Detection of viable but non-culturable Escherichia coli O157:H7 from vegetable samples using quantitative PCR with propidium monoazide and immunological assays

Quantitative differentiation of the live fraction of pathogens in raw food samples is highly critical from a public health risk perspective, as many studies have shown that under stress conditions major foodborne pathogens enter a viable but non-culturable (VBNC) state in which bacteria can remain for long periods of time and maintain the potential for virulence. The objective of this study was to evaluate the applicability of propidium monoazide (PMA) quantitative PCR (qPCR) and immunological methods for detection of Escherichia coli O157:H7 VBNC populations induced by low temperature on the surface of lettuce and spinach plants. The primer/probe set selected influenced the qPCR signal in mixtures with a defined ratio of viable and non-viable cells. The PMA qPCR used in a background of added dead pathogens and epiphytic bacteria gave a detection limit of 10³ CFU/g leaf and a linear quantitative detection range of 5 log. During quantification of VBNC cells from lettuce and spinach samples there was a good correlation between the PMA qPCR results and viable counts detected by microscopy, showing that PMA qPCR gives an accurate indication of the VBNC population. However, the commercially available immunoassay methods used to detect Shiga-like toxin production and the O157 antibody in vegetable samples with no detectable culturable pathogen underestimated the number of samples contaminated with E. coli O157:H7 VBNC cells. Results indicate that PMA qPCR is a suitable technique for the detection and quantification of VBNC cells of foodborne pathogens in contaminated raw lettuce and spinach.     

Development of a quantitative real-time PCR assay for viable Salmonella spp. without enrichment

Propidium monoazide (PMA) combined with molecular quantitative real-time PCR (qPCR) has been widely used for only detection of viable bacteria. However, recent studies indicated PMA did not fully inhibit the detection of dead Salmonella. In this study, we developed a more effective PMA Taqman-based qPCR than previous studies to quantify viable Salmonella spp. in raw shrimp. This method has high specificity by using 60 strains belonging to 23 species. The optimization of the PMA concentration showed that 100 μM was considered optimal to effectively inhibit 10⁶ CFU/mL dead cells, while only 10³–10⁴ CFU/mL dead cells could be inhibited in previous reports. This assay could detect viable Salmonella spp. at as low as 36 CFU/mL in pure culture and 100 CFU/g in raw shrimp. By comparing with PMA-qPCR, qPCR and plate counting for quantifying Salmonella in samples, this PMA-qPCR was obviously superior to qPCR and had good agreement with plate counting. In conclusion, this effective method can be used as an available tool to quantify viable Salmonella spp. in raw shrimp.     

Development of propidium monoazide combined with real-time quantitative PCR (PMA-qPCR) assays to quantify viable dominant microorganisms responsible for the traditional brewing of Hong Qu glutinous rice wine

The main disadvantage of DNA-based quantification methods is their inability to distinguish between viable and non-viable cells, as dead cells may also retain a significant amount of DNA. To overcome this limitation, propidium monoazide (PMA) treatment was applied to effectively prevent amplification of DNA from dead cells. The objectives of this study were to develop and evaluate an accurate detection method that combines PMA staining with real-time qPCR to quantify viable dominant microorganisms during the traditional brewing of Hong Qu glutinous rice wine. Firstly, three methodological factors (concentration of PMA, incubation time and photoactivation time) were optimized for representative strains of different microbial species. For the tested microbial strains (Monascus purpureus, Saccharomyces cerevisiae and Lactobacillus plantarum), a treatment of 25 μM PMA for 20 min incubation followed by 10 min photoactivation was considered optimal to achieve a compromise between minimal impact on intact cells and maximal signal reduction for the compromised cells. Compared with qPCR without PMA treatment, results of PMA-qPCR showed that the optimized PMA pretreatment can effectively exclude the dead microbial cells without any negative effects on the intact cells. The concentrations of viable cells calculated by PMA-qPCR were comparable (the same order of magnitude was obtained) with those obtained by culture-based method. Finally, the developed PMA-qPCR methods were successfully applied to monitor the total bacteria, LAB, fungi, yeasts, M. purpureus, S. cerevisiae and L. plantarum during the traditional brewing of Hong Qu glutinous rice wine. Both of the total bacterial and fungal populations increased significantly at the early brewing stage, but they decreased at the later brewing stage. PMA-qPCR results revealed that the predominant microorganisms varied in different brewing phases. L. plantarum and S. cerevisiae grew significantly and became the predominant bacterial and fungal species at the final stage of traditional brewing process, respectively. This study standardized the PMA-qPCR parameters for dominant microorganisms associated with the traditional brewing of Hong Qu glutinous rice wine, which would show great value in scientifically understanding of the brewing mechanism and provide useful information for the selection of beneficial microbial strains to improve wine quality.     

