A STUDY ON SUITABILITY OF FOUR ENRICHMENT BROTHS FOR PCR-BASED DETECTION OF LISTERIA MONOCYTOGENES FROM RAW MEAT

Four enrichment broths were evaluated for their compatibility with the polymerase chain reaction (PCR) for detection of Listeria monocytogenes from raw meat after single‐step enrichment. Standardized PCR protocols for listeriolysin O (hlyA) gene were used for the species‐specific identification of L. monocytogenes. Four broths, namely, modified University of Vermont broth (MUVM), Listeria enrichment broth (LEB), Fraser broth (FB) and polymyxin, acriflavin, lithium chloride, ceftazidime, aesculin, mannitol, egg yolk broth (PALCAM) , were inoculated with L. monocytogenes. The enriched cultures were subjected for PCR. Similarly, meat samples were artificially spiked with various concentrations of L. monocytogenes, these spiked samples were enriched in the above‐mentioned four broths and subjected to PCR to determine the medium that was most compatible for PCR‐based detection of L. monocytogenes. The aliquots taken during different incubation periods were subjected to three different procedures for the concentration of the target organism for use in PCR. Results revealed that MUVM was better than other broths for the detection of L. monocytogenes by both PCR and cultural method; moreover, it was able to support the growth of as low as 10 cfu/g of meat. Concentration of the target organisms by centrifugation and washing with PCR buffer was the most suitable method for improving PCR performance for detection of L. monocytogenes. Goat (n = 67) and buffalo (n = 45) meat samples from local markets were also screened by both PCR and cultural method to validate the results obtained from the spiking studies. Both results were in agreement in spiking studies as well as screening of market meat samples.     

EFFECT OF CHILLING AND FREEZING ON SURVIVAL OF VIBRIO PARAHAEMOLYTICUS ON FISH FILLETS1

Vibrio parahaemolyticus is a foodborne pathogen isolated from coastal waters of the United States, and from seafoods including fish. No information is available on the viability of V. parahaemolyticus on raw, chilled and frozen fish. A three‐strain mixture of V. parahaemolyticus was inoculated on fish fillets (pH 6.4) to obtain a bacterial load of 10⁴ (high) or 10³ (low) CFU/fillet, and stored at 4C or 8C for 9 days or at – 18C for seven weeks. At 4C and 8C, and at both levels of inoculation, V. parahaemolyticus survived on the fillets for the entire duration of the study. However, there was a significant reduction (P < 0.01) in V. parahaemolyticus population on the fillets by 9 days of storage. In the frozen fillets, there was a sharp decline (P < 0.01) in the population of V. parahaemolyticus by day 5 of storage. Although chilling and freezing significantly (P < 0.01) inactivated high numbers of V. parahaemolyticus on fish, they cannot be relied upon as a method to reduce V. parahaemolyticus on fish, since the time and magnitude of reduction depends on the initial load of the pathogen and the storage temperature.     

A PCR Assay for Specific Detection of the Pandemic Vibrio parahaemolyticus O3:K6 Clone from Shellfish

: The current standard method for identifying Vibrio parahaemolyticus serotype O3:K6, an emerging pathogen with apparent enhanced virulence characteristics, typically takes 4 to 6 d to complete and requires serotyping. To provide a more rapid strategy, we optimized a polymerase chain reaction (PCR)‐based assay for specific detection of V. parahaemolyticus O3:K6. Of 78 V. parahaemolyticus isolates and other related species; only strains classified into the V. parahaemolyticus O3:K6 clonal group (n= 39) showed positive results in the PCR assay. The assay detected 2.3 cells/PCR reaction and 310 cells/g using bacterial cultures and inoculated oyster samples, respectively. Sensitive and specific detection of V. parahaemolyticus O3:K6 was possible following a 6‐h enrichment.     

