The Identification And Characterization Of Francisella Isolated From Environmental Water Sample Of Salty Sources

ObjectivesTo isolate and culture Francisella-like strains from environmental water sample of salty sources. Identify and determine the taxonomic position of the suspected strains. Study the microbiological characteristics of the suspected strains and provide the basis of identifying Francisella strains in clinical.Methods(1) Water separation:The environmental water sample of salty sources were trested with concentrated acid and antibiotic tablet of BCYE-GVPCC. And then choosed the gray and translucent bacteria colony for preliminary identification.(2)Preliminary identification:The Francisella-like isolates;were tested whih catalase test and gram staining microscopy. The weakly catalase positive and comprises tiny gram-negative cocco-bacilli isolate was preliminary determinated the phylogenetic position by 16S rRNA sequencing analysis.(3) Phlyphasic taxonomy study:Inculding 16S rRNA gene phylogenetic analysis, characteristics with housekeeping genes (sdhA gene, rpoB gene and mdh gene), phenotypic, physiological and biochemical characteristics (API ZYM, API Rapid ID 32 A and API NH, drug sensitive test, DNA(G+C)mol%, whole-cell fatty acid, the type of quinone, lipid phosphate, optimum growth temperature test and virulence experiment).(4) Francisella characteristics analysis:Comprehensive analysed the Francisella characteristics and identified with the other gram-negative bacteria in clinical.Results(1) Water separation:There were 184 environmental water samples of salty sources were collected,15 samples were positive, coveraged the positive rate of 8.15%.(2) Suspected strains:There were ten isolates (except the strains which failed to save)which weakly catalase positive and comprises tiny gram-negative cocco-bacilli. Including nine isolates which from salty water:eight isolates (QH-2, QH-3、QH-4、QH-5、QH-12、QH-15、HN-3、YJ-2、D3-2) were identified as Francisella philomiragia, one isolate (D3-2) was identified as new genus of strain, one isolate (WZ-1) which isolated from the blood of pneumonia patient who comed from Wenzhou was identified as Francisella. novicida-like strain.(3) Phlyphasic taxonomy study:①16S rRNA gene phylogenetic analysis:Eight isolates (QH-2、QH-3、QH-4、 QH-5、QH-12、QH-15、HN-3、YJ-2) were located in the same branch. WZ-1 and F. novicida, F. tularensiswere located in the same branch. D3-2 was itself located in the one branch, had a faraway genetic relationship with the other Francisella strains.②haracteristics with housekeeping genes:Ten isolates were amplificated to a length of about 300bp DNA fragment of sdhA gene, a length of about 330bp DNA fragment of rpoB gene,a length of about 750bp DNA fragment of mdh gene(except D3-2).③Phenotypic characteristics:The ten Francisella-like isolates were well growing on buffered charcoal yeast extraction agar (BCYE a), but delayed growth on blood agar, with a common dependency on the amino-acid cysteine for growth. They were gram-negative coccobacilli with no flagellu and power. The colonies on the ager were pale, gray, translucent.④Pysiological and biochemical characteristics:Ten isolates were tested weakly catalase positive, positive for oxidase, negative for indole (except WZ-1). Characteristics of enzymes included alkaline phosphatase, acid phosphatase, esterase(C4), lipid esterase(C8), naphthol-AS-BI-phosphoric acid hydrolase, ArgA, AlaA, HisA, PEN, GLU, FRU, SAC. The test showed the ten isolates were sensitived to gentamicin, tetracycline, doxycycline, ciprofloxacin, levofloxacin, chloramphenicol. DNA(G+C)mol%of WZ-1 was 33.30%, and D3-2 was 38%. The main fatty acids of WZ-1 were C10:0(16.75%)、C14:0(9.79%)、C16:0(18.87%)、C16:0 3-0H(8.89%)and C18:1ω9c(11.16%), coveraged the 65.46% of the whole-cell fatty acid. The D3-2 were C16:0(5.38%)、C18:0 (20.43%)、anteiso-C15:0(34.40%)、iso-C16:0 (5.92%), anteiso-C17.0(10.73%), coveraged the 76.86% of the whole-cell fatty acid. The mager types of quinone of WZ-1 were Q8(66.89%) and Q9(33.11%) and the D3-2 was Q8(100%). The mager lipid phosphates of WZ-1 were DPG、PE、PG、PC、 PL1、ALF2、UL and the D3-2 were PL、DPG、PE、PG、PC、ALP、AL. The preliminary virulence experiment of WZ-1 showed LD50 was nearly 107CFU/mL, LD100 was nearly 109CFU/mL. We couldn’t finish the test because of the experiment conditions. The optimum growth temperature of both of the D3-2 and WZ-1 were 35℃ and didn’t grow at 42℃.⑤Isolate D3-2 was named as coccobacilli, acid bacillus coccodibacilli, and isolate WZ-1 was named as Francisella wenzhouensis.(4) The Francisella colonies on the ager were pale, gray, translucent. Growing without X/V factor, but L-cysteine could promote the bacteria growth. Francisella is gram-negative coccobacilli with no flagellu and power, weakly catalase positive, positive for oxidase, negative for indole. DNA (G+C) mol%is about 32%～34%. Fatty acids characters are even chain saturated fatty acids (C10:0, C14:0 and C16：0) and two long chain hydroxy acids (C16:0 3-OH和C18:0 3-OH) The main quinone character is Q8. The main lipid phosphates are DPG、PG、PC、 PE、PL1、PL2、PL3、APL2、APL3. The similar gram-negative coccobacilli to Francisella in clinical include Brucella, Bartonella, Acinetobacter, Sixuegan-junsu, Yersinia pestis, Pasteurella multocida. It can be identify with plate culture characteristics, gram stain microscopy, oxidase test, urease test, catalase test, the effect of X/V factor or cysteine on the growth of bacteria, dynamic test, main fatty acid composition and other aspects of the identification.ConclusionsFrancisella strains are distributed in the environmental water samples of salty sources,it is difficult to identify by using traditional methods.It is successfully to identify and determinate the phylogenetic positions of Francisella strains by using 16S rRNA gene sequencing analysis and phlyphasic taxonomy study. It could identify the similar strains with microbiological characteristics of bacteria in clinical.