PMA based real-time fluorescent LAMP for detection of Vibrio parahaemolyticus in viable but nonculturable state

Vibrio parahaemolyticus is a crucial foodborne pathogen. The viable but non-culturable (VBNC) state of V. parahaemolyticus cannot be detected by traditional culture methods. The objective of this study was to develop and evaluate a method that combines propidium monoazide (PMA) treatment with real-time fluorescent loop-mediated isothermal amplification (RF-LAMP) to detect and quantify VBNC cells of V. parahaemolyticus. Different states of cells were treated with PMA in dark for 5 min and subsequently exposed to a 650 W halogen lamp for 5 min. The cells were collected and DNA was amplified by RF-LAMP. The primers which targeted six distinct regions in the tlh gene of V. parahaemolyticus were used for the PMA-RF-LAMP assay. The results indicated that the treatment with 4 μg/mL of PMA and a further exposure to light for 5 min was suitable for PMA-RF-LAMP to distinguish viable cells from dead cells of V. parahaemolyticus. The developed assay exhibited remarkably high specificity, sensitivity and rapidity, without any cross reaction with the tested non-V. parahaemolyticus strains. There was good linear correlation between Ct values and log copy/mL in the range of 6.8–6.8 × 10⁷ copy/mL, with the reaction endpoints less than 30 cycles (30 min). It could detect as low as 14 copy/g of V. parahaemolyticus in spiked seafood samples (pomfret, shrimp, scallop, oyster and salted fish) without any interference from food matrices, dead cells and other bacteria.     

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.     

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     

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.     

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.     

Detection and quantification of pathogenic Vibrio parahaemolyticus in shellfish by using multiplex PCR and loop-mediated isothermal amplification assay

Vibrio parahaemolyticus is a halophilic bacterium that commonly inhabits the marine and estuarine environments. This organism is also one of the leading causative pathogen of gastroenteritis often related to consumption of raw or undercooked seafood. In this study, molluscan shellfish (bloody clams and surf clams) and crustaceans (shrimps) were monitored in wet markets and hypermarkets. Two molecular methods were employed and compared to detect total and pathogenic V. parahaemolyticus in MPN enrichments: multiplex PCR and LAMP assay. The multiplex PCR was optimized to detect the total (toxR+), tdh+ and trh+ V. parahaemolyticus. On the other hand, the LAMP assay was employed to target the pathogenic strains only, the tdh+ and trh+, respectively. Out of 232 samples examined, 229 (98.7%) were positive for V. parahaemolyticus with counts ranging from 30 to >110, 000 MPN/g. Positive samples for tdh+ V. parahaemolyticus were obtained in 77 out of 232 (33.1%) samples ranging from 30 to >110, 000 MPN/g. Meanwhile, positive samples for trh+ were identified in 16 out of 232 (6.9%) samples examined ranging from 30 to 9600 MPN/g. Detection of samples with presence of tdh+ genes did not vary between methods, but a significant difference was observed when the LAMP assay was compared to PCR to detect trh+ V. parahaemolyticus. Therefore, on occasions where the density of the targeted genes is low, the LAMP assay serves as a better alternative. Nonetheless, this study constitutes an assessment of presence of total and potentially pathogenic V. parahaemolyticus in shellfishes for domestic consumption revealing the potential risk of contracting vibriosis if precautions and safety measures are not properly managed.     

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.     

Prevalence,pathogenicity, and serotypes of Vibrio parahaemolyticus in shrimp from Chinese retail markets

Vibrio parahaemolyticus is one of the most common foodborne pathogens in Asian countries. V. parahaemolyticus contamination of retail shrimp in China has been reported previously, and such contaminated shrimp have been linked to outbreaks of foodborne diseases. However, to date, the prevalence of V. parahaemolyticus in retail shrimp in China has not been determined. This study aimed at identifying the prevalence of V. parahaemolyticus in shrimps in Chinese retail market. From May 2012 to April 2013, a total of 273 shrimp samples was obtained from retail markets in 16 provinces (19 cities) of China. V. parahaemolyticus was detected in 103 (37.7%) of 273 samples by the most probable number method. Bacterial densities were less than 100 MPN/g in 95.1% (98/103) of the samples. Five trh-positive isolates were identified from 247 isolates, and none of the isolates were tdh-positive. In multiplex PCR-based O-antigen serotyping of these 247 pathogenic isolates, all O-antigen serotypes, except O9, were detected, and serotype O2 was found to be the most prevalent (detected in 82 isolates). This is the first report on V. parahaemolyticus prevalence in retail shrimp in China, and the findings of this study can be used for microbiological risk assessment of shrimp in China.     

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.     

