| Vibrio parahaemolyticus is a Gram-negative halophilic bacterium widely found in estuarine and marine environments, which causes gastroenteritis and traveler's diarrhea through consumption contaminated seafoods. The main virulence factors of V. parahaemolyticus includes thermostable direct haemolysin (TDH), TDH-related toxin (TRH), thermolabile haemolysin (TLH), two type III secretion systems, which play distinct roles in cytotoxicity and enterotoxicity, respectively.Serotyping of V. parahaemolyticus isolates has identified more than 13 O antigen groups and 71 K antigen types. Before 1995, V. parahaemolyticus associated gastroenteritis was caused by many different serogroups. Since 1996, so-called"pandemic clones,"the majority of which belong to serotype O3:K6, have caused worldwide outbreaks of gastroenteritis. Previous the pulsed field gel electrophoresis (PFGE), arbitrarily primed–PCR (AP-PCR) and multilocus sequence typing (MLST) studies revealed that the new O3:K6 and its derivates (O4:K68,O1:K25 and O1:KUT ) gave very similar fingerprint patterns or sequence types (STs), suggesting that they constitute a clonal complex. These strains are collectively called the 'pandemic group' that is thought to be responsible for the pandemic outbreaks. Extraction of high-purity genomic DNALipopolysaccharide is the major component of the outer membrane in Gram-negative bacteria, and some of these bacteria produce two other kinds of polysaccharide as well, i.e., exopolysaccharide and capsular polysaccharide. V. parahaemolyticus expresses abundant exopolysaccharides, which is essential for its environmental survival and transmission by providing access to nutrients and protection from predators and adverse natural environmental conditions. Polysaccharides are problematic for DNA extraction from mucoid bacteria. In the present study, mucoid V. parahaemolyticus RIMD2210633 and K. pneumonia NTUH-K2044 were used as the model organisms, and Escherichia coli JM109 as control. Two methods were established for extracting genomic DNA from mucoid Gram-negative bacteria.In this study, genomic DNA was extracted with the three methods. Method I is a classical phenol/chloroform extraction approach. Methods II or III was modified from the method I by introducing methoxyethanol or ethyl ether for removal of polysaccharides, respectively. Experiments were performed in triplicates. The yields of DNA obtained from the three methods were different and method I got the highest yields. Polysaccharide removal procedures in methods II and III significantly reduced the yields of DNA. Electrophoresis assay of DNA samples indicated that DNA bands were clear and clean, and no signs of RNA smears and of degraded DNA were observed. In this study, the OD260/OD280 and OD260/OD230 ratios were calculated for each DNA sample to evaluate the three DNA extraction methods. All the three methods gave the OD260/OD280 values between 1.8 and 2.0 for all the three bacteria, indicating the relevant DNA samples were free of protein contamination. Both methods II and III gave the OD260/OD230 values≥2.0 for all the three strains, while the method I gave the values≥2.0 for only non-mucoid JM109. For the DNA samples isolated from RIMD2210633 and NTUH-K2044 using the method I, the OD260/OD230 values were less than 1.4 indicating severe of polysaccharides in them.In the present work, we established two reproducible and cost-effective methods for routine genomic DNA extractions from mucoid Gram-negative bacteria. And the two methods could also be applied to mucoid Gram-positive bacteria if lysozyme (e.g. lysostaphin specific for Staphylococcus) was used in the cell lysis step. Large variably-presented gene cluster(LVPC) -based genotypingTwo hundred and fifty one V. parahaemolyticus isolates were tested in this study, including193 clinical isolates and 58 non-clinical ones. From our previous M-GCH study, we indentified 18 LVPC. Single gene was chosen from each LVPC, and PCR assays were set up to screen for the presence (1) or absence (0) of each LVPC within the 251 strains tested. A total of 48 different LVPC profiles were identified.According to the binary LVPC data, we constructed an unweighted pair-group