Characterization of Pandemic O1 KUT Vibrio parahaemolyticus and Evaluation of Multiple-Locas Variable-Number Tandem Repeat Analysis (MLVA) for Clinical Strain Typing

Abstract

Vibrio parahaemolyticus is one of the important causative agents of gastroenteritis, especially 03 K6 serotype which is considered as a pandemic serotype disseminated worldwide. Recently, the outbreaks of V. parahaemolyticus infection occurred not only by 03:K6 serotype but also its serovariants, 01:K25 and 01:KUT (untypeable). However, little information of 01:KUT serotype has become available. In order to obtain more knowledge of 01:KUT isolates it is imperative to explore their characteristics, and evaluate the novel method to discriminate the strains. In this study, A total of 46 pandemic V. parahaemolyticus isolates (tdh gene positive, trh gene negative, GS-PCR positive) serotypes 01: KUT (n=32), 01:K25 (n=8), and 03:K6 (n=6) obtained from diarrhea patients in Hat Yai hospital, Songkhla during 2001-2012 were examined for their antimicrobial resistance profiles and virulence characteristics. Resistant towards ampicillin, ciprofloxacin and norfloxacin were found in all (100%), 7 (15%) and 1 (2%) isolates, respectively. Interestingly, more than 50% of the isolates were found to be intermediated susceptibility to ciprofloxacin and/or norfloxacin. The virulence-associated genes encoding type III secretion systems (T3SS1 and T3SS2), and type VI secretion systems (T6SS1 and T6SS2) were widely distributed among the isolates in all serotypes. All isolates were able to produce the similar level of thermostable direct hemolysin (TDH) on the Wagatsuma blood agar. Of 46 isolates, 21 (46%) and 15 (33%) displayed high level of swarming and twitching motilities, respectively. All isolates except one were able to use hemoglobin as the only iron source. More than 80% of the isolates were able to grow in iron-depleted medium under hemoglobin concentration as low as 50 μM. There was no difference in the antimicrobial resistance profiles and virulence characteristics observed among different serotypes. Due to the limitation of established K antisera, tracking the sources of KUT for epidemiological investigation is not available. Therefore, the effective molecular typing is required in order to discriminate the strains. This study demonstrated that arbitrarily primed PCR (AP2-PCR and AP4-PCR) and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) exhibited low resolution for typing among pandemic V. parahaemolyticus isolates. The discriminatory power of these methods ranged from 0.79, 0.30, and 0.24 for AP2- PCR, ERIC-PCR, and AP4-PCR, respectively. Pulsed-field gel electrophoresis (PFGE), a gold standard method, showed low ability to discriminate among isolates of the same serotypes. Most of the 01:KUT isolates obtained during 2001-2005 (n=12) showed similar PFGE profile. However, A minimum spanning tree (MST) based on PFGE revealed that PFGE profiles of all isolates were associated with their serotype and the period of strain isolation. In order to discriminate among all pandemic isolates, this study develops a multiplex multiple-locus variable-number tandem repeat (MLVA) assay for strain typing. The assay was based on the analysis of 4 variable number of tandem repeat (VNTR) loci. MLVA analysis of V. parahemolyticus isolates generated 38 distinct profiles whereas only 16 types were obtained from PFGE. High copy number variations of VNRT alleles in TR1, TR2, TR3, and TR4 loci were ranged from 5-44, 7-24, 5-44, and 7-38, respectively. The Nei's genetic diversity indices (DI) of the 4 VNTR loci ranged from 0.83-0.92. For stability study, some variations in TR2 were found after multiple subcultures and in stress conditions. In this work, MLVA resolved the 12 isolates of 01:KUT obtained in 2001-2005 with identical PFGE patterns into unique profiles. The multiplex MLVA developed in this study has higher discriminatory power (D=0.99) than PFGE (0.89), and is superior to PFGE for distinction pandemic V. parahaemolyticus including 01:KUT isolates. In addition, MLVA is more rapid than other typing methods used in this study. This study highlight the antimicrobial resistant and multiple virulence characteristics among pandemic V. parahaemolyticus isolates which will be useful for further epidemiological and clinical investigations of this organism. In addition, the MLVA typing technique developed in this study would significantly contribute to surveillance and outbreak investigation of pandemic V. parahaemolyticus.