Application of grape seed extract in depuration for decontaminating Vibrio parahaemolyticus in Pacific oysters (Crassostrea gigas)

This study investigated the potential application of grape seed extract (GSE) in depuration to increase its efficacy in reducing Vibrio parahaemolyticus populations in Pacific oysters (Crassostrea gigas). Pacific oysters were inoculated with five clinical strains of V. parahaemolyticus to 10⁴⁻⁵ MPN/g and depurated in UV-irradiated artificial seawater (ASW) containing GSE at a level of 1.0% (1.8 mg/mL total phenolic contents as gallic acid equivalents) or 1.5% (3.1 mg/mL total phenolic contents as gallic acid equivalents) at 12.5 °C for up to 5 days. Changes of V. parahaemolyticus populations in oysters during depuration were analyzed every 24 h using the three-tube most probable number (MPN) method. The populations of V. parahaemolyticus in inoculated oysters decreased by 3.06 and 3.71 log MPN/g, respectively, after 4 and 5 days of depuration in ASW at 12.5 °C. However, populations of V. parahaemolyticus in oysters were reduced by 3.01 and 4.18 log MPN/g after 2 days of depuration at 12.5 °C in ASW containing 1.0 and 1.5% GSE, respectively. Further studies confirmed that depuration using ASW containing 1.5% GSE with 3.1 mg/mL total phenolic contents as gallic acid equivalents at 12.5 °C for two days was capable of achieving >3.52 log MPN/g reductions of V. parahaemolyticus in Pacific oysters.     

Development of an SD-PMA-mPCR assay with internal amplification control for rapid and sensitive detection of viable Salmonella spp., Shigella spp. and Staphylococcus aureus in food products

In this work, a specific, sensitive, and accurate technique was presented for simultaneous detection of Salmonella spp., Shigella spp., and Staphylococcus aureus in food products, three of the more frequent foodborne pathogens that were usually reported in a variety of food matrices. An internal amplification control (IAC) was added in a multiplex PCR (mPCR) reaction system as an indicator of false negative result that can come from the presence of PCR inhibitors in food products. In the presence of inhibitor, no signal would result for the target genes as well as the IAC which results in a positive signal, thereby, eliminating false negative results. To ensure detection of only the viable cells, the effects of sodium deoxycholate (SD) in combination with propidium monoazide (PMA) treatment in the presence of dead cells and viable cells were investigated. Results showed that PMA treatment alone could not effectively inhibit the detection of 10⁷ CFU/mL of dead Salmonella Typhimurium, Shigella sonnei, and S. aureus from PCR amplification. However, the SD in combination with PMA treatment gave negative results for PCR amplification of dead S. Typhimurium, S. sonnei, and S. aureus in pure culture and food products. When the developed SD-PMA-mPCR assay in combination with IAC was applied to detect the spiked food (milk, ground beef), the LOD of SD-PMA-mPCR assay for S. Typhimurium, S. sonnei, and S. aureus inoculated individually or inoculated simultaneously into milk or ground beef were 10¹ CFU/mL or 10¹ CFU/g after 15 h enrichment. The results suggested that the SD-PMA-mPCR assay in combination with IAC held promise for the detection of foodborne S. Typhimurium, S. sonnei, and S. aureus.     

