Isolation And Some Biological Characteristics Identification Of Staphylococci Lentus In Bumblebee Larvae

Bumblebee is the most common bee insect on the grassland in temperate regions.It is widely used for pollinating wild plants（endangered plants）and crops because of its ability to absorb nectar and pollen from plants.Especially,fruit and vegetables（such as solanaceae,legumes,etc.）in the greenhouse have the best pollination effect.Commercial bumblebee,which is used in greenhouse pollination,is more susceptible to infection and disease than wild bumblebee with pathogens,with bacteria,virus,external parasitic mite,protozoa,etc.,the bacterium with coagulation ase-negative Staphylococci（CNS）is one of the bacterial pathogens.CNS is a zoonotic opportunistic pathogen that can cause endocarditis,septicemia and blood infection.It causes mastitis in cows,etc.CNS can also produce a variety of pathogenic factors,such as lipase,deoxyribonuclease and enterotoxin,which can cause certain damage to humans and animals.Its conserved genes,such as hsp60,gap,sod A,tuf and rpo B,are commonly used to identify species of CNS.In this study,bacterial isolation,biochemical identification,16S r RNA identification,gap gene biological analysis,hemolytic assay,mouse pathogenicity test and drug sensitivity test were performed on the larvae of dead Bumblebee from Xinjiang province.The main research contents and results are as follows:1.Isolation and identification of Staphylococci lentus（S.lentus）in Bumblebee larvae:In order to isolate the pathogens that cause the yellower,black and death of bumblebee larvae,sterile samples were collected from a sick bumblebee larvae in Shihezi,Xinjiang province,and bacterial isolation,biochemical tests,16S r RNA sequencing and identification were conducted.The results showed that a strain of Gram-positive cocci was isolated.After biochemical identification and 16S r RNA sequencing,it was preliminarily determined that the pathogen of Bumblebee larvae was S.lentus and belonged to CNS.2.Bioinformatics analysis of gap gene in Bumblebee larvae:This study amplified,cloned and sequenced the gap gene fragment from the isolated strain SHZXF1,and analyzed some of its bioinformatics characteristics.The results showed that the gap gene was 933bp and encoded 311 amino acids.Bioinformatics analysis showed that the nucleotide homology of SHZXF1 strain was 99.9%with S.lentus,and it was similar to the same branch.Gap protein was hydrophilic protein with molecular weight of 33.59 k Da and theoretical isoelectric point of 4.77.This protein has no transmembrane region and no signal peptide,and belongs to non-secretory protein.The secondary structure of 25 potential phosphorylation sites and 1glycation site was mixed,among which irregular curl accounted for the highest proportion（37.62%）.This conclusion provides a strong support for gap gene as a conservative gene to detect CNS.3.Study on some biological characteristics of S.lentus in Bumblebee larvae:To understand the antibiotic resistance and pathogenicity of SHZXF1 strain of Bumblebee larvae.The drug sensitivity test and pathogenicity analysis were carried out.The results showed that the SHZXF1 strain were highly sensitive to 21 antibiotics including gentamicin,doxycycline,penicillin,cefazolin,midecamycin,cotrimoxazole,norfloxacin and polymyxin B,etc.The survival time of mice infected with SHZXF1 strain was 4.125 days on average,and the LD₅₀was 10^6.86CFU.