Dataset Information

Preliminary Study on Rapid and Simultaneous Detection of Viable Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella by PMA-mPCR in Food.

ABSTRACT: Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella are major foodborne pathogens that are widespread in nature and responsible for several outbreaks of food safety accidents. Thus, a rapid and practical technique (PMA-mPCR) was developed for the simultaneous detection of viable E. coli O157:H7, S. aureus, and Salmonella in pure culture and in a food matrix. To eliminate false positive results, propidium monoazide (PMA) was applied to selectively suppress the DNA amplification of dead cells. The results showed the optimum concentration of PMA is 5.0 µg/mL. The detection limit of this assay by mPCR was 10³ CFU/mL in the culture broth, and by PMA-mPCR was 10⁴ CFU/mL both in pure culture and a food matrix (milk and ground beef). In addition, the detection of mixed viable and dead cells was also explored in this study. The detection sensitivity ratio of viable and dead counts was less than 1:10. Therefore, the PMA-mPCR assay proposed here might provide an efficient detection tool for the simultaneous detection of viable E. coli O157:H7, S. aureus, and Salmonella and also have great potential for the detection and concentration assessment of VBNC cells.

SUBMITTER: Liu Y

PROVIDER: S-EPMC10421101 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Preliminary Study on Rapid and Simultaneous Detection of Viable Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella by PMA-mPCR in Food.

Liu Yao Y Wei Caijiao C Wan Hui H Sarengaowa Liang Xiaoping X Jiang Tao T Dong Yuhe Y Zhao Xihong X Zhong Tian T

Molecules (Basel, Switzerland) 20230803 15

Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella are major foodborne pathogens that are widespread in nature and responsible for several outbreaks of food safety accidents. Thus, a rapid and practical technique (PMA-mPCR) was developed for the simultaneous detection of viable E. coli O157:H7, S. aureus, and Salmonella in pure culture and in a food matrix. To eliminate false positive results, propidium monoazide (PMA) was applied to selecti ...[more]

PMID: 37570805

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Project description:Multipathogen detection on a single-assay platform not only reduces the cost for testing but also provides data on the presence of pathogens in a single experiment. To achieve this detection, a multipathogen selective enrichment medium is essential to allow the concurrent growth of pathogens. SEL broth was formulated to allow the simultaneous growth of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes. The results were compared to those obtained with the respective individual selective enrichment broths, Rappaport-Vassiliadis (RV) for S. enterica, modified E. coli broth with 20 mg of novobiocin/liter for E. coli O157:H7, and Fraser broth for L. monocytogenes, and a currently used universal preenrichment broth (UPB). The growth of each pathogen in SEL inoculated at 10(1) or 10(3) CFU/ml was superior to that in the respective individual enrichment broth, except in the case of RV, in which Salmonella cells inoculated at both concentrations grew equally well. In mixed-culture experiments with cells of the three species present in equal concentrations or at a 1:10:1,000 ratio, the overall growth was proportional to the initial inoculation levels; however, the growth of L. monocytogenes was markedly suppressed when cells of this species were present at lower concentrations than those of the other two species. Further, SEL was able to resuscitate acid- and cold-stressed cells, and recovery was comparable to that in nonselective tryptic soy broth containing 6% yeast extract but superior to that in the respective individual selective broths. SEL promoted the growth of all three pathogens in a mixture in ready-to-eat salami and in turkey meat samples. Moreover, each pathogen was readily detected by a pathogen-specific immunochromatographic lateral-flow or multiplex PCR assay. Even though the growth of each pathogen in SEL was comparable to that in UPB, SEL inhibited greater numbers of nontarget organisms than did UPB. In summary, SEL was demonstrated to be a promising new multiplex selective enrichment broth for the detection of the three most prominent food-borne pathogens by antibody- or nucleic acid-based methods.

