Prevalence Of Antibiotic Resistance In Haemophilus Parasuis And Molecula Characteristic Of Florfenicol Resistance

Haemophilus parasuis(HPS)is a gram-negative bacteria,mainly leads to pig serositis,arthritis and meningitis,also known as glasser disease(Glass’ s disease).At present,the disease is worldwide distribution,and caused huge economic losses to the global pig industry.So far,there are 15 serotypes of Haemophilus parasuis.Although the disease can be prevented by vaccines,a considerable number of clinical isolates cannot be serotyped,and the cross-protection ability of vaccines is poor.Drug treatment remains the most effective way to control the disease.However,with the rapid development of pig industry,the nonstandard use of antibiotics is becoming more and more common,and the drug resistance of bacteria is becoming more and more serious.The resistance phenotype of Haemophilus parasuis isolated from south China and north China was investigated,the serotypes of florfenicol resistant strains were identified,PFGE typing was carried out,and the molecular characteristics of florfenicol resistant strains were investigated.The aim of this study was to elucidate the epidemic characteristics and molecular mechanism of florfenicol resistance gene,and to provide a scientific basis for the risk assessment and prevention and control of florfenicol resistance.In this study,the sensitivity of 20 drugs of 106 clinical isolates of Haemophilus parasuis from south China and north China from 2012 to 2016 was tested by agar dilution method.The results showed that 106 strains of Haemophilus parasuis were resistant to the quinolones levofloxacin,ciprofloxacin and enrofloxacin in the range of 2.83%-22.64%.1.89%-16.03% of the strains were resistanted to β-lactam drugs such as penicillin,ampicillin and ceftiofur.The resistance rates to tetracycline and doxycycline were 27.32% and 16.52% respectively.The rates of resistance to nalidixic acid,sulfamethoxazole and TMP were 96.23%,40.87% and 46.96% respectively.In addition,17.92% of the strains were resistanted to chloramphenicol and 7.54% to florfenicol.The multi-drug resistance of 106 strains of Haemophilus parasuis(including resistance to different kinds of drugs,and resistance to different drugs in the same kind of drugs)showed that the tested strains were mainly 7 weight(19.13%),8 weight(17.39%)drug resistance,followed by 9 weight(12.17%),5 weight(9.57%)and 6 weight(9.57%)drug resistance.Eight strains of Haemophilus parasuis resistant to florfenicol were identified by drug resistance sensitivity test.PFGE typing of Haemophilus parasuis resistant to florfenicol was carried out.The results showed that PFGE fingerprints of Haemophilus parasuis resistant to florfenicol in north China were identical,all showed the same band and belonged to the same PFGE typing.Eight strains of Haemophilus parasuis resistant to florfenicol were identified by agar diffusion test.results all the 8 strains were serotype 2.These results suggested that florfenicol resistant strains from pig farms in north China had long-term clonal transmission relationship.Gene floR,fexA,fexB and cfr were detected in 8 strains.The results showed that all 8 strains were flor positive.Amplify floR gene by PCR of eight florfenicol resistant strains,recover and connecting that product to a vector for transformation,sequencing to obtain flor gene sequence,and then translating the sequence into amino acid sequence for comparison.The result show that the amino acid sequence of the floR gene of the eight strains has the similarity of 99.8%-100%;Through GPS-PCR,plasmid extraction and large fragment electrophoresis,floR gene of 8 florfenicol resistant strains were locate on plasmids with that size of about 5 kb.Plasmid sequencing showed that the floR-carrying plasmid sequence of 8 florfenicol resistant strains were the same,which was named pHF.Plasmid transformation and drug sensitivity test showed that pHF could mediate florfenicol resistance.The plasmid pHF consist of three open reading frame,including rep,floR and lysr.The database search identified three plasmids with high sequence homology to pHF,including plasmid pB1001(GenBank,accession number: EU252517),plasmid pCCK381(GenBank,accession number: AJ87969)and plasmid pMh1405(GenBank,accession number: AB621152).pHF appear to be assembled with different elements from the above plasmids,indicating that recombinant events may occur between these small plasmids and form new resistance plasmids.