Efficacy of acidic and alkaline electrolyzed water for inactivating Escherichia coli O104:H4, Listeria monocytogenes,Campylobacter jejuni,Aeromonas hydrophila,and Vibrio parahaemolyticus in cell suspensions

This study investigated the effect of electrolyzed water on pathogenic bacteria cell suspensions. Specifically, we evaluated the efficacy of strong and weak acidic electrolyzed waters (SACEW, WACEW) and strong and weak alkaline electrolyzed waters (SALEW, WALEW) on Vibrio parahaemolyticus, Listeria monocytogenes, Aeromonas hydrophila, Campylobacter jejuni, and Escherichia coli O104:H4 in suspensions of (10⁷–10⁹ CFU/mL) in 1% NaCl. SACEW and WACEW were applied at available chlorine concentrations (ACC) of 20 and 10 mg/mL, pH 3.1 and 3.55 and oxidation-reduction potentials (ORP) of 1150 and 950 mV, respectively. Results show that no viable cells were recovered for V. parahaemolyticus, L. monocytogenes, A. hydrophila, C. jejuni within 2 min at 20 °C. However, E. coli O104:H4 was significantly more resistant to ALEW compared to ACEW. Results also show that the bactericidal activity of SACEW (20 mg/mL ACC) was more effective than WACEW (10 mg/mL ACC) in terms of inactivating E. coli O104:H4. Alkaline-electrolyzed waters were found to reduce cell numbers by 1–3 log (P < 0.05). However, alkaline electrolyzed water was less effective (P < 0.05) than acidic electrolyzed treatment.     

Effect of detergents as antibacterial agents on biofilm of antibiotics-resistant Vibrio parahaemolyticus isolates

Vibrio parahaemolyticus (V. parahaemolyticus) is a halophilic, Gram-negative human pathogen known as a leading cause of seafood-derived food poisoning. Due to high contamination rate of seafood in Asian countries, V. parahaemolyticus is considered as a food safety concern. V. parahaemolyticus is able to produce biofilm which is more resistant toward disinfectants and antibodies than its planktonic form. Thirty six V. parahaemolyticus isolates from seafood were tested for their susceptibility using 18 different antibiotics. Two V. parahaemolyticus isolates were resistant to bacitracin, chloramphenicol, rifampin, ampicillin, vancomycin, nalidixic acid, penicillin and spectinomycin. Fourteen V. parahaemolyticus isolates were found to be resistant to bacitracin, tetracycline, rifampin, ampicillin, vancomycin, penicillin and spectinomycin. The remaining two isolates were resistant to more than 2 antibiotics. Majority of the V. parahaemolyticus isolates (97.2%) showed MAR index > 0.2, indicating that these isolates were originated from high risk sources. To investigate effect of three common detergents on antibacterial-resistant V. parahaemolyticus, 16 V. parahaemolyticus isolates resistant to more than 7 antibiotics were selected. V. parahaemolyticus (ATCC 17802) was used as reference strain. Detergents were tested for their minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) and time–kill curves were constructed to assess the concentration between MIC and bactericidal activity of detergents. Detergents D1 (Linear alkyl benzene based) was found to be the most effective with MIC and MBC ranged between 97.656 and 1562.5 μg/ml and 781.25–3125 μg/ml, respectively. The time–kill curves demonstrated that the bactericidal endpoint for resistant V. parahaemolyticus isolates reached after 30 min incubation with D1 at concentration 8 × MIC. The isolate VP003 was killed at 8 × MIC within 0.5 h and the reduction in CFU/ml was 3 log units (99.9%). V. parahaemolyticus biofilms were formed in 96 wells microtiter plates at 37 °C and 24 h-old biofilm were used to test antibacterial activity of detergents. Results showed that biofilm-producing ability of antibacterial-resistant V. parahaemolyticus isolates were inhibited at 1562.5–6250 μg/ml of D1 and eradicated at 3125 – ≥50,000 μg/ml of D1. Detergents showed potential antimicrobial activity against V. parahaemolyticus     