Prevalence of Vibrio parahaemolyticus in seafoods and their processing environments as detected by duplex PCR

A duplex polymerase chain reaction (PCR) procedure targeting the genes gyrB and tl was established for specific identification of Vibrio parahaemolyticus from seafoods and processing environments. It could detect as few as 2.5 × 10² colony‐forming units mL^?1 in pure cultures. Direct detection of V. parahaemolyticus was also possible from artificially contaminated shrimp samples if combined with proteinase treatment. The homogeneous colonies on thiosulfate/citrate/bile salts/sucrose agar suspected to be V. parahaemolyticus or closely related species (n = 37) out of 259 samples of seafoods and processing environments were identified using the conventional method and duplex PCR. Both methods identified 17 isolates as V. parahaemolyticus from among the suspected isolates. Only one of the 17 V. parahaemolyticus isolates possessed the thermostable direct haemolysin gene (tdh) fragment as detected by different primer pairs in single PCR. Copyright © 2006 Society of Chemical Industry     

Detection and molecular characterization of Vibrio parahaemolyticus isolated from seafood harvested along the southwest coast of India

The levels of total and tdh⁺ Vibrio parahaemolyticus were estimated in 83 seafood samples from southwest coast of India by colony hybridization. Conventional enrichment and isolation technique was also used to study the prevalence. Polymerase chain reaction (PCR) was performed on bacterial cell lyates for detection of total and pathogenic V. parahaemolyticus by amplification of specific genes. Of 83 samples tested, V. parahaemolyticus could be detected in 74 (89.2%) samples and tdh⁺ V. parahaemolyticus in 5 (6.0%) samples by colony hybridization. V. parahaemolyticus was detected in 68 (81.9%) of 83 samples after 18 h of enrichment by PCR, and isolated from 63 (75.9%) of 83 samples by conventional isolation. The virulence genes tdh and trh could be detected in 8.4% and 25.3%, respectively, in the sample enrichment broths by PCR. Use of colony hybridization following enrichment to achieve sensitive detection of tdh⁺ V. parahaemolyticus in seafood was evaluated using another set of 58 seafood samples. Thirty pathogenic V. parahaemolyticus strains isolated during the study were screened by PCR for genetic markers to be specific for the detection of the pandemic clone. Results of this study suggest that the GS-PCR may serve as a reliable genetic marker for the pandemic clone of V. parahaemolyticus.     

COMPARISON OF A REAL-TIME POLYMERASE CHAIN REACTION ASSAY WITH A CULTURE METHOD FOR THE DETECTION OF SALMONELLA IN RETAIL MEAT SAMPLES

A real‐time polymerase chain reaction (PCR) method developed in this study was compared with a conventional International Organization for Standardization culture method for the detection of Salmonella in Irish beef, chicken, pork and turkey. This also provided a snapshot of the incidence of Salmonella in such products. After selective enrichment, all meat samples (n = 100) were: (1) subjected to DNA extraction and real‐time PCR analysis using primers against Salmonella spp. specific gene (16S rRNA) or the invA virulence gene; and (2) plated on differential media and identified as Salmonella using immunological and sub‐typing methods. Retail samples (5/100) were shown to contain Salmonella using the 16S rRNA gene‐based real‐time PCR assay and the standard culture method. However, only two (of five) samples were shown to contain Salmonella using the invA gene‐based real‐time PCR assay. For the sample set examined, the developed 16S rRNA gene‐based real‐time PCR assay demonstrated comparable specificity and sensitivity to the currently used standard culture method but was considerably more rapid.     

Comparison of molecular detection methods for Vibrio parahaemolyticus and Vibrio vulnificus

Pathogenic vibrios are a global concern for seafood safety and many molecular methods have been developed for their detection. This study compares several molecular methods for detection of total and pathogenic Vibrio parahaemolyticus and Vibrio vulnificus, in MPN enrichments from oysters and fish intestine samples. This study employed the DuPont Qualicon BAX^® System Real-Time PCR assay for detection of V. parahaemolyticus and V. vulnificus. Multiplex real-time PCR detection of total (tlh+), tdh+, and trh+ V. parahaemolyticus was conducted on the Cepheid SmartCycler II. Total (rpoD) and tdh+ V. parahaemolyticus were also detected using LAMP. V. vulnificus detection was performed using real-time PCR methods developed for the SmartCycler and the AB 7500 Fast. Recommended template preparations were compared to BAX^® lysis samples for suitability. There was no significant difference in detection of V. parahaemolyticus and V. vulnificus using the BAX^® or SmartCycler assays. The AB assay showed no difference from other methods in detection of V. vulnificus unless boiled templates were utilized. There was a significant difference in detection of tdh+ V. parahaemolyticus between SmartCycler and LAMP assays unless the total (tlh+) V. parahaemolyticus gene target was omitted from the SmartCycler assay; a similar trend was observed for trh+ V. parahaemolyticus.     

