Study On Genome And Drug Susceptibility Of Citrobacter Species

Citrobacter is a common zoonotic pathogen,which can infect weak people with low immunity and cause diseases including diarrhea,sepsis,meningitis,respiratory and urinary system infections.To study on the pathogenesis of Citrobacter is beneficial for the prevention and control of Citrobacter disease.With the continuous improvement of whole genome sequencing and the reduction of sequencing costs,whole genome sequencing analysis can be widely applied in bacterial identification,genotyping,virulence gene and drug resistance gene profiling.Combined with phenotype testing,it lays the foundation for the scientific prevention and treatment of Citrobacter disease.This study first conducted microbiological identification on the four Citrobacter isolates,followed by whole genome sequencing and Multi Locus Sequence Typing(MLST)analysis.Finally,the drug sensitivity of Citrobacter to 20 drugs and 41 Chinese herbal medicines was determined,providing reference for the clinical prevention and treatment of Citrobacter disease.The main findings are as follows:(1)Isolation and identification of Citrobacter and whole genome sequencing analysis.Through morphological and biochemical characterization of the four isolates named B1,C1,M1,and M4,they are determined as Citrobacter.Then the bacterial genomic DNA was extracted,the whole genome sequencing was performed.The four strains were identified by 16 S r RNA gene sequence,NR database and ribosome multi site sequence typing(r MLST)analysis.The results indicate that Citrobacter B1,C1,and M1 strains are Citrobacter braakii,while M4 strain is Citrobacter werkmanii.Then in silicon MLST analysis of the four tested strains based on whole genome sequence showed that the four strains had a novel allele profile and generated four new sequence types(ST).(2)Study on the virulence and resistance gene profiles of Citrobacter.Whole genome sequence analysis of four strains of Citrobacter was performed to obtain the virulence gene profile of Citrobacter using the Virulence Factors Database(VFDB).Citrobacter strains B1,C1,M1,and M4 carried 134,120,145,and 121 virulence genes,respectively.According to their function,these virulence genes were divided into 7categories,namely adhesion,nutrition/metabolism,immune regulation,secretion system,regulation,invasion,and biofilm formation genes.In addition,the M1 strain also contains Vi antigen related genes related to innate immune escape.Meanwhile,the resistance gene profile of four strains of Citrobacter was also investigated using the Antibiotic Resistance Genes Database(ARDB).B1 strain carried 21 resistance genes,mainly aiming at common antibiotics such as tetracyclines,macrolides,chloramphenicols,and chloroquinolones;C1 strain carried 20 resistance genes,mainly aiming at drugs such as tetracyclines,cephalosporins,and chloramphenicols;M1carried 21 drug resistance genes,mainly aiming at tetracycline,aminoglycoside,lactam;M4 carried 21 drug resistance genes,mainly aiming at glycopeptides,tetracycline,aminoglycosides,lactam and other drugs.(3)The susceptibility analysis of Citrobacter to 20 antibiotics,including neomycin,cephalosporin,cefoperazone,clindamycin,ofloxacin,norfloxacin,doxycycline,minocycline and cefuroxime,was studied by disk diffusion method.The experimental results indicated that B1 strain was moderately sensitive to cefoperazone and sensitive to most other bacteria β-Lactam antibiotics,while resistant to quinolones,tetracyclines,lincomycin,macrolides and other common antibiotics;C1strain was moderately sensitive to cefoperazone,and meanwhile resistant to tetracycline,quinolone,macrolides,and sulfonamide antibiotics;M1 was moderately sensitive to cefoperazone and resistant to tetracycline,quinolone,macrolides,and sulfonamide antibiotics;M4 was moderately sensitive to minocycline and sensitive toβ-Lactam,tetracycline,quinolone antibiotics resistance.(4)Agar diffusion method was used to determine in vitro antibacterial effect of 41 Chinese herbal medicines against Citrobacter.The experimental results showed that Chinese nutgall and black plum had the strongest inhibitory effects on Citrobacter B1;Red peony,Ligustrum lucidum,and Coptis chinensis had moderate inhibition on Citrobacter B1.Chinese gallnut and honeysuckle had the strongest inhibitory effects on Citrobacter C1;Dark plum,red peony,and ligustrum lucidum had moderate inhibition on Citrobacter C1.Chinese gallnut,black plum,and honeysuckle had the strongest inhibitory effects on Citrobacter M1;Peony bark,red Gou,and Huanglian had moderate inhibitory effects on Citrobacter M1.Chinese gallnut had the strongest inhibitory effect on Citrobacter M4;Wumei,Honeysuckle,Peony Peel,Chigou,and Huanglian had moderate inhibitory effects on Citrobacter M4.Based on the results of antibiotic susceptibility tests,it was found that Citrobacter B1,C1,M1,and M4 developped resistance to various antibiotics such as tetracyclines,quinolones,macrolides,and sulfonamides,but were highly sensitive to gallnuts.Secondly,red peony also had significantly inhibitory effects on four strains of Citrobacillus.Therefore,Chinese gallnut and red peony have good application prospects in the treatment of citric acid bacteria disease,and their advantages are prominent when used to treat drug-resistant citric acid bacteria.