Selective turn-on fluorescence detection of Vibrio parahaemolyticus in food based on charge-transfer between CdSe/ZnS quantum dots and gold nanoparticles

Vibrio parahaemolyticus (V. parahaemolyticus) is one of the most common food-borne pathogens found along the east coast of China, Japan, and several southeast Asian countries, where marine foods are frequently consumed. Thus it is of great interest to establish a fast, sensitive and robust analytical assay for V. parahaemolyticus detection. Herein, we report a V. parahaemolyticus-specific egg yolk antibody (IgY), extracted from the egg yolks of immunized hens with inactivated V. parahaemolyticus bacteria. By immobilizing IgY onto the surface of gold nanoparticles (AuNPs) through electrostatic self-assembly, a detection system was developed based on charge-transfer quenching fluorescence, which contains core/shell CdSe/ZnS quantum dots (QDs), IgY-AuNPs and free AuNPs. In this strategy, the IgY-AuNPs were induced to aggregate quickly in the presence of target V. parahaemolyticus, resulting in a rapid fluorescence response of the QDs. More importantly, the convenient turn-on fluorescent detection system exhibited excellent selectivity over the other bacteria. Furthermore, this facile one-step method can detect V. parahaemolyticus at low concentrations of 10 cfu/mL without pre-enrichment and can be applicable for the detection of V. parahaemolyticus in real food samples. With the advantage of simplicity, rapidness, sensitivity, and specificity, this proposed approach shows promise as a successful application of V. parahaemolyticus in bioanalysis.     

Detection of non-emetic and emetic Bacillus cereus by propidium monoazide multiplex PCR (PMA-mPCR) with internal amplification control

Bacillus cereus is an etiological agent of food-borne disease that can cause a type of emesis. To develop a sensitive and reliable diagnosis technique for detecting all the species of the B. cereus group, specific primers were designed to target a recently discovered part of the cereulide synthetase gene (cesB) for emetic B. cereus and 16S rRNA for non-emetic B. cereus. To detect PCR signals only from viable cells, propidium monoazide (PMA) was selected to eliminate the false-positive results. In addition, an internal amplification control (IAC) was applied to meet diagnostic multiplex PCR requirements that will prevent the occurrence of false-negative results. The inclusivity and exclusivity of the mPCR assay were estimated using a panel of 100 strains, including 2 emetic B. cereus, 77 non-emetic B. cereus and 21 non-Bacillus strains. The limit of detection (LOD) for dead B. cereus without PMA treatment in pure bacteria culture was 4.0 × 10² CFU/mL, as low as 7.5 × 10⁰ CFU/mL for viable B. cereus without PMA treatment, and 7.5 × 10¹ CFU/mL for viable B. cereus with PMA treatment. B. cereus in spiked food produce was detected specifically and sensitively at 1.0 × 10³ CFU/g which was the lowest concentration detected. This novel PMA-mPCR-IAC assay is rapid and reliable, providing an efficient diagnostic tool with promising application in monitoring food samples.     

