| Non-O1/O139 Vibrio cholerae naturally presents in aquatic ecosystems, especially in the water environment and seafood, and may cause sporadic cholera-like diarrhea and local outbreaks. However, up to now, there have no molecular detection method for non-01/0139 V.cholera. The distribution of virulence associated genes, and genetic relationship among aquatic isolates are largely undetermined. In view of the above issues, we carried out a series of studies on non-O1/O139 V. cholera on the following aspects.Firstly, we developed the methodology of both multiple PCR and real-time SYBR green PCR for the detection of non-01/0139 V. cholera. According to the specific electrophoretic bands(multiple PCR) and the specific melt curve temperature (real-time SYBR green PCR), both methods could specifically detect the non-01/0139 V.cholerae, and to differentiate them from O1,O139 V. cholera, other five Vibrios and 3 intestinal bacterias. The detection limits were 7X104 cfu/ml (multiple PCR) and 7×102 cfu/ml (real-time SYBR green PCR), with statistically significant difference seen (P<0.05). For the reproducibility of real-time SYBR green PCR, the external coefficient variation ranging from 0.22% to 0.92%, while the internal coefficient variation ranging from 0.27% to 1.41%.370 strains of non-01/0139 V. cholerae, were detected, with both consistency rates as 100%.Secondly, a total of 295 aquatic isolates of V. cholerae non-01/0139 serogroups, isolated from different regions in China were investigated for virulence genes. Only one isolate was toxigenic (ctxB+) and harboring a rare ctxB genotype; ten (3.4%) isolates carried several rare types of pre-CTX prophage (ctxB- but rstR+), of which eight (2.7%) isolates carried several rare types of toxin-coregulated pili subunit A (tcpA); meanwhile,16 (5.4%) isolates carried intact (partial ORFs) of Vibrio seventh pandemic island ? (VSP-?) or VSP-? clusters, which were further identified as thirteen novel types. PCR-based analyses revealed remarkable variations in the distribution of putative accessory virulence factors, including mshA (95.6%), hlyA (95.3%), rtxC (89.8%), rtxA (82.7%), IS1004 (52.9%), chxA (30.2%), SXT (15.3%), type ? secretion system (TTSS,18.0%), NAG-ST (3.7%), respectively. No correlation between the prevalence of putative virulence genes and CTX prophage or TCP genes, whereas there were correlation among the putative virulence genes.Finally, Multilocus sequence typing (MLST) placed selected isolates (n=70) into 70 unique sequence types (STs), which different from that of toxigenic O1 and 0139 counterparts, and scattered into the different position of the MLST tree. In summary, the V.cholerae non-01/0139 aquatic isolates predominant in China presence high degree of genotypic diversity, while the presence of diverse CTX prophages and other virulence factors amongst environmental non-01/0139 constitute a reservoir of potential pathogenic isolates, which may cause to either sporadic or outbreak of cholera-like diarrhea.Aeromonas can infect human and animals by food chain causing diarrhea and food poisoning to endanger peoples'health. But the research on Aeromonas is not relatively enough, large quantities of aquatic animals die because of not identifying pathogency quickly and correctly, and not taking effective prevention and cure measures in time. In recent years, diarrhea caused by aeromonas has become a problem that can not be ignored. There is no a molecular method for Aeromonas to correctly be classified at the species level, the distribution of virulence associated genes, especially the differences between environmental and clinical isolates is not clear, resistance patterns of Aeromonas is not explicit. Aimed at this practical problem, a total of 257 stains isolated aquatic environment (n=64) and patients (n=193) in our laboratory was studied. So we can proven the molecular epidemiology characters of Aeromonas strains comprehensively, which will provide scientific evidences for surveillance, prevention and control of Aeromonas infection, accumulate the data of molecular biology characteristics of Aeromonas.Firstly, PCR tests aimed at the seven housekeeping genes gyrB-rpoD-recA-d naJ-gyrA-dnaX-atpD were carried out and PCR products were sequenced. By a multilocus phylogenetic analysis, we classified the Aeromonas at species level, compared the difference between a single genotype level and MLPA level, and found, the results of rpoD and MLPA were almostly identical, all strains were further identified as ten species, the most commonly species were A. veronii (42.5%) and A. caviae (37.5%) from stools and water, respectively. Notably,5 strains can't be classified, this suggested that they might be some new specie-s.Meanwhile, all strains were tested of eleven potential virulence genes via PCR, Eleven virulence genes were presented at following:Elastase (85.9%), fla (66.5%), act (64.5%), lip (60.3%), ast (34.2%), hlyA (33.1%), alt (31.5%), ahp (29.5%), laf (12.8%), aer (10.5%), ASCF-G (10.1%). Five genes(Elastase, Lipase, ast, alt and laf) were presented significant different between stools and water (P<0.05), the most commonly combinations of virulence genes were Fla/Ela/ast/act (8.6%) and Fla/Lip/Ela/act/ahp (8.2%).Furthermore, all strains were resistant to twenty-four tested antibiotics in different degree, the resistant rate of five antibiotics (ampicillin, amoxicillin clavulanic acid, erythromycin, cefalotin and nalidixic acid) accounts for over 50% (55.3-89.1%), notably, the resistant rates of nine antibiotics from water were significantly higher than that of stools (P?0.003), in contrast, only two antibiotics (amoxicillin clavulanic acid and ampicillin) from stools were significantly higher than that of aquatic water. Further,250 strains contained 193 multiple antibiotics resistance (MAR) patterns, the MAR rate of over 10 (including) antibiotics account for 8.6% (16/187) from stools and 30.2%(19/63) in water. This study emphasizes the multifactors in the pathogenesis of Aeromonas, and environmental Aeromonas has acquired a wide range of MAR before entering into human bodies. |
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