Incidence, Antibiotic Susceptibility and Characterization of Vibrio parahaemolyticus Isolated from Seafood in Selangor, Malaysia

Vibrio parahaemolyticus is one of the major foodborne pathogens owing to its cause of infectious diseases such as gastroenteritis. These diseases are often associated with the consumption of contaminated seafood. This study aims to investigate the presence of V. parahaemolyticus, their virulence, antibiotic profiles, and plasmid profiles from 77 different kinds of shellfish samples collected from wet markets and supermarkets in Selangor, Malaysia. High densities of Vibrio species ( > 5 log CFU/g) were found in 14/16 groups of shellfish. Among 77 presumptive V. parahaemolyticus isolates, 43 (55.8%) were positive for the toxR gene, confirming the identity of the isolates at the species level. However, none of the V. parahaemolyticus isolates harboured the virulence tdh and trh genes. The antibiotic susceptibility of the V. parahaemolyticus isolates revealed that most of them were resistant to ampicillin (95.3%), ampicillin-sulbactam (81.4%), cefotaxime (37.2%) and imipenem (23.3%). The plasmid profiles of the V. parahaemolyticus isolates showed that 41.9% (18/43) possess at least one plasmid. Our results indicate the V. parahaemolyticus isolates are continuously exposed to various antibiotics in the environments, thus consuming the seafood carries a potential health risk to consumers. The antibiotic resistance conferred by the species necessitates an immediate plan to approach the usage of antibiotics differently.

Deteksi Vibrio harveyi dengan Metode Amplifikasi DNA pada Gen toxR

Abstrak -Infeksi hewan akuakultur oleh Vibrio harveyi dapat menyebabkan kematian serta kerugian ekonomi. Kemampuan untuk mendeteksi bakteri tersebut secara dini dapat mencegah penyebarannya dalam akuakultur. Penelitian ini bertujuan untuk mengembangkan metode untuk mendeteksi Vibrio harveyi melalui amplifikasi gen toxR. Amplifikasi DNA dilakukan dengan dua metode, yakni amplifikasi isotermal termediasi loop (loop-mediated isothermal amplification, LAMP) dan reaksi berantai polimerase (PCR). Amplifikasi menggunakan metode LAMP menunjukan perlu dilakukan optimasi protokol maupun desain primer untuk mencegah perolehan hasil false positive. Amplifikasi menggunakan metode PCR menghasilkan produk berukuran 229 pasang basa yang spesifik pada Vibrio harveyi dengan batas deteksi hingga 0,526 ng.µL-1 (setara 2,09 × 106 CFU.mL-1). Kata kunci: Akuakultur, LAMP, PCR, toxR, Vibrio harveyi Abstract -Vibrio harveyi infection in aquacultures may cause death and economical loss. Rapid detection of this bacteria may prevent its dispersal in aquacultures. The goal of this research was to develop method in detection of Vibrio harveyi via amplification of toxR gene. DNA amplification was carried out with two methods, loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). Amplification with LAMP suggest optimization of either protocol or primer design was needed to prevents false positive results. Amplification with PCR yields 229 bp-length product specific to Vibrio harveyi with detection limit up to 0.526 ng.µL-1 (equals to 2.09 × 106 CFU.mL-1). Keywords: Aquaculture, LAMP, PCR, toxR, Vibrio harveyi

ISOLATION AND IDENTIFICATION OF Vibrio parahaemolyticus FROM FLOWER CRAB (Portunus pelagicus) IN NEGERI SEMBILAN AND SELANGOR

This study was conducted to determine the presence of Vibrio parahaemolyticus on 30 samples of flower crab (Portunus pelagicus) collected from Negeri Sembilan (n = 15) and Selangor (n = 15) from March to July 2014. A total of 193 isolates of Vibrio sp. were isolated from the thiosulphate citrate bile-salt (TCBS) agar medium. Of 79 Vibrio isolates were identified as V. parahaemolyticus through biochemical testing methods. In V. parahaemolyticus identification by detection of regulatory gene toxR, 51 (64.6%) of 79 isolates contained toxR gene while none of the isolates contained virulence gene of tdh dan trh. by targeting toxR gene which produced amplicons of 368 bp molecular weight. All V. parahaemolyticus isolates were negative for tdh and trh genes. Keywords: Vibrio parahaemolyicus, flower crab, toxR, trh, tdh

