This study examined the relationship between NaCl sensitivity and stress response of Listeria monocytogenes. Nine strains of L. monocytogenes (NCCP10805, NCCP10806, NCCP10807, NCCP10808, NCCP10809, NCCP10810, NCCP10811, NCCP10920 and NCCP 10943) were exposed to 0%, 1%, 2% and 4% NaCl, and then incubated at 60℃ for 60 min to select strains that were heat-sensitized (HS) and non-sensitized (NS) by NaCl exposure. After heat challenge, L. monocytogenes strains were categorized as HS (NCCP 10805, NCCP10806, NCCP10807, NCCP10810, NCCP10811 and NCCP10920) or NS (NCCP10808, NCCP10809 and NCCP10943). Total mRNA was extracted from a HS strain (NCCP10811) and two NS strains (NCCP10808 and NCCP10809), and then cDNA was prepared to analyze the expression of genes (inlA, inlB, opuC, betL, gbuB, osmC and ctc) that may be altered in response to NaCl stress, by qRT-PCR. The expression levels of two invasion-related genes (inlA and inlB) and two stress response genes (opuC and ctc) were increased (p<0.05) in NS strains after NaCl exposure in an NaCl concentration-dependent manner. However, only betL expression was increased (p<0.05) in the HS strains. These results indicate that the effect of NaCl on heat sensitization of L. monocytogenes is strain dependent and that opuC and ctc may prevent NS L. monocytogenes strains from being heat sensitized by NaCl. Moreover, NaCl also increases the expression of invasion-related genes (inlA and inlB).
This study assessed the quantitative microbial risk of non-enterohemorrhagic Escherichia coli (EHEC). For hazard identification, hazards of non-EHEC E. coli in natural and processed cheeses were identified by research papers. Regarding exposure assessment, non-EHEC E. coli cell counts in cheese were enumerated, and the developed predictive models were used to describe the fates of non-EHEC E. coli strains in cheese during distribution and storage. In addition, data on the amounts and frequency of cheese consumption were collected from the research report of the Ministry of Food and Drug Safety. For hazard characterization, a doseresponse model for non-EHEC E. coli was used. Using the collected data, simulation models were constructed, using software @RISK to calculate the risk of illness per person per day. Non-EHEC E. coli cells in natural- (n=90) and processed-cheese samples (n=308) from factories and markets were not detected. Thus, we estimated the initial levels of contamination by Uniform distribution ${\times}$ Beta distribution, and the levels were -2.35 and -2.73 Log CFU/g for natural and processed cheese, respectively. The proposed predictive models described properly the fates of non-EHEC E. coli during distribution and storage of cheese. For hazard characterization, we used the Beta-Poisson model (${\alpha}=2.21{\times}10^{-1}$, $N_{50}=6.85{\times}10^7$). The results of risk characterization for non-EHEC E. coli in natural and processed cheese were $1.36{\times}10^{-7}$ and $2.12{\times}10^{-10}$ (the mean probability of illness per person per day), respectively. These results indicate that the risk of non-EHEC E. coli foodborne illness can be considered low in present conditions.
This study evaluated if vitamin E consumption affects gut microbiota. Mice were grouped into control, low vitamin E (LV), and high vitamin E (HV). LV and HV were fed DL-α-tocopherol at 0.06 mg/20 g and 0.18 mg/20 g of body weight per day, respectively, for 34 days. Body weight of mice was measured before and after vitamin E treatment. Animals were sacrificed, liver, spleen, small intestine and large intestine collected, and weight and length were measured. Composition of gut microbiota was determined by microbiome analysis. Spleen weight index of LV was the highest. However, liver weight indices and intestinal lengths were not different. Body weights of LV group were higher than those of control. Ratio of Firmicutes to Bacteroidetes was different in LV compared to control and HV. These results indicate that low-level consumption of vitamin E increases spleen and body weight, and changes gut microbiota.
