Multiple-Locus Variable-Number Tandem-Repeat Analysis for Clonal Identification of Vibrio parahaemolyticus Isolates by Using Capillary Electrophoresis

Epidemics of Vibrio parahaemolyticus in Chile have occurred since 1998. Direct genome restriction enzyme analysis (DGREA) using conventional gel electrophoresis permitted discrimination of different V. parahaemolyticus isolates obtained from these outbreaks and showed that this species consists of a highly diverse population. A multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) approach was developed and applied to 22 clinical and 91 environmental V. parahaemolyticus isolates from Chile to understand their clonal structures. To this end, an advanced molecular technique was developed by applying multiplex PCR, fluorescent primers, and capillary electrophoresis, resulting in a high-resolution and high-throughput (HRHT) genotyping method. The genomic basis of this HRHT method was eight VNTR loci described previously by Kimura et al. (J. Microbiol. Methods 72:313-320, 2008) and two new loci which were identified by a detailed molecular study of 24 potential VNTR loci on both chromosomes. The isolates of V. parahaemolyticus belonging to the same DGREA pattern were distinguishable by the size variations in the indicative 10 VNTRs. This assay showed that these 10 VNTR loci were useful for distinguishing isolates of V. parahaemolyticus that had different DGREA patterns and also isolates that belong to the same group. Isolates that differed in their DGREA patterns showed polymorphism in their VNTR profiles. A total of 81 isolates was associated with 59 MLVA groups, providing fine-scale differentiation, even among very closely related isolates. The developed approach enables rapid and high-resolution analysis of V. parahaemolyticus with pandemic potential and provides a new surveillance tool for food-borne pathogens.Food-borne infections by Vibrio parahaemolyticus cause gastroenteritis, which is the most common clinical manifestation (38). An increasing number of V. parahaemolyticus infections and outbreaks caused by strains belonging to a pandemic clonal complex have been observed throughout the world since 1996 (2, 6, 9, 12, 13, 31, 32, 36, 40). Epidemics of Vibrio parahaemolyticus in Chile have occurred since the summer of 1998 and were caused by the pandemic clone O3:K6 that had emerged in Southeast Asia in 1996 (12, 13, 15). However, this strain was only a minor component of a highly diverse V. parahaemolyticus population in shellfish, as demonstrated by an improved method for restriction enzyme analysis, using total bacterial DNA, named direct genome restriction enzyme analysis (DGREA), in combination with conventional gel electrophoresis (12). This method has a discrimination index similar to that of restriction fragment length polymorphism-pulsed-field gel electrophoresis (PFGE) (12, 13, 19).A variety of molecular typing methods have been applied to V. parahaemolyticus, such as ribotyping (3, 10, 14), PFGE (3, 30), group-specific PCR (32), arbitrarily primed PCR (18, 32, 36), and multilocus sequence typing (7, 16). The use of DGREA permitted discrimination of different V. parahaemolyticus Chilean isolates and showed that these bacteria consist of a highly diverse population comprising at least 23 different genotypic groups among the environmental isolates obtained from shellfish and 5 different groups of clinical isolates (19).Epidemiological analyses of infections caused by pathogenic bacteria depend on the accurate identification of strains, preferably at the clonal level. Variable-number tandem repeats (VNTRs) comprising short sequence repeats constitute a rich source of genetic polymorphism and have been used extensively as markers for discrimination between strains of many different bacterial genera (27, 46). VNTRs have been used to discriminate among individual strains within several food- or waterborne pathogens with little genetic variation, including Escherichia coli O157:H7 (25, 35), Pseudomonas aeruginosa (37), Staphylococcus aureus (41), and Salmonella enterica subsp. enterica serovar Typhimurium (26), and to characterize other important human pathogens, such as Neisseria meningitidis (42), Listeria monocytogenes (28), Legionella pneumophila (34, 39), Leptospira interrogans (43), and Mycobacterium tuberculosis (45). VNTR loci have even been found in genetically highly homogenous pathogens, such as Bacillus anthracis (1, 21, 29). Multiple-locus VNTR analysis (MLVA) is defined as the analysis of a set of loci spread throughout the bacterial genome (23). Individual strains within a bacterial species often maintain the same sequence elements but with different copy numbers due to variations introduced by slipped-strand mispairing during DNA replication (33).Recently, a study of the polymorphism of tandem repeats in V. parahaemolyticus showed the utility of the MLVA approach for characterizing recently emerged and highly homogeneous pandemic strains of serotype O3:K6 (22). These authors reported a scheme of eight genomic VNTR loci, comparing PFGE results for clinical strains of V. parahaemolyticus serotype O3:K6. The study by Kimura et al. (22) comprised only strains of serogroup O3:K6 and used conventional gel electrophoresis to evaluate VNTRs. In epidemiological studies, a more rapid technique is needed for mass application of MLVA that also provides improved resolution and has been validated for nonserogroup O3:K6 isolates. Capillary electrophoresis has become the preferred technology to improve resolution and accuracy in bacterial VNTR analysis due to the availability of multiple fluorescent labels and better accuracy and reproducibility (27).In our study we describe the use of an improved MLVA for discriminating genotypically a diverse collection of clinical and environmental V. parahaemolyticus isolates from Chile. These very closely related isolates have been analyzed and grouped by DGREA previously (12). To this end, we developed and applied multiplex PCR of 10 VNTR loci, tagged with multiple fluorescent dyes, and analyzed the amplicons by capillary electrophoresis. The results demonstrated that MLVA typing is able to distinguish between V. parahaemolyticus isolates that have different DGREA patterns and isolates that belong to the same group, allowing accurate sizing of amplicons by assignment of the fragment size. Validation of this typing method with 113 Chilean isolates demonstrated the utility of this technique also for nonserogroup O3:K6 clinical isolates, thereby providing a new tool for the study of the molecular epidemiology of V. parahaemolyticus.