The Diversity And Pathogenicity Of Burkholderia Cepacia Complex Isolates In Water Of West Lake And Other Environment In China

The bacteria of Burkholderia cepacia complex (Bcc) were commonly found in water bodies, hospitals and agricultural environments. They were known as phytopathogens and opportunistic human pathogens although some Bcc strains were considered as beneficial agents. It was very important to understand the Bcc species and their genetic diversity in water bodies, hospitals and some plants for utilization of the Bcc strains and environmental safety of Bcc in China. However, little information is available for Bcc populations in water bodies, hospitals and plants in China. A survey of distribution of Bcc species and seasonal variation during one year period were conducted in water of West Lake at present study. The Bcc strains from some hospitals in China were isolated and identified. The rotted fruit of apricot——a new plant disease caused by Bcc isolates was studied simultaneously. All Bcc isolates from water of West Lake, hospitals and apricot were compared for their genomovars, pathogenicity and virulence genes. As the intrinsic resistance of Bcc to antibiotics and the difficulty to control when infected by some Bcc strains, a new chemical for control of Bcc was explored in this study. The main results were as follows:(1) The cultured and uncultured-molecular methods were performed for examination of Bcc species and genetic variety in West Lake during one year by fixed locations and time for sample collection. The Bcc isolates from the lake were analyzed and compared with those recovered from hospitals and apricot. Total 776 putative Bcc isolates was recovered on PC AT selected medium and 309 of them were identified as Bcc by recA gene-specific PCR amplification, of which 265,42,2 were from water of West Lake, hospitals, apricot, respectively. The restriction fragment length polymorphism (RFLP) assay with enzyme HaeⅢwas performed to detect genetic variability among 309 Bcc isolates. The results showed that 7 different RFLP patterns (F, G, I, H, J, B, A) were found among 265 Bcc isolates from water of West Lake, and three different patterns (K, G, H) were also detected among 42 Bcc isolates from hospitals, the 2 phytobacterial strains of apricot belong to RFLP pattern H. It was the first report about Bcc recovered as apricot fruit pathogen. Our results revealed considerable variability among the Bcc isolates.(2) The identification of Bcc species was performed among 309 Bcc isolates from the water of West Lake, hospitals and apricot fruits by a combination of recA-HaeⅢRFLP assays, species-specific PCR tests, recA gene sequence analysis and multilocus sequence typing (MLST) scheme. Five species including B. multivorans, B. cenocepacia, B. stabilis, B. vietnamiensis and B. seminalis were recovered from the water of West Lake for the first time and B. cenocepacia was the dominant species from water of West Lake with 51% isolation frequecncy. Three species including B. cepacia, B. cenocepacia and Burkholderia contaminans were recovered from hospital environments and B. cenocepacia was also predominant with 73.8% of the total isolates tested. The phytobacterium of apricot fruit was identified as B. seminalis. It is a first report about B. seminalis recovered as phytobacterium. Our result showed that MLST scheme could not only differentiate the Bcc species but also distinguished the difference strains within a species.(3) The pathogenicity of 93 Bcc representatives recovered from the water of West Lake, hospitals and apricot fruit were evaluated. Seventeen of them were non-pathogenic while 76 isolates induced rot symptoms on onion bulbs in varying severity index, indicating these isolates were onion phytopathogens. B. multivorans and B. vietnamiensis didn't show protease activity in all 7 species tested, while most isolates of other 5 species showed protease activity. It was noted that the isolates from water of West Lake and apricot friut shared the same virulence factors with the isolates from hospital, indicating no significant difference in pathogenicity among them. B. cepacia, B. cenocepacia and B. contaminans isolates from hospital induced symptoms on alfalfa seedlings and B. cenocepacia isolates from water of West Lake did produce symptoms similar as formers, indicating that these isolates from water of West Lake may be potential human opportunistic pathogens. Virulence gene BCESM was detected from two isolates A8 and Y5 which had a high risk of the Bcc strains to human beings.(4) The susceptibility of 29 Bcc representatives recovered from the water of West Lake, hospital and apricot to 4 antibiotics (Cefoxitin, Tetracycline, Polymycin B sulfate, Vancomycin) and chitosan solution was evaluated. The Bcc isolates tested were shown resistant to these 4 antibiotics, but susceptible to chitosan solution. Chitosan was considered the best effective one for Bcc inhibition in this study and could be served as a good candidate for novel antimicrobial agents against Bcc infections in CF patients and plant diseases.