Combining basic electrolyzed water pretreatment and mild heat greatly enhanced the efficacy of acidic electrolyzed water against Vibrio parahaemolyticus on shrimp

The objective of this study was to investigate the efficacy of acidic electrolyzed water (AEW) against Vibrio parahaemolyticus on shrimp. The shrimp was initially inoculated with V. parahaemolyticus(7-8 log CFU/g), and treated with AEW (AEW1 containing 51 mg/L of chlorine or AEW2 containing 78 mg/L of chlorine) or organic acids (2% AA and 2%LA) for 1 min or 5 min under different treated conditions. The effect of AEW was better than that of organic acids, the number of survival V. parahaemolyticus cells on shrimp was reduced by 0.9 log CFU/g after treatment for 5 min with AEW without vibration, while 1.0 log CFU/g bacteria cells reduced with vibration. No significant difference (p > 0.05) was observed between AEW and organic acids in the bactericidal activity with or without vibration. The effective order of temperatures on bactericidal activities of AEW was 50 °C > 20 °C > 4 °C, and a 3.1 log CFU/g reduction of V. parahaemolyticus cells on shrimp was detected with treatment of AEW at 50 °C. Mild heat greatly enhanced efficacy of electrolyzed water against V. parahaemolyticus. Basic electrolyzed water (BEW) (50 °C) pretreatment combined with AEW (50 °C) treatment remarkably reduced bacterial cells by 5.4 log CFU/g on shrimp after treatment for 5 min. There was a significant change in physicochemical properties (pH, ORP, ACC) of AEW, after it was used to wash shrimp (P < 0.05). This study suggests that BEW (50 °C) pretreatment followed by AEW (50 °C) treatment could be a possible method to effectively control V. parahaemolyticus contamination on shrimp.     

Rapid detection of viable Bacillus cereus emetic and enterotoxic strains in food by coupling propidium monoazide and multiplex PCR (PMA-mPCR)

Bacillus cereus can cause emetic and diarrheal food poisoning. It is widespread in nature and therefore, considered a major foodborne pathogen. To develop a sensitive and reliable assay for detecting enterotoxin genes (nheA, entFM, hblD, cytK) and emetic toxin (ces), specific primers each targeting one individual gene were designed. Propidium monoazide (PMA) was coupled with the developed multiplex PCR (mPCR) for the detection of viable B. cereus. The inclusivity and exclusivity of the PMA-mPCR was confirmed using a panel of 44 strains including 17 emetic and 9 enterotoxic B. cereus reference strains and 18 non-target strains. The limit of detection (LOD) without PMA treatment in pure DNA was 2 pg/reaction tube. The LOD of mPCR assay in pure heat-killed dead bacteria was 4.0 × 10² CFU/mL. Also, the LOD on the viable bacteria with or without PMA treatment was similar (3.8 × 10² CFU/mL) showing that the PMA treatment did not significantly decrease sensitivity. Finally, the newly developed PMA-mPCR successfully detected 4.8 × 10³ and 3.6 × 10³ CFU/g of viable B. cereus F4810/72 (emetic) and B. cereus ATCC 12480 (enterotoxic) reference strains, respectively, in food samples. Hence, this study combines PMA and mPCR to detect viable B. cereus with a wide range of toxin detection (5 toxins). Thus, the novel PMA-mPCR assay developed in this study is a rapid and efficient diagnostic tool for the monitoring of viable B. cereus in food samples and potentially other samples via appropriate DNA extraction.     

Pomegranate peel (Punica granatum L) extract and Chinese gall (Galla chinensis) extract inhibit Vibrio parahaemolyticus and Listeria monocytogenes on cooked shrimp and raw tuna

Vibrio parahaemolyticus and Listeria monocytogenes are bacterial pathogens associated with raw or ready-to-eat seafood products. Many compounds extracted from plant material have shown promise for inhibiting bacterial pathogens when applied to some foods. In this study, aqueous methanol extracts from pomegranate peel (Punica granatum L.) and Chinese gallnut (Galla chinensis) were tested against V. parahaemolyticus and L. monocytogenes on cooked shrimp and raw homogenized tuna. The extracts were applied to the shrimp by soaking for 2 min (5 mg/ml). The extracts (1.7 mg ml) were added to homogenized tuna and stirred. The antimicrobial assay on V. parahaemolyticus was conducted at 12 °C, and the assay on tuna was conducted at both 4° and 12 °C. Both Chinese gall and pomegranate peel extracts significantly inhibited the growth of V. parahaemolyticus in both shrimp and tuna. Only Chinese gall extract significantly inhibited growth of L. monocytogenes. Overall V. parahaemolyticus was more sensitive to both plant extracts compared with L. monocytogenes. Both plant extracts had stronger antimicrobial activity on shrimp compared with the tuna. Neither extract completely inhibited the growth of V. parahaemolyticus or L. monocytogenes.     

