Brazil.

Effects of partial replacement of NaCl with KCl on bacterial communities and physicochemical characteristics of typical Chinese bacon.

This work aimed to determine the effects of partial substitution of NaCl with 0% (control), 30%, 50%, and 70% of KCl on the bacterial communities, proteolysis and lipid oxidation of Chinese bacon during processing. The proportion of genus Lactobacillus increased from 22.45% (fresh meat) to 72.78%, 81.64%, 76.53% and 85.63% at the end of processing for 0%, 30%, 50% and 70% KCl replacement samples, respectively. During the processing, Lactobacillus gradually became the dominant one, and higher the KCl ratio, more rapid was the process. After salting, the TBARS of control was markedly higher (P < 0.05) than that of the others, while a similar lipid oxidation level (P > 0.05) was observed at the end of processing for different groups. After salting, there was no difference in total free amino acids (TFAA) content among four treatments (P > 0.05), whereas KCl replacement samples shared significantly higher (P < 0.05) values than control at the end of processing. Redundancy analysis and Pearson correlation showed positive correlation between Lactobacillus versus TBARS and TFAA. Partial replacement of NaCl with KCl could, directly or subsequently by promoting the growth of Lactobacillus, influence proteolysis and lipid oxidation over the manufacturing process.

Genomic diversity and characterization of Listeria monocytogenes from dry-cured ham processing plants.

Genomic diversity of Listeria monocytogenes isolates from the deboning and slicing areas of three dry-cured ham processing plants was analysed. L. monocytogenes was detected in 58 out of 491 samples from the environment and equipment surfaces, all from the deboning area, with differences in prevalence among facilities. The most frequent PCR-serogroup was IIa (74.1%) followed by IIb and IIc, and only one isolate was serogroup IVb. Twenty different pulsotypes and 11 sequence types (STs) grouped into 10 clonal complexes (CCs) were determined. ST121 (CC121) and ST9 (CC9) were the most abundant. Premature stop codons (PMSC6 and PMSC19) associated with attenuated virulence were found in the inlA sequence in 7 out of 12 selected strains. CC121 strains were strong biofilm formers and some harboured the transposon Tn6188, related with increased tolerance to quaternary ammonium compounds. L. monocytogenes clones considered hypovirulent resulted predominant in the deboning areas. The clonal structure and potential virulence of the isolates could help to establish adequate control measures and cleaning protocols for the comprehensive elimination of the pathogen in dry-cured ham processing environment.

High-throughput sequencing-based characterization of the predominant microbial community associated with characteristic flavor formation in Jinhua Ham.

Our purpose was to investigate the main bacterial microbiota and volatile profiles in the Chinese traditional dry-cured product-Jinhua ham during different processing stages and to analyze the role of the main microbiota in the formation of characteristic flavor. We determined the microbiota of Jinhua ham by using 16 S high throughput sequencing, and found that Staphylococcus constituted the predominant microbiota throughout the flavor formation process. Based on the volatile profiles of Jinhua dry-cured products from 11 different processing via SPME-GC-MS analysis, Aldehydes were the main groups of volatiles, with the most abundant ones being hexanal (13.89%) and nonanal (3.96%). To further investigate the relationship between predominant microbiota and the major volatile compounds in Jinhua ham, we screened and isolated genus Staphylococcus with high protease and lipase activities. The main Staphylococcus isolates, S. saprophyticus (53.4%) and S. equorum (31.0%) are related to the yields of aldehydes by producing hexanal, nonanal, benzaldehyde, and phenylacetaldehyde, indicating their contributions on the formation of characteristic flavor substances in Jinhua ham.

Prevalence and Antimicrobial Susceptibility of Salmonella Serovars Isolated from U.S. Retail Ground Pork.