Isolation of Vibrio Parahaemolyticus from Beef Marketed in Padang, Identification Targeted on ToxR Gene and Amplification of TDH and TRH Genes on Isolates Using Polimerase Chain Reaction

Sebanyak 40 isolat V. parahaemolitycus telah berhasil diisolasi dari sejumlah sampel daging sapi mentah yang dikoleksi di Pasar Raya Padang. Isolasi dilakukan dengan menggunakan media CHROMagarTM Vibrio. Terhadap 40 isolat tersebut dilakukan identifikasi menggunakan metoda Polymerase Chain Reaction (PCR) untuk mengamplifikasi gen toxR. Gen toxR merupakan gen yang sangat spesifik pada spesies V. parahemolyticus. Selanjutnya, dengan metoda yang sama juga dilakukan amplifikasi terhadap gen pengkode produksi toksin hemolisin (tdh dan trh) yang merupakan faktor virulen utama pada bakteri tersebut. Hasil penelitian menunjukkan bahwa seluruh V. parahaemolyticus hasil isolasi memiliki gen tox-R, namun tidak ada satu isolatpun yang memiliki gen pengkode produksi toksin hemolisin baik tdh maupun trh.

Prevalence and Antimicrobial Resistance of Vibrio parahaemolyticus Isolated from Raw Shellfish in Poland

Vibrio parahaemolyticus is a marine bacterium recognized as an important cause of gastroenteritis in humans consuming contaminated shellfish. In recent years, increasing resistance to ampicillin and aminoglycosides has been observed among V. parahaemolyticus isolates. However, the first-line antimicrobials such as tetracyclines and fluoroquinolones remained highly effective against these bacteria. The aim of this study was to evaluate the occurrence of V. parahaemolyticus in live bivalve molluscs available on the Polish market and to determine the antimicrobial resistance of the recovered isolates. A total of 400 shellfish samples (mussels, oysters, clams, and scallops) from 2009 to 2012 were tested using the International Organization for Standardization standard 21872-1 method and PCR for the species-specific toxR gene. Antimicrobial susceptibility of the isolates was determined using a microbroth dilution method. V. parahaemolyticus was identified in 70 (17.5%) of the 400 samples, and the toxR gene was confirmed in 64 (91.4%) of these isolates. Most of the isolates were recovered from clams (31 isolates; 48.4% prevalence) followed by mussels (17 isolates; 26.6% prevalence). More V. parahaemolyticus–positive samples were found between May and September (22.7% prevalence) than between October and April (11.4% prevalence). Antibiotic profiling revealed that most isolates were resistant to ampicillin (56 isolates; 87.5%) and to streptomycin (45 isolates; 70.3%), but all of them were susceptible to tetracycline and chloramphenicol. Forty-one isolates (64.1%) were resistant to two or more antimicrobials; however, only one isolate (1.6%) was resistant to three antimicrobial classes. The antimicrobials used in treatment of human V. parahaemolyticus infection had high efficacy against the bacterial isolates tested. This study is the first concerning antibiotic resistance of V. parahaemolyticus isolates in Poland, and the results obtained indicate that these bacteria may pose a health risk to consumers.

IDENTIFICATION TARGETED ON toxR GENE AND DETECTION OF VIRULENT FACTOR ENCODING GENES ON Vibrio parahaemolyticus ISOLATED FROM RAW CHICKEN MEAT

A number of V. parahaemolyticus isolates has been isolated from raw chicken meat samples collected in Pasar raya Padang. Isolation was done using ChromagarTM Vibrio media. Identification of toxR gene was then done to these isolates. toxR is a very specific gene in V. parahaemolyticus species. Detection for the presence of genes encoding virulence factors, in this case were the gene encoding thermostable direct hemolysin (tdh) and TDH related hemolysin (trh) was performed on toxR positive V. parahaemolyticus isolates. Identification of toxR gene and detection of tdh and trh genes were done trough amplification using polymerase chain reaction PCR (method). The results showed that all of the tested V. parahaemolyticus isolates (22 isolates) had toxR gene, but none of isolates has gene encoding the production of virulence factors both tdh and trh.