FATTY ACID COMPOSITION OF DIFFERENT MARGARINES AND BUTTERS FROM PAKISTAN WITH SPECIAL EMPHASIS ON TRANS UNSATURATED CONTENTS

Fatty acid (FA) composition and isomeric trans unsaturated contents in selected brands of margarines and butter, which are more popular among general consumers of Pakistan, has been reported in this study. A sum of 10 butter and 10 margarine samples, collected from different bakers, confectioners and retail outlets, was analyzed. FA methyl esters were prepared, and quantitative measurements were made using a methyl lignoserate‐coated stationary phase, capillary column (SP‐2340, 60 m × 0.25 mm, 0.20 μm), with flame ionization detection. The contents of saturated FA, cis monounsaturated FA and cis polyunsaturated FA were in the following ranges: margarines 38.9–53.1, 21.9–35.8, 7.45–21.5% and butter 63.7–68.5, 23.0–27.0 and 1.20–2.94%, respectively. Results showed significantly higher amounts of trans FA (TFA) in margarines ranged from 2.45 to 21.1%, whereas butter samples contained less than 5.00% of TFA.     

Validation of a method for the detection of five species,serogroups, biotypes and virulence factors of Vibrio by multiplex PCR in fish and seafood

In this work a sequential multiplex PCR system was designed and validated for the detection of most frequent foodborne pathogen Vibrio species in fish and seafood (Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginoliticus and Vibrio mimicus). The method proposed functions in a hierarchical way, being composed of an end-point multiplex PCR to detect the presence of DNA belonging to the studied species, followed by multiplex PCR and fragment analysis allowing the viability assessment of the detected strains. The final multiplex PCR step of the method may be applied if identification of the serogroup, biotype and/or virulence factor level is necessary. Forty samples of commercial fish and seafood products were used at the method validation stage. Sixty three marine organism samples obtained from various estuarine areas of Spain including shrimps, crabs, bivalve mollusks and fishes were screened for presence of Vibrio species and 2 mussel samples were found positive for V. parahaemolyticus. On the whole, the proposed method is robust and readily adaptable in routine molecular diagnostic laboratories, allowing monitoring and simultaneous detection of all these bacterial pathogens in seafood samples, reducing the expenses and time consumed by other analytical methods.     

A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus

A multiplex PCR assay using three collagenase-targeted primer pairs for the species-specific detection of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus was developed. The results highlight the species specificity of the three primer sets designed. Because of the increasing importance of Vibrio spp. in human foodborne diseases, molecular approaches for routine microbial screening and monitoring of clinical, environmental, and food samples also have become more important. The results of this study indicate that the gene coding for collagenase should be used as an alternative molecular target to discriminate among the three Vibrio species.     

Improving Detection of Vibrio vulnificus in Octopus variabilis by PCR

PCR methods can detect foodborne pathogenic bacteria with simplicity, specificity and speed. In order to improve sensitivity and speed of PCR methods for detection of Vibrio vulnificus in small octopus homogenate, several media and culture conditions were compared. Modified brain heart infusion media containing 2% NaCl and adjusted to pH 8.0 and 30°C was most effective for enrichment of the bacteria. Procedures affecting the efficiency of template DNA extraction and target DNA amplification were also optimized. By these combined efforts, a PCR procedure capable of detecting V. vulnificus as low as 10 cells/mL within 10h was developed.     

Evaluation of a Double Layer Agar Plate For Direct Enumeration of Vibrio parahaemolyticus

A double layer agar plate (DLAP) was developed according to the thin agar layer (TAL) method as a 1‐step procedure for direct enumeration of injured Vibrio parahaemolyticus cells based on the formation of unique purple colonies by V. parahaemolyticus. The DLAP was prepared by overlaying an equal volume (10 mL) of a nonselective medium (tryptic soy agar supplemented with 1.5% NaCl) onto a selective medium (Bio‐Chrome Vibrio medium). The DLAP was capable of detecting V. parahaemolyticus in mixed cultures containing non‐Vibrio bacteria. Production of purple colonies by V. parahaemolyticus on DLAP was not affected by the growth of other bacteria, even when V. parahaemolyticus was only a small fraction (5%) of the entire bacterial population. Direct plating on DLAP was found as effective as the most probable number (MPN) method for recovering heat‐and cold‐injured V. parahaemolyticus cells, which could not be detected by direct plating on Bio‐Chrome Vibrio medium or thiosulfate‐citrate‐bile salts‐sucrose agar. The DLAP offers an alternative to the MPN method for detecting injured V. parahaemolyticus cells and can be used as a simple 1‐step procedure for quick screening of V. parahaemolyticus in foods.     