One objective of this study was to determine overall prevalence of Salmonella in ground pork from U.S. retail stores over three seasons including both case-ready and store-ground packages. Package types collected included: overwrap, chub, modified atmosphere packaging, and other (plastic or wax paper wrapped). Because package type represents different production systems and are subject to varied microbiological government regulation and testing methodologies, both USDA-FSIS and FDA Salmonella isolation protocols were performed. Another objective of the study was to determine serotypes and antimicrobial susceptibility profiles of the isolates obtained from the ground pork samples. Ground pork aliquots were subjected to real-time PCR. Recovered isolates were serotyped and minimum inhibitory concentration analysis to 15 antimicrobials was determined using microbroth dilution. Overall prevalence of Salmonella in ground pork from the 865 samples collected was 1.39%. Prevalence was not affected by package type (p = 0.29) nor grind location (case-ready vs. store-ground; p = 0.17). Season affected Salmonella prevalence (p = 0.05) with most isolates found during fall, and there was a tendency for geographic region to affect prevalence (p = 0.07). The USDA Salmonella isolation method was more effective at recovering isolates (p = 0.01) compared with the FDA methodology and yielded a kappa statistic of 0.26 as a measure of agreement. The serotypes isolated included: Infantis, 4,5,12:i:-, Brandenburg, Typhimurium var 5-, Seftenberg, and Johannesburg with only two packages containing multiple serotypes. No isolates were resistant to antibiotics commonly used to treat human Salmonella infections including extended spectrum cephalosporins or fluoroquinolones. Although the recovery of Salmonella from retail ground pork samples was rare, Salmonella Typhimurium (and its monophasic variant 4,5,12:i:-), which are among the most common serovars recovered from human infections, were recovered. Therefore, more effective strategies to further reduce or eliminate these pathogens from retail pork products are warranted.

Comparison of China's and the European Union's Approaches to Antimicrobial Stewardship in the Pork Industry.

Antimicrobial resistance (AMR) is a recognized global public health concern. Although the link between antimicrobial usage in food animals and AMR in humans is established, the detailed interactions are unclear. Antimicrobial stewardship (AMS) in livestock was first implemented in Europe with Sweden as the pioneer in 1986. Despite this head start, AMR is still an ongoing challenge for Europe. The European Union (EU) is an established agriculture producer, the second largest pork producer globally, and one of the largest markets for organic food. China is the global leader in both production and consumption of pork. China's rise in prosperity has led to an increase in its pork demand. Chinese producers commonly use antimicrobials during production for disease treatment and prevention to meet this increased demand. China's rising prosperity together with recent publicized food safety scandals, disease outbreaks in domestic livestock products, and increased AMR awareness have resulted in an increased willingness to pay and demand for organic food by Chinese consumers. Responding to the growing concerns of AMR by consumers and the World Health Organization (WHO), the Chinese government introduced a national pilot program in 2016 to reduce unnecessary antimicrobial use. Compared with China, the EU is a different entity as it is a political union comprising diverse countries and although it may have more experience in AMS, both entities face similar issues with AMR and increasing demand for organic food. Increased interest in organic food has arisen due to concerns about AMR, food safety, outbreaks of bacterial food contamination, and animal welfare. This article aims to compare the different AMS strategies employed by each entity, China and the EU, and how the increased demand for organic produce globally also influences the effort to reduce antimicrobial use in these entities' pork industries.

Colorimetric determination of Listeria monocytogenes using aptamer and urease dual-labeled magnetic nanoparticles and cucurbit[7]uril-mediated supramolecular assembly of gold nanoparticle.

A host-guest colorimetric strategy is described for the detection of Listeria monocytogenes (L. monocytogenes). The optical probes were self-assembled based on the supramolecular interactions between the carbonyl groups of cucurbit[7]uril portals and gold nanoparticles (CB[7]-AuNPs). Aptamer and urease modified magnetic nanoparticles were used to specifically recognize and binding to L. monocytogenes, simultaneously hydrolyzing urea to produce ammonium ion (NH4+) that can reverse CB[7] induced AuNPs aggregation. In the presence of L. monocytogenes, the above-mentioned magnetic conjugates preferentially bind to the bacterial surface, which results in blocking the catalytic active sites, thus inhibiting the production of ammonium ions. The normalized absorbance ratio of A700 nm/A525 nm was proportional to the L. monocytogenes concentration ranging from 10 to 106 cfu·mL-1, and the visual determination can be done down to 10 cfu·mL-1. For spiked food samples analyzed without pre-enrichment, recoveries of 98.4% to 99.3% were achieved could be verified and RSD were less than 10%. This work may offer a broad prospect for sensitive and specific determination  of pathogens.