This study evaluated a combined method for the detection of Listeria monocytogenes in mushrooms, involving enrichment and quantitative real-time polymerase chain reaction (qPCR), to improve sensitivity and reduce detection time. The growth of L. monocytogenes was evaluated in Listeria enrichment broth (LEB) with modified carbon and nitrogen sources, increasing sodium concentrations, and added micronutrients. Primers targeting the L. monocytogenes iap (iap1 and iap2), hlyA (hlyA1-hlyA6), and prfA (prfA1-prfA4) genes were developed and their sensitivity and specificity were evaluated. The greatest increase in L. monocytogenes cell count was observed after 6-h incubation at 30°C in LEB+2 × FAC (LEB plus 20 mL/L ferric ammonium citrate), where cell count increased by 1.4 log CFU (colony-forming unit)/mL, compared with 0.9 log CFU/mL in LEB (p < 0.05). iap2 primers targeting the iap gene showed high specificity and were the most sensitive among those tested, with a detection limit of 2 log CFU/mL in LEB medium, 3.1 log CFU/g in golden needle mushroom, and 3.5 log CFU/g in large oyster mushroom. When applied to detection in golden needle mushrooms, a combination of 3-h incubation in LEB+2 × FAC medium and qPCR analysis with iap2 primers permitted detection of L. monocytogenes, even at an inoculum of 1 log CFU/g. Similarly, in large oyster mushrooms, 10-h enrichment in LEB+2 × FAC medium resulted in a cell count of 3.7 log CFU/g. These results indicate that a combined detection method, using LEB+2 × FAC medium for enrichment followed by qPCR with iap2 primer pair, can reduce enrichment time and improve the sensitivity and specificity of L. monocytogenes detection in mushrooms.
The objective of this study was to investigate the pathogenicity and antimicrobial resistance of foodborne pathogens isolated from farmstead cheeses. Twenty-seven isolates, including 18 Bacilluscereus, two Escherichiacoli, and seven Staphylococcusaureus, were subjected to polymerase chain reaction (PCR) to detect virulence genes and toxin genes, and the antibiotic resistances of the isolates were determined. All E. coli isolates were determined by PCR to be non-pathogenic. Among the 18 B. cereus isolates, 17 isolates (94.4%) were diarrheal type, as indicated by the presence of nheA, entFM, hbIC, cytK and bceT genes, and one isolate (5.6%) was emetic type, based on the presence of the CER gene. Among the seven S. aureus isolates, three (42.9%) had the mecA gene, which is related to methicillin-resistance. Most B. cereus isolates (94.7%) showed antibiotic resistance to oxacillin and penicillin G, and some strains also showed resistance to ampicillin (26.3%), erythromycin (5.3%), tetracycline (10.5%), and vancomycin (5.3%). These results indicate that microbial food safety measures for farmstead cheese must be implemented in Korea because antibiotic resistant foodborne pathogens, with resistance even to vancomycin, harboring virulence genes were found to be present in the final products of farmstead cheese.
This study evaluated Clostridium perfringens risk in fermented bean pastes. The prevalence in fermented bean pastes, development of the predictive model, temperature, time, and consumption data were investigated. A simulation model was prepared in @RISK with the obtained data to calculate the probability of foodborne C. perfringens foodborne illness from fermented bean paste consumption. Only 74 of the 1,097 samples were positive for C. perfringens. The initial contamination level was estimated to be 0.7 log CFU/g. It showed the probability of foodborne C. perfringens infection per person per day upon consumption of fermented bean pastes was 8.0×10−12, which is considered low risk.
Abstract This study elucidated the effect of NaCl on the thermal resistance and cell morphology of Escherichia coli strains. E. coli O157:H7 NCCP11142, E. coli O111 ATCC12795 and E. coli O26 ATCC43887 were incubated in tryptic soy broth formulated with 0% NaCl (nonhabituated) as well as broth formulated with 2% and 4% NaCl (NaCl‐habituated) at 35C. Nonhabituated, 2% NaCl‐habituated and 4% NaCl‐habituated E. coli O157:H7, E. coli O111 and E. coli O26 were heat‐challenged at 50C. The transcriptomes (osmotic stress‐related: betA , ompC , proV , proW and heat stress‐related: clpB , dksA , dnaJ , dnaK , grpE , msbB , rpoE ) were analyzed using real‐time polymerase chain reaction and the morphology of E. coli strains was also observed by field emission scanning electron microscopy. The survival of E. coli O157:H7 was higher ( P < 0.05) than those of other strains after the heat challenge. The expression levels of proV , proW and rpoE increased ( P < 0.05) as NaCl concentration increased, but only proV expression levels were significantly different ( P < 0.05) among E. coli strains. The results indicate that the effect of NaCl on the thermal resistance and the cell morphology depends on the E. coli strain, and increased proV expression level by NaCl may cause the increased thermal resistance of E. coli O157:H7. Practical Applications NaCl is widely used as an antimicrobial and a preservative agent in food processing. However, Escherichia coli O157:H7 exposed to 2% and 4% NaCl showed thermal resistance, upregulated gene expression on osmotic‐ and heat‐related genes and changed cell morphology. Thus, NaCl concentration should be limited in food not to increase E. coli O157:H7 tolerance to other stresses.
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