Study Of Antibiotic Resistance And Resistance Mechanisms Of Brucella Isolates

Brucella(phylum Proteobacteria,class Alphaproteobacteria,order Rhizobiales,family Brucellaceae)is a gram-negative bacillus,without pods and flagella,which does not release exotoxins and is a parthenogenic intracellular parasitic bacterium Currently,the genus Brucella is divided into 12 species.Brucella can cause brucellosis,which poses a serious threat to the health of animals and humans.Brucella infection can lead to abortion or even infertility in females and orchitis in males.The population susceptible to brucellosis is clearly occupational,with no significant gender or age differences,and the infection depends mainly on the frequency of exposure and can occur throughout the year.Brucellosis is widespread worldwide,mainly in countries without effective vaccines for humans and animal health programs,and is endemic in most provinces and cities in China.Antibiotics are currently the most effective treatment for brucellosis,but the lack of timely treatment and irrational use of antibiotics has also lead to the development of bacterial antibiotic resistance,making the diagnosis and treatment of brucellosis difficult.Of the 111 Brucella isolates in this study,61 Brucella isolates were from clinical patients in Heilongjiang Province and 50 Brucella isolates were from clinical patients in Hebei Province.The DNA of Brucella isolates was extracted and the bacterial species was identified by genusspecific PCR and AMOS-PCR,and the results showed that all of them were Brucella melitensis.The E-test strips were used to test the Brucella isolates for sensitivity to 13 antibiotics.The results showed that 61 Brucella isolates from Heilongjiang Province were 86.9%,65.6%,27.9%,24.6%,3.3% and 1.6% resistant to azithromycin,cefepime,cefoperazone/sulbactam(2/1),rifampicin,cefotaxime and trimethoprim/sulfamethoxazole(1/19),while susceptible to doxycycline,tetracycline,minocycline,levofloxacin,ciprofloxacin,gentamicin,and streptomycin.50 Brucella isolates from Hebei Province were 100%,100%,82%,20% and 14%resistant to azithromycin,cefepime,cefoperazone/sulbactam,cefotaxime and rifampicin,while susceptible to doxycycline,tetracycline,minocycline,levofloxacin,ciprofloxacin,gentamicin,streptomycin and trimethoprim /sulfamethoxazole.In this study,B.melitensis resistant to cephalosporin antibiotics was identified for the first time in China.Short-read whole-genome sequencing and bioinformatics analysis were performed on Brucella isolates.The results of the Comprehensive Antibiotic Research Database(CARD)showed that there were 32 antibiotic resistance genes with >40% similarity in Brucella isolates from Heilongjiang Province and 41 antibiotic resistance genes with >40% similarity in Brucella isolates from Hebei Province.According to the different functions of the resistance genes,about60% of the antibiotic resistance genes were associated with the efflux pump system,mainly the resistance nodulation division family(RND).Genetic evolutionary analysis and multi-locus sequence analysis(MLST)showed that all 111 Brucella isolates were of type ST8,group CC4,with minimal genetic variation from each other and distinct geographical characteristics.Three β-lactamase inhibitors(sulbactam,potassium clavulanate,and tazobactam sodium)and two efflux pump inhibitors(carbonyl cyanide m-chlorophenylhydrazone(CCCP)and phenylalanine-arginine β-naphthylamide(PAβN)),were selected for the study of cephalosporin antibiotic resistance mechanisms by combining the results of cephalosporin antibiotic resistance phenotypes and WGS analysis.The results of the resistance inhibition assay using micro-broth dilution showed that sulbactam and CCCP reduced the MIC values of Brucella isolates against cephalosporin antibiotics by 2-4 concentration gradients,with minimum effective concentrations of 48 μg/m L and 0.25 μg/m L,respectively.The protein profiles of Cephalosporin-resistant Brucella isolates before and after inhibitor action using a fully automated rapid microbial mass spectrometry system.Antibiotics are selected at sub-inhibitory concentrations and inhibitors are selected at minimum effective concentrations.The results showed that at the minimum effective concentration of the inhibitor,there were significant differences in the peaks of the protein profiles in the same interval,indicating that the size and content of some protein ions were changed.In summary,in this study,111 Brucella isolates from two provinces were subjected to species identification,antibiotic sensitivity testing,whole-genome sequencing,genetic evolutionary analysis,multi-locus sequence analysis,antibiotic resistance gene analysis,inhibitor effect detection and protein profile difference analysis.The epidemiological characteristics and antibiotic resistance of Brucella isolates in northern China were obtained,and the preliminary investigation of the resistance mechanism of Brucella isolates cephalosporin antibiotics was carried out.In order to ensure the efficacy and safety of antibiotics for the treatment of brucellosis,there is an urgent need to monitor the resistance of Brucella in different regions of China and to conduct in-depth studies on the resistance mechanisms of their antibiotics for clinical treatment,so as to provide guidance for the clinical use of brucellosis and avoid ineffective prescriptive use.