A novel in situ methodology for visual detection of Clostridium perfringens in pork harnessing saltatory rolling circle amplification.

Clostridium perfringens (C. perfringens), a prolific toxin-producing anaerobe is an important foodborne pathogen with a huge public health concern. Rapid and on-site detection of C. perfringens is of specific importance in developing countries. In the present study, saltatory rolling circle amplification (SRCA) assay was developed for culture-independent, rapid and visual detection of C. perfringens and evaluated in meat with pork as a model. The specificity of the SRCA assay was ascertained by using 62 C. perfringens and 18 non- C. perfringens strains. The analytical sensitivity of the developed SRCA, conventional and real-time PCR assays were 80 fg, 800 fg and 800 fg DNA per tube, respectively. The limit of detection of the SRCA assay was 80 CFU/g of pork in the absence of enrichment and 8 CFU/g after short enrichment of 6 h. The detection limits of 80 CFU/g and 8 CFU/g of pork were attained within 120 min and 8 h, respectively. Real-world or field relevancy of the developed assay was evaluated by screening 82 raw and processed pork samples. As the developed assay is simple, user-friendly, cost-effective and sophisticated-equipment free, it would be more suitable for on-site testing of C. perfringens in foods. To our information, this is the first report to apply SRCA for the detection of C. perfringens.

Properties of Banana (Cavendish spp.) Starch Film Incorporated with Banana Peel Extract and Its Application.

The objective of this study was to develop an active banana starch film (BSF) incorporated with banana peel extract. We compared the film's properties with commercial wrap film (polyvinyl chloride; PVC). Moreover, a comparison of the quality of minced pork wrapped during refrigerated storage (7 days at ±4 °C) was also performed. The BSF with different concentrations of banana peel extract (0, 1, 3, and 5 (%, w/v)) showed low mechanical properties (tensile strength (TS): 4.43-31.20 MPa and elongation at break (EAB): 9.66-15.63%) and water vapor permeability (3.74-11.0 × 10-10 g mm/sm2 Pa). The BSF showed low film solubility (26-41%), but excellent barrier properties to UV light. The BSF had a thickness range of 0.030-0.047 mm, and color attributes were: L* = 49.6-51.1, a* = 0.21-0.43, b* = 1.26-1.49. The BSF incorporated with banana peel extracts 5 (%, w/v) showed the highest radical scavenging activity (97.9%) and inhibitory activity of E. coli O157: H7. The BSF showed some properties comparable to the commercial PVC wrap film. Changes in qualities of minced pork were determined for 7 days during storage at ±4 °C. It was found that thiobarbituric acid reactive substances (TBARS) of the sample wrapped with the BSF decreased compared to that wrapped with the PVC. The successful inhibition of lipid oxidation in the minced pork was possible with the BSF. The BSF incorporated with banana peel extract could maintain the quality of minced pork in terms of oxidation retardation.

Evaluation of the Membrane Damage Mechanism of Chlorogenic Acid against Yersinia enterocolitica and Enterobacter sakazakii and Its Application in the Preservation of Raw Pork and Skim Milk.