Behavior of Vibrio parahemolyticus cocktail including pathogenic and nonpathogenic strains on cooked shrimp

Pathogenic Vibrio parahaemolyticus, an important foodborne bacterium, coexists with nonpathogenic strains of V. parahaemolyticus in the environment. However, current predictive models for V. parahaemolyticus usually focused on single strain without considering the pathogenic and nonpathogenic bacteria cocktail coexist situation. In this study, the behavior of four V. parahaemolyticus strains (F18: tlh⁺/trh⁺/tdh⁻, ATCC 17802: tlh⁺/trh⁺/tdh⁻, F36: tlh⁺/trh⁻/tdh⁻, ATCC 33847: tlh⁺/trh⁻/tdh⁺) and their cocktails on cooked shrimp were investigated at different temperatures (4, 7, 10, 15, 20, 25 and 30 °C). Total bacteria counts were enumerated by traditional plate count method, periodically. Results showed both V. parahaemolyticus cocktails and four single strains grew in a temperature range of 15–30 °C and died at 4–7 °C. At 10 °C, for the cocktails, it inactivated at initial 100 h and then grew rapidly; but for single strain, only one strain F18 displayed the similar growth tendency, while the others grew all the time. Compared with single strains, the primary and secondary model analysis showed that the cocktails displayed a better growth activity with higher maximum growth rate and shorter lag time. The above results indicated that modeling bacterial growth by using a cocktail of V. parahaemolyticus strains may be a better option for simulating growth in the real environment. This study will fill in the gap of predictive microbiology and improve significantly the accuracy of microbial risk assessment.     

Comparison of toxR and tlh based PCR assays for Vibrio parahaemolyticus

Vibrio parahaemolyticus is a Gram-negative bacterium found in marine and estuarine environments and is globally the leading cause of bacterial seafood-related illness. A real-time PCR assay for V. parahaemolyticus was developed for the marker toxR, with a large-scale and direct comparison of its applicability as a species-specific marker compared to the tlh gene carried out. Assays for tlh and toxR were used for 255 presumptive V. parahaemolyticus strains from our strain library, utilising both real-time (toxR) and conventional PCR assays (tlh). Of the 255 strains test, 254 results were in concordance; 255 strains were identified as being toxR positive (100%) and 254 strains were tlh positive (99.6%). The single discordant strain (isolate V12/023) was of interest, because it represented a presumptive V. parahaemolyticus strain, isolated from a clinical case. Whole genome sequence analysis and multi locus sequence typing of this single discordant strain was carried out, which unambiguously identified that the isolate was indeed V. parahaemolyticus. Genome analysis identified mismatches in the primer binding sites for the established tlh assay is likely responsible for the assay failing on this particular strain. The identification of false-negative results in strains that are implicated in human infections using the tlh assay and clearly highlights the relevance of the comparison with a toxR assay which showed 100% identification for the V. parahaemolyticus strains tested.     

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.     

Real time loop-mediated isothermal amplification using a portable fluorescence scanner for rapid and simple detection of Vibrio parahaemolyticus

We evaluated the usefulness of the loop-mediated isothermal amplification (LAMP) using a portable ESE Quant tube scanner as a rapid and simple method for the detection of Vibrio parahaemolyticus, an important pathogen causing seafood-borne gastroenteritis. The real time LAMP (RT-LAMP) assay using a hemolysin gene (tlh/ldh)-specific primers was verified using V. parahaemolyticus strains (n = 91) from different countries and other non-target strains to check the utility of this method. Both the sensitivity and specificity of the RT-LAMP using 3 pairs of tlh/ldh-specific primers developed in this study were excellent (100%). The detection limit of the RT-LAMP was as low as 7 Colony forming unit per reaction and detection time was only 20 min. Comparative evaluation of the target bacterial strains with the RT-LAMP using ESE Quant tube scanner, API 20E system and conventional RT-PCR method revealed that the RT-LAMP assay developed in this study is simpler and more rapid than the latter two methods. Therefore, the RT-LAMP method using the easily portable ESE Quant tube scanner can be considered as an effective tool for the rapid screening of V. parahaemolyticus strains in environmental and clinical samples, especially, in remote areas of developing countries during epidemic periods.     

