| Klebsiella is an important pathogen causing pneumonia and mastitis in humans and animals,and it is also one of the critical multi-drug resistance（MDR）microorganisms in the world,causing serious damage to global public health security and the breeding industry.In order to fully understand the distribution of Klebsiella from Tibetan yaks in milk,and to grasp its biological characteristics,virulence genes,and drug resistance genes,this study selected 15 cattle farms in 5cities in Tibet.On each farm,10 female yaks with symptoms of respiratory tract,mastitis or diarrhea were collected in each farm,and a total of 150 milk samples were collected for the isolation and identification of Klebsiella.The growth curve,biochemical characteristics and in vitro antibiotic susceptibility tests were carried out on the isolates,and their biological characteristics were preliminarily grasped.The virulence genes and drug resistance genes of the isolates were detected by PCR method to grasp their drug resistance characteristics.On this basis,isolates K6,K11 and K12 were selected for pathogenicity test in mice.The isolate K11 was selected for whole genome sequencing,whole genome and bioinformatics analysis.This research mainly includes the following four aspects:1.For 150 milk samples,through the isolation,cultivation and identification of Klebsiella,and the observation of colony morphology,PCR amplification of 16S r RNA and specific khe gene was performed on similar strains.A fragment matching the size of the target band was successfully amplified.The amplified products were compared with NCBI database Blast,and the similarity with Klebsiella was more than 98%.The 12 isolates obtained were all Klebsiella oxytoca,with an isolation rate of 8.0%.2.The amplification results of virulence genes and drug resistance genes showed that 7 virulence genes were successfully amplified,namely uge,mrk D,fim H,ent B,ure A,all S,ycf M.The virulence genes with the highest detection rate were ure A（100%）,all S（91.67%）,mrk D（66.67%）.Seven drug-resistant genes were successfully amplified,namely blaTEM-1,blaCTX-M1,blaOXA-48,sul1,sul2,tet A,tet B,and the highest detection rates of drug-resistant genes were blaTEM-1,blaCTX-M1 and sul2,the lowest detection rate is the tet A gene.3.Biological characteristics and pathogenicity studies show that the isolates have a 4-hour lag phase,a 6-hour logarithmic growth phase,a 4-hour stable phase,and then a decline phase.Drug susceptibility tests showed that the isolates were widely resistant to antibiotics such as first-generation and second-generation cephalosporins and aminoglycosides,and were sensitive to polymyxin and imipenem.After challenge with isolates K6,K11 and K12,the mice showed severe curling up,convulsions and tremors,and all had diarrhea and incontinence,and the anus of the dead mice had fecal adhesions.After dissection,there was fluid in the abdominal cavity,and there were obvious intestinal flatulence,large intestinal edema and hemorrhage.Some mice had heart and liver necrosis,and spleen enlargement.Pathogenicity tests in mice showed that Klebsiella from Tibetan yaks had strong pathogenicity.4.Bioinformatics analysis showed that the full-length genome of the isolate K11 was 5775088bp,and the GC content was 56.0%.the number of protein-coding genes is 5519.the shortest gene length is 39bp.the longest gene length is 9947bp.the average gene length is 934.1bp.the number of t RNA is 87,r RNA 25,and CRISPR is 3.Annotate databases such as GO,KEGG,and COG,and annotate the pathogen-host interaction database（PHI）.Further predict signal peptide,gene island and other information.In summary,this study isolated and identified 12 strains of Klebsiella oxytoca from Tibetan yak milk samples for the first time,and studied their biological characteristics to understand their growth characteristics,virulence and drug resistance.Conducting pathogenicity research and selecting representative strains for genome-wide bioinformatics analysis provided a theoretical basis for the prevention and control of Klebsiella animal diseases from Tibetan yaks and subsequent related research. |