Production and characterization of a monoclonal antibody against recombinant thermolabile hemolysin and its application to screen for Vibrio parahaemolyticus contamination in raw seafood

Thermolabile hemolysin gene (tlh) is regarded as a species-specific marker for Vibrio parahaemolyticus. To assess the utility of the tlh gene product, thermolabile hemolysin (TLH), as a marker to screen for V. parahaemolyticus-contamination in raw seafood, we generated a monoclonal antibody (MAb) against recombinant TLH and established with an enzyme-linked immunosorbent assay using the MAb (TLH-ELISA). TLH-ELISA testing of broth cultures for 78 V. parahaemolyticus strains showed positive results all around. In contrast, most broth cultures of 53 non-V. parahaemolyticus species tested yielded negative results.We devised a screening method using TLH-ELISA to check for low-level contamination of V. parahaemolyticus in raw seafood within 24 h and evaluated its ability. In testing of V. parahaemolyticus-spiked raw seafood, results suggested that our screening method can detect 100 most-probable-number (MPN) of V. parahaemolyticus/g. Further, on testing 119 commercial raw seafood samples with our screening method, 117 samples were determined to contain less than 100 MPN of V. parahaemolyticus/g. All of the 117 samples were also estimated by the MPN method to contain less than 100 MPN of V. parahaemolyticus/g. Taken together, these results suggest that our screening method using TLH-ELISA is useful to check for low-level (<100 MPN/g) of V. parahaemolyticus in raw seafood.     

An insight into the inhibitory activity of dihydromyricetin against Vibrio parahaemolyticus

The objective of this study is to investigate the antibacterial activity of dihydromyricetin (DMY) against Vibrio parahaemolyticus. The dilution method indicated that the minimum inhibitory concentration (MIC) of DMY against V. parahaemolyticus was at 0.625 mg/mL. The inhibitory effects of DMY against V. parahaemolyticus was further studied by analyzing cell morphology, cell injury, cell permeability, cell surface hydrophobicity (CSH) and antibacterial rate. The results showed bacterium cells are completely inactivated in a higher concentration (10 MIC). DMY treatment also lead to an increase in cell membrane permeability, cell injury as well as CSH. A good correlation between antibacterial rate and CSH was also observed. These findings indicated DMY could be used as a new alternative natural antibacterial agent for control pathogen growth in aquatic food.     

Mathematical quantification of inactivation of Vibrio parahaemolyticus on two types of surface soiled with different substrates

Survivability of foodborne pathogens on food-processing surfaces is an important factor in understanding and quantifying bacterial transfer to foods (i.e. cross-contamination). This study examined the survival of Vibrio parahaemolyticus on two different surfaces in a laboratory-based simulation. V. parahaemolyticus was inoculated onto both polypropylene and stainless steel surfaces following contamination with saline solution (SS), tryptone soya broth (TSB), or seafood purge. V. parahaemolyticus remained viable on polypropylene for 10 days, but was undetectable within 24 h on the stainless steel surface. The survivability was similar on polypropylene in the presence of all contaminating substrates, as shown by data from the Weibull and biphasic models (adjusted-R² > 0.91). However, for stainless steel, SS and TSB prolonged the survival of V. parahaemolyticus to 144 and 120 h, respectively. Survivability data revealed a shoulder period in the first 4 h and a slight tailing effect at the end of the survival curve in the presence of seafood purge, which suggested that the biphasic model might be appropriate (adjusted-R² = 0.9442). These results indicate that the biphasic model may accurately estimate pathogen survival for cross contamination exposure. Integrating the survivability model into quantitative studies will help understand cross contamination.     

