Isolation And Identification Of Bacteroides Fragilis From Giant Panda,Screening Of Sensitive Drugs

The giant panda(Ailuropoda melanoleuca)is a unique species in China and is a national first-class protected animal.In order to maintain the stable development of this population,breeding work is a key link that cannot be ignored.Bacterial infections and imbalances in the flora of the reproductive system can affect the reproductive health of giant pandas and can cause miscarriages in severe cases,which is one of the reasons for reproductive disorders in giant pandas.Therefore,research on the reproductive system health of giant pandas is particularly important for their protection.In this study,we isolated a strain of fragile bacteria from the vagina of giant pandas with vaginitis to explore its biological characteristics,screen sensitive drugs and analyze drug-resistant genes.Experiment 1,isolation and identification of fragile bacteria.In this experiment,a suspected strain of fragile bacteria was isolated from the reproductive tract of giant pandas with vaginitis.The strain was identified through morphological observation,culture characteristics,physiological and biochemical tests,16 S r RNA PCR amplification and construction of an evolutionary tree comparison.Identification was also performed through animal acute toxicity experiments and screening of virulence genes(bft).The results showed that the colony was circular,gray-white,slightly convex,without spores,smooth on the surface and had a neat edge.It hydrolyzed seven saponins,had no motility and good oxygen tolerance.The morphological characteristics and physicochemical properties of the strain were consistent with the description of the Bacteroides genus in Bergey ’ s Manual of Systematic Bacteriology.The 16 S evolutionary tree showed that it had a 99.99%affinity with Bacteroides fragilis strain S14(CP012706.1),indicating that it was a fragile bacterium.When mice were continuously injected with a bacterial solution at a concentration of 109 CFU/m L for 3 days,no abnormal clinical manifestations or deaths were observed.There were no significant differences in body weight,organ index and fragile bacterium(ATCC9394)control group compared with the experimental group.This experiment showed that the bacteria were non-toxigenic bft NTBF and did not have strong pathogenicity to mice.Experiment 2: whole-genome sequencing of fragile bacteria.In this experiment,high-throughput sequencing technology was used for whole-genome sequencing.Seven housekeeping genes were selected and the strain type was determined by multilocus sequence typing(MLST).Various bioinformatics analysis software and the NCBI gene database were used to analyze the obtained gene information.The results showed that the GPBF01 gene sequence was 5,006,113 bp in length,with an average GC content of 44.25%.The average length of the coding genes was 1,076 bp and the total length of the coding region accounted for 89.05% of the whole genome.It contained 12 gene islands with a total length of 193,595 bp;it contained 10 prophages with a total length of 470,454 bp;there were five CRISPRs with a total length of 3145 bp.It carried 22 virulence-related genes,but it is currently unclear whether they are related to virulence expression.Its drug-resistant genes were all located on the chromosome without plasmid distribution and horizontal transmission risk.Experiment 3: screening of sensitive drugs and study of drug-resistant genes.In this experiment,the broth microdilution method was used to select 14 different types of antibiotics and set different concentration gradients.The MIC value of each antibiotic was determined and whether it was resistant was judged.Fluorescence quantitative PCR was used to screen for drug-resistant genes.The drug-resistant phenotype results showed that the strain exhibited varying degrees of resistance to cephalosporins such as ceftriaxone sodium and ceftazidime,quinolones such as moxifloxacin hydrochloride,aminoglycosides,glycopeptides and sulfonamides.Fluorescence quantitative PCR detected drug-resistant genes including cfx A,cep A and cop A;12 drug-resistant genes were detected by whole-genome sequencing,including tet T,ade C,ade G,mex I,cep A,Mfd,cde A,ala S,mex K,mef C,Pmr E and Tri C.The drug sensitivity phenotype results for β-lactams and aminoglycosides were consistent with the genotype results.Conclusions:(1)An anaerobic bacterium was isolated and cultured from the vagina of giant pandas and named GPBF01.The strain was not acutely lethal to mice.(2)The whole genome was predicted to carry 22 virulence-related genes and 12antibiotic-resistant genes,which are mostly bacterial capsules,flagella and surface antigens.It is currently unclear whether they are related to virulence expression.All drug-resistant genes of GPBF01 are located on the chromosome without plasmid distribution and horizontal transmission risk.(3)The drug sensitivity phenotype and drug-resistant genotype both indicate that this experimental strain exhibits certain resistance to β-lactams and aminoglycosides,while being sensitive to meropenem in β-lactams,macrolides,tetracyclines,levofloxacin in quinolones and metronidazole in other synthetic nitroimidazoles.