Plant-derived antimicrobial agents have adequate antimicrobial effects on food-borne pathogens, which can be used as food preservatives. The purpose of this study was to evaluate the antibacterial mechanism of chlorogenic acid (CA) against Yersinia enterocolitica and Enterobacter sakazakii. The minimum inhibitory concentration (MIC) of CA was determined by employing the broth microdilution method. Then, the cell function and morphological changes of Y. enterocolitica and E. sakazakii treated with CA were characterized. Finally, the growth inhibition models of Y. enterocolitica in raw pork and E. sakazakii in skim milk were constructed through the response surface methodology. The results demonstrated that CA has a satisfactory inhibitory effect against Y. enterocolitica and E. sakazakii with a MIC of 2.5 mg/mL. In addition, CA inhibited the growth of Y. enterocolitica and E. sakazakii via cell membrane damage, such as depolarization of the cell membrane, reduction in intracellular adenosine triphosphate (ATP) and pH levels, and destruction of cell morphology. Moreover, CA reduced two log cycles of Y. enterocolitica in raw pork and E. sakazakii in skim milk at a certain temperature. According to the corresponding findings, CA has the potential to be developed as an effective preservative to control Y. enterocolitica and E. sakazakii-associated foodborne diseases.

Seasonal Prevalence of Shiga Toxin-Producing Escherichia coli on Pork Carcasses for Three Steps of the Harvest Process at Two Commercial Processing Plants in the United States.

Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen that has a significant impact on public health, with strains possessing the attachment factor intimin referred to as enterohemorrhagic E. coli (EHEC) and associated with life-threatening illnesses. Cattle and beef are considered typical sources of STEC, but their presence in pork products is a growing concern. Therefore, carcasses (n = 1,536) at two U.S. pork processors were sampled once per season at three stages of harvest (poststunning skins, postscald carcasses, and chilled carcasses) and then examined using PCR for Shiga toxin genes (stx), intimin genes (eae), aerobic plate count (APC), and Enterobacteriaceae counts (EBC). The prevalence of stx on skins, postscald, and chilled carcasses was 85.3, 17.5, and 5.4%, respectively, with 82.3, 7.8, and 1.7% of swabs, respectively, having stx and eae present. All stx-positive samples were subjected to culture isolation that resulted in 368 STEC and 46 EHEC isolates. The most frequently identified STEC were serogroups O121, O8, and O91 (63, 6.7, and 6.0% of total STEC, respectively). The most frequently isolated EHEC was serotype O157:H7 (63% of total EHEC). Results showed that scalding significantly reduced (P < 0.05) carcass APC and EBC by 3.00- and 2.50-log10 CFU/100 cm2, respectively. A seasonal effect was observed, with STEC prevalence lower (P < 0.05) in winter. The data from this study show significant (P < 0.05) reduction in the incidence of STEC (stx) from 85.3% to 5.4% and of EHEC (stx plus eae) from 82.3% to 1.7% within the slaughter-to-chilling continuum, respectively, and that potential EHEC can be confirmed present throughout using culture isolation.IMPORTANCE Seven serogroups of STEC are responsible for most (>75%) cases of severe illnesses caused by STEC and are considered adulterants of beef. However, some STEC outbreaks have been attributed to pork products, although the same E. coli are not considered adulterants in pork because little is known of their prevalence along the pork chain. The significance of the work presented here is that it identifies disease-causing STEC, EHEC, demonstrating that these same organisms are a food safety hazard in pork as well as beef. The results show that most STEC isolated from pork are not likely to cause severe disease in humans and that processes used in pork harvest, such as scalding, offer a significant control point to reduce contamination. The results will assist the pork processing industry and regulatory agencies to optimize interventions to improve the safety of pork products.

An FcεRI-IgE-based genetically encoded microfluidic cell sensor for fast Gram-negative bacterial screening in food samples.