Quantitative detection of viable foodborne E. coli O157:H7, Listeria monocytogenes and Salmonella in fresh-cut vegetables combining propidium monoazide and real-time PCR

The increase of foodborne outbreaks associated with fresh vegetables has highlighted the importance of developing rapid and specific methods for the detection and quantification of foodborne pathogens. In this sense, real-time PCR (qPCR) fulfills these requirements although it may detect dead cells. Recently, a potential strategy to specifically detect viable cells has been proposed relying on the use of DNA binding molecules as sample pretreatment previous to the qPCR. In this study propidium monoazide (PMA) and reagent D, combined with qPCR, were evaluated for the detection and quantification of viable Escherichia coli O157:H7, Salmonella and Listeria monocytogenes. Initially, the optimal concentration of both reagents was determined for discrimination between viable and dead bacteria in cell suspensions. Although both reagents showed similar reductions for the three pathogens, reagent D was toxic to L. monocytogenes and Salmonella and therefore only PMA was used to evaluate the applicability of this technique on food samples. A final concentration of 50 μM PMA was assayed in artificially inoculated spinach and mixed salad. PMA-qPCR signal was negative for all dead cell concentrations tested except for mixed salad inoculated with L. monocytogenes at the highest concentration. These results demonstrate that PMA-qPCR is a suitable technique for the detection and quantification of viable pathogens in fresh-cut vegetables at the levels normally found in vegetable samples.     

Optimization and validation of a PMA qPCR method for Escherichia coli quantification in primary production

The microbial requirements defined in many quality assurance guidelines and standards of primary production demand the establishment of microbial sampling programs. Recently, a q-PCR Escherichia coli assay has been reported as a good method to quantify the presence of fecal indicator bacterial in groundwater samples. This study focuses on the optimization, validation and application of a qPCR method combined with propidium monoazide (PMA) treatment to exclude DNA from dead cells. A first screening consisting of six primer sets targeting single and multi-copy of E. coli were tested to evaluate the sensitivity of the assay. After that, four primer sets were selected, combined with PMA treatment and their capacity to distinguish viable cells when combined with a background of dead cells was assessed. A primer set targeting the 23S rRNA gene was 10-fold more sensitive than the rest of primers, enabling the detection of low concentrations of viable E. coli cells. This assay also exhibited good repeatability and reproducibility, which indicates the robustness of the method. Optimized and validated PMA-qPCR was used to enumerate E. coli in environmental samples including irrigation water and fresh produce. The results were compared with the levels quantified using qPCR and cultivation-based techniques. Counts of E. coli using plate count assay were significantly lower than the levels obtained by molecular techniques (PMA-qPCR and qPCR) in both irrigation water and fresh produce. E. coli PMA-qPCR enumeration method showed similar results as qPCR quantification, although the PMA-qPCR treatment seemed to a good alternative to distinguish between viable and dead cells. It can be concluded that the optimized PMA-qPCR assay can be used by the industry in microbial sampling programs, helping them with the implementation of Good Agricultural Practices (GAP).     