Project description:Escherichia coli O157:H7 and Salmonella enterica are leading causes of foodborne outbreaks linked to fresh produce. Both species can enter the "viable but nonculturable" (VBNC) state that precludes detection using conventional culture-based or molecular methods. In this study, we assessed propidium monoazide-quantitative PCR (PMA-qPCR) assays and novel methods combining PMA and loop-mediated isothermal amplification (LAMP) for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce. The performance of PMA-LAMP assays targeting the wzy gene of E. coli O157:H7 and the agfA gene of S. enterica and the performance of PMA-qPCR assays were compared in pure culture and spiked tomato, lettuce, and spinach. No cross-reaction was observed in the specificity tests. The values representing the limit of detection (LOD) seen with PMA-LAMP were 9.0 CFU/reaction for E. coli O157:H7 and 4.6 CFU/reaction for S. enterica in pure culture and were 5.13 × 103 or 5.13 × 104 CFU/g for VBNC E. coli O157:H7 and 1.05 × 104 or 1.05 × 105 CFU/g for VBNC S. enterica in fresh produce, representing results comparable to those obtained by PMA-qPCR. Standard curves showed correlation coefficients ranging from 0.925 to 0.996, indicating a good quantitative capacity of PMA-LAMP for determining populations of both bacterial species in the VBNC state. The PMA-LAMP assay was completed with considerable economy of time (30 min versus 1 h) and achieved sensitivity and quantitative capacity comparable to those seen with a PMA-qPCR assay. PMA-LAMP is a rapid, sensitive, and robust method for the detection and quantification of VBNC E. coli O157:H7 and S. enterica in fresh produce.IMPORTANCE VBNC pathogenic bacteria pose a potential risk to the food industry because they do not multiply on routine microbiological media and thus can evade detection in conventional plating assays. Both E. coli O157:H7 and S. enterica have been reported to enter the VBNC state under a range of environmental stress conditions and to resuscitate under favorable conditions and are a potential cause of human infections. PMA-LAMP methods developed in this study provide a rapid, sensitive, and specific way to determine levels of VBNC E. coli O157:H7 and S. enterica in fresh produce, which potentially decreases the risks related to the consumption of fresh produce contaminated by enteric pathogens in this state. PMA-LAMP can be further applied in the field study to enhance our understanding of the fate of VBNC pathogens in the preharvest and postharvest stages of fresh produce.

Project description:The formation of viable but nonculturable (VBNC) Escherichia coli O157:H7 induced by high-pressure CO2 (HPCD) was investigated using RNA sequencing (RNA-Seq) transcriptomics and isobaric tag for relative and absolute quantitation (iTRAQ) proteomic methods. The analyses revealed that 97 genes and 56 proteins were significantly changed upon VBNC state entry. Genes and proteins related to membrane transport, central metabolisms, DNA replication, and cell division were mainly downregulated in the VBNC cells. This caused low metabolic activity concurrently with a division arrest in cells, which may be related to VBNC state formation. Cell division repression and outer membrane overexpression were confirmed to be involved in VBNC state formation by homologous expression of z2046 coding for transcriptional repressor and ompF encoding outer membrane protein F. Upon VBNC state entry, pyruvate catabolism in the cells shifted from the tricarboxylic acid (TCA) cycle toward the fermentative route; this led to a low level of ATP. Combating the low energy supply, ATP production in the VBNC cells was compensated by the degradation of l-serine and l-threonine, the increased AMP generation, and the enhanced electron transfer. Furthermore, tolerance of the cells with respect to HPCD-induced acid, oxidation, and high CO2 stresses was enhanced by promoting the production of ammonia and NADPH and by reducing CO2 production during VBNC state formation. Most genes and proteins related to pathogenicity were downregulated in the VBNC cells. This would decrease the cell pathogenicity, which was confirmed by adhesion assays. In conclusion, the decreased metabolic activity, repressed cell division, and enhanced survival ability in E. coli O157:H7 might cause HPCD-induced VBNC state formation. Escherichia coli O157:H7 has been implicated in large foodborne outbreaks worldwide. It has been reported that the presence of as few as 10 cells in food could cause illness. However, the presence of only 0.73 to 1.5 culturable E. coli O157:H7 cells in salted salmon roe caused infection in Japan. Investigators found that E. coli O157:H7 in the viable but nonculturable (VBNC) state was the source of the outbreak. So far, formation mechanisms of VBNC state are not well known. In a previous study, we demonstrated that high-pressure CO2 (HPCD) could induce the transition of E. coli O157:H7 into the VBNC state. In this study, we used RNA-Seq transcriptomic analysis combined with the iTRAQ proteomic method to investigate the formation of VBNC E. coli O157:H7 induced by HPCD treatment. Finally, we proposed a putative formation mechanism of the VBNC cells induced by HPCD, which may provide a theoretical foundation for controlling the VBNC state entry induced by HPCD treatment.

Dataset Information

Preliminary Study on Rapid and Simultaneous Detection of Viable Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella by PMA-mPCR in Food.

Publications

Preliminary Study on Rapid and Simultaneous Detection of Viable <i>Escherichia coli</i> O157:H7, <i>Staphylococcus aureu</i>s, and <i>Salmonella</i> by PMA-mPCR in Food.

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