An FcεRI-IgE-based genetically encoded microfluidic cell sensor was constructed for fast Gram-negative bacterial screening in food samples. CD14-Fcε IgE, produced by the gene engineered antibodies (GEAs) technology, was used for the recognition of the target bacteria or lipopolysaccharide (LPS). Stable cell lines expressing GCaMP6s, a genetically encoded indicator of calcium flux, were first established for monitoring mast cell activation and improving detection sensitivity. The microfluidic system was designed to improve automation and control the reaction time. Once Gram-negative bacteria bound to the CD14-Fcε IgE on the RBL-2H3 cell surface, RBL-2H3 cell receptor (FcεRI)-induced Ca2+ signaling pathway was immediately activated to release Ca2+. The elevated intracellular Ca2+ triggers GCaMP6s for reporting the presence of Gram-negative bacteria. The developed biosensor was able to detect 80 CFU mL-1 Gram-negative bacteria within 2.5 min in pure culture samples. The biosensor was used to detect Gram-negative bacteria in pork samples. With its short screening time and easy operation, the proposed biosensor shows promise in future applications of foodborne pathogen testing in 1 h to 1 day.

Development of a novel and rapid polymerase spiral reaction (PSR) assay to detect Salmonella in pork and pork products.

The polymerase spiral reaction (PSR), a novel isothermal method for targeted DNA amplification, was effectively applied to detect Salmonella in artificially spiked pork. The specificity of the developed PSR was tested using 16 Salmonella and 15 non-Salmonella strains. The PSR assay was 10-fold more sensitive than conventional end-point PCR, having a sensitivity comparable to real-time PCR. The limit of detection of the developed assay was 4 × 103 per gram of pork without enrichment and 4 CFU per gram after a 6 h enrichment. The detection of 4 CFU per gram of pork was achieved within 8 h. The PSR assay was successful, and accurate in comparison to microbiological methods, in detecting Salmonella in 11 of 76 commercial pork samples. Therefore the positive predictive value, negative predictive value and accuracy rate of the developed assay were 100%. Considering its rapidity, user-friendliness, simplicity, cost-effectiveness and equipment-free nature, this PSR assay is a promising tool for the food industry for the detection of Salmonella and prevention of Salmonella outbreaks and recalls.

Isolation and characterization of Salmonella in pork samples collected from retail and wholesale markets in each season from 2016 to 2018 in Wuhan, China.

AIMS: To investigate the prevalence and characteristics of Salmonella in pork on sale in Wuhan, Central China. METHODS AND RESULTS: A total of 4744 pork samples were collected from retail or wholesale markets in each season from 2016 to 2018. The samples showed an overall Salmonella prevalence of 19·54% (927/4744), among which the samples collected in 2017 (21·67%, 428/1975) possessed a significantly higher prevalence than those collected in 2016 (18·61%, 209/1123) (P = 0·047) or 2018 (17·51%, 290/1656) (P = 0·002), and the samples collected in winter showed the lowest prevalence (15·86%, 177/1116). The Salmonella prevalence was significantly higher among samples from retail markets (25·68%, 283/1102) than wholesale markets (17·68%, 644/3642) (P = 0·000). Antimicrobial resistance of 922 Salmonella strains was tested by determining the minimal inhibitory concentrations using a broth microdilution method. The strains revealed that 98·92% (912/922) were resistant to at least one of the antimicrobial agents, and 80·04% (738/922) were resistant to three or more antimicrobials (MDR). Resistance to sulfamethoxazole/trimethoprim (89·91%), tetracycline (87·20%) and ampicillin (71·69%) was predominant. The proportion of MDR strains in 2017 (93·62%, 396/423) was significantly higher than that in 2016 (63·16%, 132/209) (P = 0·000) or 2018 (69·66%, 202/290) (P = 0·000). No significant difference was observed in the proportions of MDR strains between wholesale markets (76·07%, 213/280) and retail markets (80·53%, 517/642) (P = 0·075). Multi-locus sequence typing for 554 of the isolates revealed 20 different sequence types (STs), among which ST40 (38·27%, 212/554), ST34 (18·41%, 102/554) and ST469 (14·46%, 79/554) were dominant. CONCLUSIONS: A high risk of Salmonella prevalence and antimicrobial resistance was observed in pork in Wuhan. The risk varies between different sampling years, seasons and market types. SIGNIFICANCE AND IMPACT OF THE STUDY: Providing baseline data on Salmonella contamination in pork on sale in Central China.