Evaluation of Typing of Vibrio parahaemolyticus by Three PCR Methods Using Specific Primers

Abstract

Vibrio parahaemolyticus is a halophilic bacterium frequently involved in human outbreaks of seafoodassociated gastroenteritis. For epidemiological purposes, different molecular typing methods, such as pulsedfield gel electrophoresis (PFGE) or ribotyping, have been developed for this pathogen; however, these methods are mostly labor-intensive and time-consuming. In this work, we designed and evaluated three rapid PCR typing methods for this pathogen using primers designed on the basis of the following specific sequences: conserved ribosomal gene spacer sequence (RS), repetitive extragenic palindromic sequence (REP), and enterobacterial repetitive intergenic consensus sequence (ERIC). Typing patterns and clustering analysis indicated that these methods apparently differentiated V. parahaemolyticus strains from reference strains of interspecific Escherichia coli, V. cholerae, and V. vulnificus and were also valuable in subspecies typing of this pathogen. Forty domestic strains of V. parahaemolyticus, representing a wide range of PFGE patterns, were grouped into 15, 27, and 27 patterns, with discrimination indexes of 0.91, 0.97, and 0.98, by RS-, REP-, and ERIC-PCR, respectively. The discriminative abilities of these PCR methods closely approached or even exceeded those of PFGE and ribotyping. REP-PCR is preferable to ERIC-PCR because of the greater reproducibility of its fingerprints, while RS-PCR may be a practical method because it generates fewer amplification bands and patterns than the alternatives.Vibrio parahaemolyticus is a halophilic gram-negative bacterium that causes acute gastroenteritis in humans. Food poisoning caused by this pathogen is generally associated with the consumption of contaminated seafood; this organism is a crucial food-borne pathogen in Taiwan, Japan, and other coastal countries with high rates of seafood consumption (17). Clinical manifestations include diarrhea, abdominal cramps, nausea, vomiting, headache, fever, and chills, with incubation periods ranging from 4 to 96 h (4, 9).Isolates of V. parahaemolyticus can be differentiated by serotyping, and 13 O groups and 71 K types have been identified (7). Serotyping is generally unable to differentiate all isolates originating from different regions or sources. Reliable molecular methods for strain typing would significantly aid epidemiological investigations. Recently, several molecular methods were developed for the subspecies typing of V. parahaemolyticus, namely, pulsed-field gel electrophoresis (PFGE) (33), ribotyping (29), and random amplified polymorphic DNA (RAPD) analysis (30). The PFGE method using SfiI digestion is reliable, achieves high discrimination efficiency, and has been applied to typing of V. parahaemolyticus strains in many situations, such as the first pandemic O3:K6 strains (32), food poisoning outbreaks (28), environmental strains from seafood (31), and nosocomial outbreaks (12). However, the whole process takes several days to complete. Compared with PFGE, RAPD analysis has the merits of being less labor-intensi...

Facilitated Molecular Typing of Shigella Isolates Using ERIC-PCR

“…The technique requires no specialized equipment or reagents in a laboratory with PCR capabilities. Because the number of organisms that are successfully typed with ERIC-PCR continues to grow to include diarrheogenic 22 and uropathogenic E. coli, 23 Pseudomonas aeruginosa, 24 Aeromonas hydrophila, 25 Vibrio parahemolyticus, 26 Stenotrophomonas, 24 Klebsiella pneumoniae, 27 Yersinia enterocolitica, 28 Bartonella henselea, 29 and Helicobacter pylori, 20 the advantage of adopting this technique at the hospital and regional level also increases. Experience with this simplified technique allows for the preliminary investigation of many important nosocomial or regional outbreaks without the need for a disease-specific approach after primary culture.…”

Random Amplified Polymorphic Dna-PCR Typing of Vibrio parahaemolyticus Isolated from Local Cockles (Anadara granosa)

“…A rapid typing method based on the random amplification of polymorphic DNA segments (RAPD-PCR) has been described [10,11] . This method has been successfully used in the characterization of several organisms including V. parahaemolyticus and other vibrios [1,[12][13][14] . In this study, a library of 10 primers (50% G+C) was examined for suitability.…”

Revisión y recomendaciones para el manejo de diarrea por Vibrio parahaemolyticus

“…Actualmente existen kits comerciales que reconocen 13 grupos O y 71 tipos K. En relación a los métodos moleculares, existe una gran variedad, destacando la determinación de genes tdh y trh por RPC, de gran utilidad para diferenciar cepas patógenas de las no patógenas, ya que ambos genes representan los mayores factores de virulencia de V. parahaemolyticus 18,23 . Para la determinación de genotipos se puede realizar electroforesis de campo pulsado (PFGE) [24][25][26][27] , ribotipificación y diversas técnicas basadas en amplificación como RPC con partidores arbitrarios (AP-PCR), amplificación de elementos palindrómicos extragénicos repetitivos (REP-PCR), amplificación de secuencias intergénicas de consenso repetitivas (ERIC-PCR) [26][27][28][29][30][31] , etc. La PFGE representa la técnica de tipificación genética de elección por su poder discriminatorio, estandarización y capacidad de almacenamiento de los distintos patrones genéticos en bases de datos.…”

“…En febrero de 1996 en Calcuta, India, se detectó por primera vez un nuevo clon de V. parahaemolyticus perteneciente al serotipo O3:K6, TDH positivo, TRH negativo, siendo responsable de un gran aumento de los casos de enfermedad diarreica en esa zona 29 . Posteriormente este clon pandémi-co 25,28,[29][30][31][32][33][34] ha sido el responsable del aumento de casos en otras regiones de Asia, llegando incluso a Norteamérica y finalmente a Chile, donde se ha detectado su presencia desde el año 1998; desde entonces ha sido el responsable de todos los grandes brotes ocurridos 35 (comunicación verbal ISP). El fundamento de la expansión de este clon permanece sin dilucidar, puesto que su nivel de producción de TDH y su susceptibilidad antimicrobiana no es distinta a la de otras cepas patógenas de V. parahaemolyticus.…”