Polyphasic Taxonomy Of Francisella-like Strains Isolated From Air Condition Systems And Establishment Of A Novel Genus Of Allofrancisella

ObjectiveThe Francisella are facultative intracellular pathogen and tiny Gram-negative cocco-bacilli. F. tularensis, the type species of the genus Francisella, is known to be highly pathogenic form and regarded as potential bioterror bacteria. Human infections caused by F. tularensis occur via inhalation of infectious aerosols, the other species are often regarded as environmental and non-pathogenic bacteria or opportunistic pathogens. In recent years, the study found, Francisella are widely existed in the nature of fresh water, salt water and soil. The cooling water reservoir of central air-conditioning system is a novel niche for Francisella surviving. In this study, a systematic analysis is conducted by following the principle of polyphasic taxonomy on the five Francisella –like strains(SYSU 10HL1970T?10HP457?10HL1938?10HP82-10 and SYSU YG23T) recently isolated from the cooling water towers of central air-conditioning system in Guangdong area. Methods 1. Classic taxonomyThe biochemical and enzymatic activities, such as oxidase, catalase, H2 S reduction, were tested with the traditional methods, and the comparative analysis in microbiological characteristics was done by use of Rapid ID 32 A, API 20 NH and API ZYM on the novel and reference trains(F. philomiragia ATCC25015, F. guangzhouensis 08HL01032T). The antibiotic susceptibilities were detected by E-test. 2. ChemotaxonomyThe compositions of Quinones, polar lipids and cellular fatty acid from the novel and reference trains were analyzed by thin layer chromatography(TLC), high performance liquid chromatography(HPLC) and gas chromatography. 3. Molecular taxonomyThe DNA G+C content was determined by reverse-phase HPLC, the protein spectrum was revealed by mass spectrometer, and the genome features were examined by PCR, 16 S r RNA sequencing and DNA-DNA hybridization in order to yield the molecular taxonomy for the trains. Results 1. Classic taxonomyAll the five isolates, SYSU YG23 T, SYSU 10HL1970 T, 10HP457, 10HL1938 and 10HP82-10 were Gram-negative cocco-bacilli, showing catalase weakly-positive, oxidase negative, H2 S reduction negative. The strains in vitro growth was enhanced by cysteine supplementation. Colonies on chocolate agar(with cysteine) and CHAB were obviously larger than those on sheep blood agar, which were identical to F. guangzhouensis. In general, the five Francisella-like strains showed an overall similar phenotype to F. guangzhouensis, except for a few tests(eg. sucrose fermentation, leucine arylamidase, tyrosine arylamidase and phenylalanine arylamidase). The five Francisella-like strains were susceptible to aminoglycosides, tetracyclines, and fluoroquinolones. 2. ChemotaxonomyThe polar lipid profiles of the five Francisella-like strains were nearly identical, composed of the predominant lipids phosphatidylethanolamine(PE), diphosphatidylglycerol(DPG), phosphatidylglycerol(PG), phosphatidylcholine(PC), an unidentified aminophospholipid(APL4, APL2), three unidentified phospholipids(PL2, PL3 and PL4), and an unidentified glycolipid(GL2), and the furthermore minor amounts of two unidentified glycolipids(GL1, GL3); this polar lipid profile was similar to that of F. guangzhouensis. The five Francisella-like isolates were rich in three even chain saturated fatty acids, C10: 0, C14: 0 and C16:0, and two long chain hydroxyl fatty acids, C18:1 ?9c and C18:1 3-OH, which were different from the reference strains. On the other hand, the isolate SYSU YG23 T could be distinguished from the other four Francisella-like strains in the compositions of C10:0, C16:0 and C18:0. 3. Molecular taxonomySequence comparison of 16 S r RNA gene: the five Francisella-like strains were similar by 98.6%; SYSU YG23 T and SYSU 10HL1970 T were shown 98.87% and 98.82% similarities with F. guangzhouensis 08HL01032 T respectively. The isolate SYSU YG 23 T was similar to SYSU 10HL1970 T by 99.6%. The strains shared <95.3% 16 S r RNA sequence similarities with members of the family Francisellaceae, except for Francisella guangzhouensis. Despite the high sequence similarity in 16 S r RNA gene, the MP(Maximum parsimony) tree with bootstrap analysis of 1000 replicates showed the well separated cluster for SYSU YG 23 T, SYSU 10HL1970 T and F. guangzhouensis. This topology was entirely consistent with neighbor-joining and maximum likelihood-based reconstructions also done MEGA 5.05.Sequence alignment of house-keeping genes mdh, rpo B and sdh A: the molecular dendrograms also supported that five Francisella-like isolates could be grouped into two separated clusters and mostly related to F. guangzhouensis, where analysed individually and as concatenated sequences. Therefore, the 16 S r RNA gene, mdh, rpo B and sdh A sequences all indicated that the phylogenetic position of the five Francisella-like strains were related to F. guangzhouensis species within the family Francisellaceae.The analysis results of MALDI-TOF MS shows that the peaks of 5110m/z?6123m/z?9360m/z are in the spectrum of SYSU 10HL1970T?10HL1938?10HP82-10 and 10HP457. The peaks of 5110m/z ?4754m/z?6123m/z?9360m/z and 4754m/z in the spectrum of SYSU YG23 T. Molecular dendrogram of cluster analysis demonstrated a clear separation of the respective mass spectra between F. philomiragia, F. noatunensis and F. guangzhouensis. The five novel strains were also grouped into two clusters, represented by SYSU YG23 T and SYSU 10HL1970 T, 10HP82-10, 10HL1938 and 10HP457, respectively. The MALDI-TOF MS cluster analysis was identical with molecular dendrograms based on gene.DNA-DNA re-association show that DNA:DNA re-association values among the five novel strains were <70% and those of four isolates SYSU 10HL1970 T, 10HP82-10, 10HL1938, 10HP457 were >80%. Therefore, the genomic study also showed that the five Francisella-like strains belonged to two novel species of a novel genus within the family Francisellaceae. DNA-DNA re-association values between the five Francisella-like strains and the reference F. guangzhouensis 08HL01032 and F. philomiragia subsp. philomiragia ATCC25015 were <70%, which was lower than the species delineation of 70%. The DNA G+C content of strains SYSU 10HL1970 T, 10HL1938, 10HP82-10 and 10HP457 were 30.0% and the isolate SYSU YG23 T was 32.6%? ConclusionDepending upon the characteristics of classic taxonomy, chemotaxonomy and molecular taxonomy obtained from this investigation, we proposed that strains SYSU YG23T(= KCTC 42968= DSM 101834) and SYSU 10HL1970T(=KCTC 42969= DSM 101835) merit recognition as representative of two novel species of a novel genus for which the name Allofrancisella inopinata gen. nov. sp. nov. and Allofrancisella frigidaquae gen. nov. sp. Nov. are proposed. In addition, the strain F. guangzhouensis 08HL01032 T was found to considerably diverse from all recognized Francisella species by both sequence comparison and phylogenetic analysis, while it shares many common traits with the proposed new genus. It was therefore considered that the strain should be reclassified as separate taxa within the newly proposed genus for which the name Allofrancisella guangzhouensis comb. nov. is proposed.