Effects of green tea extract on reducing Vibrio parahaemolyticus and increasing shelf life of oyster meats

This study investigated effects of tea extract on growth of pathogenic Vibrio parahaemolyticus and potential utilization in post-harvest treatment to extend shelf life of Pacific oysters (Crassostrea gigas). Longjing Tea, which exhibited strong bactericidal activity against V. parahaemolyticus, was selected to use in this study. Tea extract containing equal or higher than 4.6 g/l total phenolic contents (TPC) as gallic acid equivalents (GAE) determined by Folin-Ciocalteau method could reduce a mixture of five clinical V. parahaemolyticus strains in tryptic soy broth plus 1.5% NaCl from 4.5 log CFU/ml to non-detectable level (<1 log CFU/ml) within 8 h. A treatment of shucked oysters with tea extract containing 9.1 g/l TPC as GAE for 2 h at 23 ± 1 °C with oyster/tea extract ratio of approximate 0.9 g/ml resulted in greater (p < 0.05) V. parahaemolyticus reductions (0.8 log MPN/g) compared to controls (0.2 log MPN/g). The following shelf life study indicated that green tea treatment at oyster/tea extract ratio of approximate 0.7 g/ml could enhance reducing V. parahaemolyticus while retarding the growth of total bacteria in oysters during 5 ± 1 °C storage. Therefore, green tea might be utilized as a natural antimicrobial agent to inactivate V. parahaemolyticus in oysters and extend the shelf life during refrigeration storage.     

The efficacy of grape seed extract,citric acid and lactic acid on the inactivation of Vibrio parahaemolyticus in shucked oysters

The efficacy of grape seed extract (GE), citric acid (CA) or lactic acid (LA) on the inactivation of Vibrio parahaemolyticus in shucked oysters was studied. The minimum inhibitory concentration (MIC) of GE, CA or LA against V. parahaemolyticus in TSB-1% NaCl was also determined. The shucked oysters were artificially inoculated with V. parahaemolyticus, the inoculated shucked oysters (25 g) were then dipped in solution of GE (0.0, 10.0, 20.0, 50.0, 100, 200, 300 and 500 mg mL⁻¹), CA (0.0, 5.0, 10.0, 15.0, 20.0, 50.0, 100, 200 and 300 mg mL⁻¹) or LA (0.0, 1.0, 5.0, 10.0, 15.0, 20.0, 50.0, 100 and 150 mg mL⁻¹) for 10 min. The population of V. parahaemolyticus in shucked oysters was determined. The MICs of GE, CA or LA against V. parahaemolyticus were 10.0, 5.0 or 1.0 mg mL⁻¹, respectively. A 500, 300 or 150 mg mL⁻¹ GE, CA or LA solutions were needed to reduce the population of V. parahaemolyticus to below the detection level (1.0 log g⁻¹) in shucked oysters.     

Epidemiology of foodborne disease outbreaks caused by Vibrio parahaemolyticus during 2010–2014 in Zhejiang Province,China

Vibrio parahaemolyticus is one of the most commonly reported species causing foodborne diseases in China, to gain understanding of epidemiology characteristics of V. parahaemolyticus outbreaks in Zhejiang Province, China, we summarized data on V. parahaemolyticus outbreaks from 2010 to 2014 in Zhejiang Province reported to China National Foodborne Diseases Surveillance Network. A food item was implicated if V. parahaemolyticus was isolated from food or based on epidemiologic evidence. From 2010 to 2014, 71 outbreaks due to V. parahaemolyticus were reported, resulting in 933 illnesses and 117 hospitalizations without death. The median annual incidence rate of confirmed V. parahaemolyticus outbreaks reported was 2.75 per million population (rang, 1.11–5.60). About 90.1% (64/71) of outbreaks caused by V. parahaemolyticus occurred during May and October. Aquatic products (27 outbreaks, 38.0%), meat and meat products (9 outbreaks, 12.7%), plant-based foods (6 outbreaks, 8.4%), mixed foods (5 outbreaks, 7.0%) were the most commonly implicated foods. Outbreaks most frequently occurred in restaurants (50.7%), private residents (21.1%), and cafeteria (12.7%). Cross contamination (39.4%), improper cooking (21.1%), improper storage (16.9%) were main reasons. To prevent V. parahaemolyticus outbreaks caused by cross contamination, improper cooking and improper storage in high-temperature seasons, regulations for seafood safety from the production stage to the consumption stage should be strengthened.