| Objective The genus Francisella a group of non-motile, Gramnegative, strictly aerobic,facultatively intracellular coccobacilli found in all kinds of environments and associated with disease in both vertebrates and invertebrates. To date, it has been known that as many as 100 species of animals can be readily infected by the bacterium. Tularemia is one of the severe zoonotic diseases and has high fatality rate.Thus, Francisella tularensis, the causative agent of Tularemia, is known to be one of the most infectious pathogenic bacterium, Due to the diverse routes of dissemination and transmission and its high virulence, Francisella tularensis is defined as A category of bioterrorism agent by America Center for Disease Control and Prevention.Now there are six acceptable nominal species and six subspecies, in addition to Francisella tularensis, the study revealed that the F. philomiragia and F. hispaniensis are also associated with human disease, but the bacterial cultivation is so difficult that the very few domestic research are achieved. This investigation is aimed to establish a reliable and efficient procedure to isolate and identify the Francisella strains obtained from environmental water sampling; to analyze the genotypic distribution of the bacteria in the different environmetnal water of China; to create the phylogenetic linkage of thebacterial isotypes.Methods The drugs sensitivity analysis of Francisella bacteria and selectivity of self-made selective medium.The type strain F.philomiragia and F.guangzhouensis and Legionella pneumophila saved in laboratory was revived to reserve, the drug sensitivity tests of three strains were tested by micro-dilution method, the interpretation on the result of drug sensitivity tests was based on CLSI M100-A24.The selectivity of self-made selective medium(BCYEα-GVPCC medium and CHAB-PACCV medium) was tested by observe and analyse the growth of the rests of bacterium and fungus collected in laboratory, then make comparison between BCYEα-GVPCC medium and CHAB-PACCV medium to screened out the better one.Acid resistance in F. philomiragia and F.guangzhouensisCell suspensions were centrifuged and resuspended in one of three KCL-HCL(p H=2.2) solutions, and make an investigation and contrastive analysis of the growing states of F. philomiragia and F.guangzhouensis on two mediums, the data was used toestimate the probability of survival states,To evaluate whether the self-made medium is susssuccessful in isolating the target bacteria from simulation and environmental water.First, simulation water sample containing two standard strains are filter concentrated, then mix with acid solution(1:1), plated on two mediun(CHAB-PACCV BCYEα-GVPCC) respectively after 15~20 minutes, to observe whether it successfully isolated the target strains.Phylogenetic analyses of the 16 S r DNA and housekeeping genes(sdh A、mdh、rpo B) of 19 isolates, and to study the diversity of Francisella isolated from environment water in China.Applying DNA-DNA hybridization technology to decide weather the possible species is true.Results The inhifertbition zone of F. philomiragia, F.guangzhouensis and Legionella pneumophilatoare is respectively 6mm, 6mm and 38 mm, according to CLSIM100-A24, F. philomiragi and F.guangzhouensis are resistance to Cefepime,on the contrary, Legionella pneumophilatoare show sensitive to Cefepime. Two kinds of culture medium on basic no bacterial growth or there is very little.In acid solution(p H = 2.2), Francisella can be able to stay the activity unchanged up to 20 min, once the time surpassed 20 min, its activity will significantly reduce.Two kinds of medium both can be able to successfully isolate target bacteia from s simulationwater and environmental water, the colonies of target bacteia presented smaller on BCYEα-GVPCC than CHAB – PACCV, the counts of contaminating microorganism is also less. 19 isolates were stored at-80℃.Phylogenetic analyses based on 16 S r DNA and housekeeping gene sequences indicated that 19 isolates consisted of 11 strains of F. philomiragia, 7 strains of unclassified Francisella species, 1strain of Candidatus Nebulobacter yamunensis,mainly distributed in sea water(47.37%, 9/19), central air conditioning water(26.32%, 5/19), brackish water(21.05%, 4/19), rarely distributed in fresh water(5.26%, 1/19).The DNA-DNA relation between possible species(10HL1970, 10HP457,10HP82-10, 10HL1938)and 08HL01032 is respectively 25.59%,29.26%,25.61%,31.54%.Conclusion There is difference about the sensitivity to Cefepime between Francisella and Legionella, and it can be used to distinguish with each other; the selectivity of the self-made medium with Cefepime is better. Francisella present a certain amount of resistance to acid,it can be able to stay the activity unchanged up to20 min in acid solution(p H = 2.2).BCYEα- GVPCC and CHAB- PACCV medium can be used to isolate Francisella from environmental water, the former is relatively near their optimal choice.F. philomiragia is the key components of Francisella isolated from environment water in china. At present, there are few scholars to study Francisella and a large amount of new specices waiting to discover.10HP457, 10HP82-10, 10HL1970, and 10HL1938, the four strains is highly similar to 08HL01032, were onfirmed as the novel Francisella by DNA-DNA hybridization technology, and temporarily called as Francisella frigidaquae based on information of the sample. |
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