Detection and differentiation of Vibrio spp. in seafood and fish samples with cultural and molecular methods

Vibrio spp. as natural inhabitants of sea- and brackwater of both tropical and temperate regions of the world are commonly found in different kinds of seafood. Even among the three main human pathogenic species Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus most of the isolates from seafood do not carry the different virulence factors responsible for foodborne infections. Therefore, the risk assessment of Vibrio spp. in seafood is currently based mainly on the knowledge of the genetic setting of foodborne strains. For the detection and differentiation of Vibrio spp. (V. parahaemolyticus, V. cholerae and V. vulnificus) three probe-based multiplex real-time PCR systems were developed and validated. One real-time PCR system simultaneously detects V. parahaemolyticus, V. cholerae and V. vulnificus on genus level combined with an Internal Amplification Control. The detection limit for the system was between 1 cfu/mL and 10 cfu/mL in pure culture and in different artificially contaminated sample material, e. g. prawns or Alaska Pollock. The other two PCR systems were implemented for the detection of different virulence genes of V. parahaemolyticus and V. cholerae isolates. The molecular detection systems were applied for the investigation of 338 raw and cooked seafood and fish samples for the presence of the different Vibrio spp. The collected data indicate that the PCR systems can be useful for rapid detection and differentiation of Vibrio spp. in different food matrices as basis for a preventive consumer protection policy.     

Quantitative Detection ofVibrio vulnificus in Shellfish by Competitive Polymerase Chain Reaction

Quantitative detection of V. vulnificus in shellfish via competitive PCR was herein developed. We designed a forward primer, a reverse primer and a hybrid forward primer based on the V. vulnificus specific hemolysin gene (vvh), which was used to amplify target DNA and an internal competitive PCR standard. The specificity of the primers for V. vulnificus was proven by gel electrophoresis. The detection sensitivity for V. vulnificus was 220 CFU per PCR reaction with cells from a pure culture and 270 CFU/g of tissue without enrichment. After 10 hours of enrichment at 37° C the minimum detection level was reduced to 7 CFU/g of tissue.     

Incidence and Enumeration of Vibrio parahaemolyticus in Shellfish from Two Retail Sources and the Genetic Diversity of Isolates as Determined by RAPD-PCR Analysis

A total of 83 shellfish samples from two local retail sources (A and B) yielded 38 samples positive for the presence of Vibrio parahaemolyticus based on 3 tube MPN enrichments and isolation from thiosulfate-citrate-bile salts-sucrose agar (TCBS) and biochemical tests. The 38 positive samples yielded 133 biochemically presumptive isolates of V. parahaemolyticus. Among these 133 presumptive isolates, 104 were confirmed by the polymerase chain reaction (PCR), which yielded more reliable identification results than the biochemical tests. The 38 biochemically presumptive samples yielded 29 samples that were confirmed by PCR to be positive for the presence of V. parahaemolyticus. RAPD analysis with three random primers was performed to examine the genetic diversity of 64 strains among the PCR confirmed V. parahaemolyticus isolates from both retail sources. 52 of 56 composite RAPD types consisted of single strains, indicating that most of the V. parahaemolyticus isolates were genetically quite heterogeneous. No strains representing the same RAPD type occurred in both retail outlets, implying that contamination of the shellfish by V. parahaemolyticus from the 2 retail sources was from different environmental locals and shellfish harvesting areas. Eight genomic clusters were generated at the 25% similarity level in a dendrogram based on RAPD profiles. With few exception, isolates with close genetic relationships grouping into an individual cluster tended to be derived from the same retail source.     

In-house validation of a real-time PCR method for rapid detection of<Emphasis Type="Italic"> Salmonella</Emphasis> ssp. in food products

A real-time polymerase chain reaction (PCR) method for Salmonella ssp. detection in food samples has been developed and validated in-house. The specificity of the assay was confirmed by tests with 295 different Salmonella strains, including four strains of Salmonella bongori. When tested with extracted Salmonella DNA the lowest detected amount was found to be 5 fg, which is equivalent to approximately one genome copy. The detection limit was further determined by artificial contamination of minced meat with S. Typhimurium cells and of pastry with S. enteritidis using the most probable number approach for cell dose dilutions. It was calculated that even one colony forming unit of Salmonella was still detectable in 25 g food after enrichment culture for 18 h. An additional PCR system for internal positive control, which was included in each reaction and detected in parallel via another reporter fluorescence dye, has no negative impact on the sensitivity of the assay. The method was evaluated with 1,293 naturally contaminated food samples and compared to the conventional cultural method. Of 55 positive PCR samples, 45 were confirmed by the cultural method. The statistical comparison revealed a correlation of 99.2% for specificity, of 100% for sensitivity and of 99.2% for trueness. The results of the comparative analysis and the advantages of the real-time PCR method for detection of Salmonella ssp. under routine laboratory conditions are discussed.     