Epidemiology of foodborne disease outbreaks caused by Vibrio parahaemolyticus,China, 2003–2008

Vibrio parahaemolyticus is a ubiquitous marine bacterium that has become an important public-health concern worldwide. Consumption of food contaminated with V. parahaemolyticus causes acute gastroenteritis in humans. To gain understanding of epidemiological characteristics of V. parahaemolyticus gastroenteritis outbreak in China, we summarized data on 2003–2008 V. parahaemolyticus gastroenteritis outbreaks in 12 provinces 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 2003 to 2008, 322 gastroenteritis outbreaks due to V. parahaemolyticus were reported, resulting in 9041 illnesses and 3948 hospitalizations. Outbreak size ranged from 2 to 244 cases, with a median of 20 cases. Most of the outbreaks occurred during April and October. A single food commodity was implicated in 187 (58%) outbreaks, of which 58 (31%) involved meat and meat products, and 52 (28%) involved aquatic products. Outbreaks most frequently occurred in restaurants (39%), cafeterias (30%), and private residences (15%). Outbreaks most frequently attributed to cross contamination (50%). To prevent and control V. parahaemolyticus gastroenteritis outbreaks, food workers and consumers should receive training on avoiding cross contamination of ready-to-eat foods with uncooked seafoods, particularly in warm weather months.     

Development of a multiplex real-time PCR method for simultaneous detection of Vibrio parahaemolyticus,Listeria monocytogenes and Salmonella spp. in raw shrimp

Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella spp. are important pathogens contaminating seafood in China. In this study, we developed an efficient multiplex real-time PCR for the simultaneous detection of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in raw shrimp without a prior enrichment step. In a test using 28 target and non-target strains only the targets were detected and two calibration curves, for pure cultures and artificially contaminated samples, were used to evaluate the efficiencies of this method. Amplification efficiencies of this multiplex real-time PCR were excellent in pure cultures and artificially contaminated shrimps. The limits of detection in artificially contaminated shrimps were 112 CFU/g for V. parahaemolyticus, 158 CFU/g for L. monocytogenes and 103 CFU/g for Salmonella spp. We validated this multiplex real-time PCR method on 48 commercial samples and the results were comparable to standard culture methods. This efficient multiplex real-time PCR, where each test takes only 50 min after DNA extraction, is a useful tool for high-throughput surveillance of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in seafood products.     

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.     

Prevalence and antimicrobial susceptibility of Vibrio parahaemolyticus isolated from retail shellfish in Shanghai

Vibrio parahaemolyticus is a marine and estuarine bacterium that poses the greatest threat to human health worldwide. It has been the leading bacterial cause of seafood-borne illness. This study investigated the prevalence and drug resistance of V. parahaemolyticus isolated from retail shellfish in Shanghai. A total of 140 shellfish samples were collected from February 2014 to February 2015. The occurrence of V. parahaemolyticus in shellfish was 34.3%, which has increased compared to previous reports. In addition, discernible differences of total presumptive V. parahaemolyticus counts (TPVPC) were also observed in shellfish between market A and B. The results from PCR assays indicated that thermostable direct hemolysin (tdh) gene was positive in two isolates (2.1%), and the thermostable direct hemolysin-related hemolysin (trh) gene was not detected in all isolates. Antibiotic resistance profiles of those isolates were as follows: ampicillin (87.5%), cephazolin (31.3%), cephalothin (6.3%), amoxicillin/clavulanic acid (6.3%), piperacillin (6.3%), and amikacin (3.2%). Thirty-three out of 96 isolates were resistant to two or more antimicrobial agents. It is suggested that V. parahaemolyticus in retail shellfish could be a potential risk to consumers in Shanghai.     

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.     

Development of a rapid and sensitive quantum dot-based immunochromatographic strip by double labeling PCR products for detection of Staphylococcus aureus in food

Staphylococcus aureus was listed as the 2nd common food-borne pathogens, resulting in diseases such as pneumonia, pseudomembranous colitis, pericarditis and even sepsis. A rapid, simple and sensitive detection method is required to monitor food in cases of contamination by S. aureus. Hence, a novel immunochromatographic test strip (ICTS), based on the two-component reaction (i.e. digoxigenin and anti-digoxigenin, quantum dots-conjugated streptavidin and biotin) in test line with QDs as indicator, was developed for the rapid and sensitive detection of S. aureus. Genomic DNA from bacteria lysis by boiling was extracted easily and rapidly with silica-coated magnetic nanoparticles, and a species-specific gene was amplified by PCR using digoxigenin/biotin-labeled primers. The fluorescence of captured QD labels on the test line and control line served as signals was observed by UV light. The sensitivity and specificity of the ICTS were estimated using bacteria spiked food samples (artificially mixed with cell counts (10⁸ CFU/mL for each bacterium) of Escherichia coli, Listeria monocytogenes and Lactobacillus plantarum). The limit of detection for S. aureus was 3 × 10⁰ CFU/mL and 3 × 10¹ CFU/g in spiked milk powder and meat samples, respectively, which was not affected by those non-S. aureus strains. Our results showed that QDs-based ICTS was promising for rapid and sensitive detection of S. aureus within 2 h. Hence, this protocol might be useful for screening and monitoring the contamination of S. aureus in food products, and helpful for promoting the prevention and control of communicable disease caused by food-borne pathogen.