Studies On The Species Of The Culturable Bacteria From The Air Of An Enclosure Of Sub-adult Giant Panda And The Polyphasic Taxonomy Of Two Novel Strains

As the important place of captive giant pandas and popular science education base for teenagers, research base of giant panda breeding attracts a large number of domestic and foreign tourists every day. Tourists often have a visit to enclosures of giant panda, the air quality of which is related to the number of visitors. Air quality can directly affect the health of giant panda, and even may induce the occurrence of certain diseases. In the past, people paid little attention to air micro-organisms from enclosures of captive giant pandas. This study collected culturable bacteria from the air of an enclosure of captive giant panda through natural sedimentation method, and then classified and identified these bacteria in order to assess whether air microorganism have an adverse influence on giant panda.28 bacteria strains were isolated, and we identified two novel strains using polyphasic taxonomy approach, including evaluation of its morphological, biochemical and phylogenetic characteristics. The results are as follows:1. Diversity of the eulturable bacterium species from an enclosure of sub-adult giant panda and drug resistance analysis.Based on differences in colony morphology,28 pure cultures were selected for 16S rRNA gene sequencing and phylogenetic analysis. Species included members of the genera Micrococcus, Chryseobacterium, Leuconostoc, Staphylococcus. Pseudomonas, Kocuria, Bacillus, Exiguobacterium, Acinetobacter, Escherichia. Shigella, Rothia, Dietzia, Morganella, Macrococcus. Cedecea and Lysobacler. Thereinto, Staphylococcus, Shigella and Chryseobacterium etc., are conditional pathogenic bacteria; these bacteria are easy to infect animals when they are under stress or low immunity, and verified that sub-adult giant pandas had the potential risk of infection.Susceptibility test results showed that 17 genera of bacteria had different levels of drug resistance to 23 kinds of antibiotics, and it was not surprising for multidrug resistance, at least resisting three kinds of antibiotics, and up to resisting 14 kinds of antibiotics. The resistance of penicillin G, lincomycin. furazolidone, sinomin and ampicillin were severe, at least 10 kinds of bacteria were resistant to these antibiotics, but florfenicol. doxycycline, amikacin, enrofloxacin, gentamicin, ciprofloxacin and streptomycin were almost sensitive to all bacteria. Thus, the enclosure of sub-adult giant panda is the store for certain drug-resistant bacteria.2. Polyphasic taxonomic study of strain CF21^TStrain CF21^T were gram-negative, aerobic, non-motile, and rod-shaped. It grew at 10-40℃ （optimum 28-30℃） and pH 6.0-9.0 （optimum pH 7.0-8.0）, and in the presence of NaCl concentrations ranging from 0.0% （w/v） to 2.0% （optimum 0.0-1.0%）. Catalase and oxidase are produced, but not indole or H2S. Starch and gelatin are degraded but not tween 80. Nitrate is not reduced to nitrite. It is negative for arginine decarboxylase, ornithine decarboxylase, β-galactosidase and varieties of carbon sources. The primary polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and an unknown phospholipid. And the prominent isoprenoid quinone was ubiquinone 8 （Q-8）. Major cellular fatty acids of CF21’were iso-C_16:0 （30.22%）, iso-C_15:0 （25.70%）, and the sum of 10-methyl C_16:0 and/or iso-C_17:1ω9c （21.94%）. The genomic G+C DNA content was 68.72 mol%. 16SrRNA and gyrB gene sequence analysis indicated that strain CF21^T belonged to genus Lysobacter and was most closely related to L. ruishenii CTN-1^T （95.1%）. DNA sequence relatedness between strain CF21 and L. ruishenii CTN-1 was 56%, which was clearly below the 70% threshold for prokaryotic species delineation.These analyses indicated that CF21 is a novel member of genus Lysobacter. for which the name Lysobacter chengduensis sp. nov. is proposed. The type strain is CF21^T （=CGMCC1.15145^T-DSM 100306^T）.3. Polyphasic taxonomic study of strain 25-1Strain 25-1^T was Gram-negative, aerobic, non-motile, rod-shaped. It grew optimally at pH 7.0-8.0, at 28-30℃ and in the presence of NaCl concentrations from 0.0 to 0.5%. Catalase and oxidase are produced, but not indole or H2S. Starch and tween 80 are degraded but not gelatin. Nitrate is not reduced to nitrite. Acid is not produced from salicin, acetamide, mannose, β-galactosidase, trehalose, cellobiose, arabinose and lactose. It is negative for adipic acid and arginine decarboxylase, and positive for the production of ornithine decarboxylase. And it is unable to utilize a variety of carbon sources. The major cellular fatty acids were iso-C_15:0 （44.0%）, iso-C_17:03OH （19.8%） and C_16:1ω7c/（16:1）ω6c （12.7%）. The only isoprenoid quinone was MK-6. The major polar lipids were phosphatidylethanolamine, two unidentified amino lipids and two unidentified lipids. Its genomic DNA G+C composition was 36.2 mol%.16S rRNA gene sequence analysis indicated that strain 25-1^T belongs to the genus Chryseobacterium within the family Flavobacteriaceae and is related most closely to Chryseobaclerium carnis G81^T （96.4% similarity）, Chryseobacterium lathyri RBA2-61 （95.8%）. The DNA-DNA relatedness between strain 251^T and C. lathyri RBA2-6^T was 38%.Phenotypic, genotypic and phylogenetic characteristics indicated that strain 25-1^T is a novel member of the genus Chryseobacterium, for which the name Chryseobacterium chengduensis sp. nov. is proposed. The type strain is 25-1^T （CCTCC AB2015133T= DSM 100396^T）.This study found that there were diverse microbial species in the enclosure of sub-adult giant panda, and some of them were conditional pathogenic bacterium that may have a potential hazard to the health of giant panda. Furthermore, some bacteria existed serious multiple drug resistance. Therefore, it is necessary to strengthen microorganism monitoring in the enclosure of giant panda, in order to evaluate the potential harmfulness to giant panda, and provide a reference for protecting this endangered species.