THERMAL RESISTANCE OF ANTIBIOTIC-RESISTANT AND ANTIBIOTIC-SENSITIVE SALMONELLA SPP. ON CHICKEN MEAT

The aim of this research was to investigate the potential relationship, if any, between the acquisition/possession of antibiotic resistance genes in strains of Salmonella and its resistance to heat stress. Chicken pieces were inoculated with antibiotic sensitive (AS) strains of S. Enteritidis and S. Typhimurium, its laboratory‐acquired antibiotic‐resistant (AR) mutant strains (nalidixic acid and streptomycin), or a multiresistant strain of S. Typhimurium DT104. Half of these samples were heat‐shocked (48C for 30 min) and all were heat‐challenged at 55C for up to 30 min. Samples were then plated on xylose lysine desoxycholate (XLD) and tryptone soya agar (TSA) overpoured with XLD. Heat‐shocked cultures of S. Typhimurium DT104 had significantly higher D‐values (the time required for a 1 log reduction in the number of bacteria) than their non‐heat‐shocked counterparts (P < 0.05). No significant differences were observed between AR and their AS. However, the D‐values for S. Typhimurium DT104 were significantly higher than the D‐values for S. Typhimurium (AS) and S. Enteritidis (AS) (P < 0.05). This study concluded that laboratory‐acquired antibiotic‐resistant mutation did not affect heat resistance of the Salmonella strains studied and suggested a potential link between multiantibiotic resistance and heat resistance in S. Typhimurium DT104.     

Design and validation of a novel multiplex real-time PCR assay for Vibrio pathogen detection

Three species--Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus--account for the majority of vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species. Additionally, biochemical identification and confirmation require 2 or more days to complete. Rapid and sensitive molecular techniques for the detection of vibrio pathogens would be useful for the surveillance and management of outbreaks. To facilitate the identification of human-pathogenic species, we designed and validated a highly sensitive, specific, and robust multiplex real-time PCR assay to identify V. cholerae, V. parahaemolyticus, and V. vulnificus using a four-dye configuration in a convenient lyophilized format. Multiple Vibrio strains were sequenced to verify candidate target TaqMan sites. Several individual assays within the multiplex contain multiple primers or probes to ensure detection of polymorphic variants. V. cholerae, V. parahaemolyticus, and V. vulnificus were detected either individually or in mixtures at ≤30 genomic copies. V. cholerae was specifically detected in the presence or absence of Vibrio mimicus. The Vibrio multiplex assay showed 100% specificity to all targets analyzed and no detection of nearest neighbor strains. Each assay exhibited 100% ± 10% efficiency. Multiplex real-time PCR can simplify pathogen detection and reduce costs per test since three species can be analyzed in a single reaction tube. Rapid and accurate detection of pathogenic vibrios in shellfish or seawater samples will improve the microbiological safety of seafood for consumers.     

A real-time PCR for Detection of Pathogenic Yersinia enterocolitica in food combined with an Universal Internal Amplification Control System

A real-time PCR system with an internal amplification control was developed for detection of pathogenic Yersinia) enterocolitica in food samples. The chromosomally encoded ail gene was chosen as PCR target. Sequences of plasmid pUC19 served as target for the internal amplification control. The method was validated in combination with sample enrichment in PSB and TSB broth using different food matrices spiked with Y. enterocolitica and naturally contaminated slaughterhouse samples. The results of the real-time PCR with internal control were verified by the cultural method according to EN ISO 10273:2003. The sensitivity of the real-time PCR with internal control is about 5 genome copies per reaction. Artificial contamination of food samples resulted in a detection level of 5 cfu per 25 g Y. enterocolitica in food samples. 100% of porcine tonsils and about 22% meat from pig heads were contaminated. The screening of samples by PCR prior to cultural analysis allows focusing on positive samples in routine analysis. This could result in a higher detection rate by cultural analysis.