method with arithmetic means(UPGMA)-based Minimum Spanning Tree (MS tree) that depicted the phylogentic structure of the 251 strains. Based on the MS tree structure in conjunction with the frequency profiles of virulence markers, these 251 strains could be assigned into six complexes, LVPC-C1 to LVPC-C6. Notably, the MS trees of LVPC and M-CGH shared a similar topologic structure. In addition, LVPC-C1 to LVPC-C5 generally corresponded to the five M-CGH complexes (C1 to C5), respectively. LVPC-C6 contained only a single strain S093 (GS-PCR+, tdh-, T3SS2α-, trh- and T3SS2β-), which was not assigned to any complex in the M-CGH study.All the 14 LVPC-C2 strains were the pre-1996 ?old'O3:K6 ones (GS-PCR-, tdh-, T3SS2α-, trh+ and T3SS2β+); and12 of them were characterized previously by MLST to constitute a clonal complex namely the old-O3:K6 clone. All the 63 pandemic strains (GS-PCR+, tdh+, T3SS2α+, trh- and T3SS2β-) tested were assigned into the LVPC-C3 complex . S093 was most likely a phylogenetic intermediate between the pandemic and old-O3:K6 clones. We proposed it as the ?intermediate-O3:K6 clade'. In addition, the genetic relationship in the LVPC MS tree herein supported the proposed phylogenetic relationship between the old-O3:K6, pandemic, and intermediate-O3:K6 groups. Variable number of tandem repeat(VNTR)-based genetypingA total of ten VNTR loci were tested in our strain collection. VPTR7 gave negative PCR amplification for 184 of the 251 strains, and was thus discarded from further VNTR assay.The rest nine VNTR loci were polymorphic among the 251 strains. VP2-07 and VP1-10 gave the highest and lowest allelic diversity, respectively.The categorical VNTR dataset of the 251 strains was directly analyzed to build a MS tree. The 251 strains were subdivided into 222 distinct genotypes. Only the strains from LVPC-C2 or LVPC-C3 were grouped together in the VNTR MS tree of the 251 strains, while those from LVPC-C1, LVPC-C4, and LVPC-C5 showed scattered distribution in the MS tree.Overall, the VNTR-based genotyping method had a much higher resolution than the LVPC-based one in discriminating the V. parahaemolyticus strains. Although certain VNTR loci seem to mutate too rapidly to be useful in determining global phylogenetic relationships, they are useful for strain identification and may identify rapidly evolving polymorphisms that are useful for discriminating very closely related strains, such as V. parahaemolyticus serotype O3:K6 strains. Two-step genotype approachAn optimal typing method should provide the ability to distinguish among both distantly and closely related strains. Thus, multiple methods are often required to adequately discriminate among closely related isolates. In fact, each of the methods utilizes different genetic markers to differentiate different strains, and each method has its weaknesses that can be complemented by another. Given the disadvantages and advantages of both LVPC- and VNTR-based genotyping methods, we proposed the combined used of LVPC and VNTR. For this purpose, PCR-screening of 18 LVPCs and five virulence markers (GS-PCR, tdh, T3SS2α, trh and T3SS2β) should be carried out for the test strains, which will primarily group the strains into various complexes with different pathogenicity characteristics. The strains from each LVPC-based complex would be further analyzed with the nine VNTRs loci, as the VNTR-based genotyping worked well to give a much more detailed discrimination of the strains.The VNTR-based method was applied to 251 V. parahaemolyticus isolates that were genotyped previously by LVPC-based method. The isolates of V. parahaemolyticus belonging to the same LVPC complex were distinguishable by the size variations in the 9 informative VNTRs loci. The LVPC-C1 gave 34 VNTR allelic profiles, the LVPC-C2 gave 13, the LVPC-C3 gave 55, the LVPC-C4 gave 70, and the LVPC-C5 gave 49 profiles respectivly.This combined scheme could be used to construct a genetic fingerprint-like database based on a large collection of global strains, which would be very useful for identification, genotyping, source-tracking, and risk evaluation of V. parahaemolyticus. |
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