Genotyping And In Vitro Drug Susceptibility Of Brucella Isolates From Ulanqab Of Inner Mongollia

Brucellosis has become a serious public health problem in Ulanqab.In order to reveal the cause of the high incidence of brucellosis,and to improve the effect of Brucellosis treatment and control,we have done this study.Brucella species were isolated and identified by three methods.MLST was applied to investigate population structure of the isolates,and MLVA-16 scheme was used to type the isolates,which also to be used to determine the genotyping characteristics and epidemiological association.The sensitivity of Brucella to ten antibiotics was detected,and the resistant mechanisms of B.melitensis to azithromycin and rifampicin were also anylized.Moreover,susceptibility of Rifampicin and cotrimoxazole to 149 B.melitensis from different regions of China have been detected.1.116 Brucella strains were isolated and all of these strains were identified as B.melitensis biovars,115 of them isolated from human blood and 1 from sheep spleen.94 strains were identified as B.melitensis biovars 3 and 22 were B.melitensis biovars 1.111 strains were isolated from 11 counties of Ulanqab and 5 isolates were from three neighboring regions.There were 84 were first visit cases(73%),The main clinical symptom were pain,fever,weakness and sweating.77%(88/114)of patients hadn't received any antibiotic drugs before diagnosis,and more than 30% patients were misdiagnosed and mistreated.These date provide a comprehensive reference for brucellosis control and supervise in this area.2.In this study,we analyzed the evolution of 16Sr DNA gene in B.melitensis.The result confirmed that 16 Sr DNA of Brucella was highly conserved,the similarity of 16 Sr DNA was 99%,which was a desirable target gene for Brucella rapid identification.The genetic distance and evolutionary degree between Brucella field strains and standard strains were closer,so it unable to be used to discriminate the relationship between the strains,and also 16 SrDNA was unsuitable for genetic evolution analysis of B.melitensis.There were closer genetic relationship between Brucella and Ochrobactrum anthropi,and it was distantly related to other test strains(Vibrio cholerae,Yersinia enterocolitis,et al)based on 16 SrDNA.These data provided reference for classification study of Brucella.3.Population structure of Brucella melitensis was analyzed by multilocus sequence typing.The result showed that all strains had the exactly same 9 MLST allelotype.116 strains were all belonged to ST8 sequence type,despite ST8 strains were wide distribution in diferent areas,but their population structure was great simple.ST8 strains was the mainly epidemic isolates in this regions,which had a close genetic relationship and the same evolution degree with the strains of other regions of Inner Mongolia.4.Multiple-locus variable-number tandem repeat analysis(MLVA-16)was applied for genotyping and molecular epidemiological investigation of Brucella melitensis.MLVA-8 has identified three genotypes(42,63 and 114),87%(n=101)of them were genotype 42.MLVA-11 has identified eight genotypes,all of them were identified as the East Mediterranean group,81%(n = 94)were genotype 116,which is the mostly predominant genotype of Ulanqab and neighboring regions.In this study,four new MLVA-11 genotypes were first discovered all over the world,and were named as 342-345 by MLVAbank.The MLVA-16 showed 69 genotypes,46 were represented by single isolates.It revealed that brucellosis cases had unrelated and sporadic characteristics in Ulanqab.There were 23 sharing genotypes with each genotype containing two to eight isolates.It indicated that these cases may be epidemiological related.MLVA genotyping confirmed the occurrence of a multipoint outbreak and familial brucellosis,and cross infection from different regions.Extensive genotype sharing events were observed among isolates of Ulanqab and other provinces in China,indicate a possible epidemiological relationship.Frequently,disorder transportation and trafficking of infectious animal(sheep)could be the main reason for high incidence of brucellosis in this region.5.MLVA was applied to investigation the transmission pattern of B.melitensis of Inner Mongolia.The results indicated that strains from different host(including in human,sheep,cattle and camel)shared identical or very similar genetypes.This indicate that B.melitensis were circulated transmission between sheep(cattle)and camel(other wildlife animals)and eventually infection humans,which was a kind of transmission pattern of B.melitensis in Inner Mongolia.Infected sheep was the mainly source of brucellosis,infected by directly or indirectly contact with infected sheep was mainly infectious modes for human brucellosis in this area.Camel maybe act as reservoirs of B.melitensis.6.Assays to discriminate three biotypes of B.melitensis with PCR methods were studied.Base on the polymorphisms of Rpo B gene and Bru42 gene loci of B.melitensis standard reference strain 16 M,two methods were had been established,and these two tests can be used to correctly identify the three biotypes of B.melitensis reference strains.The results showed that 100 clinical isolates(including 34 B.melitensis bv.1,6 B.melitensis bv.2 and 60 B.melitensis bv.3)were all identified as RpoB-2 genetype by RpoB-PCR,only two B.melitensis bv.1 strain was identified as Rpo B-3 genetype.Moreover,Bru42-PCR result displayed that all of 102 clinical B.melitensis isolates belonged to Bru42-2 genotype.These data indicated that there were significant different between B.melitensis standard reference strains and clinical isolates within RpoB gene and Bru42 gene loci.That confirmed that the two methods were unsuitable for typing of clinical B.melitensis isolates.7.The susceptibilities of 85 Brucella melitensis isolates to ten common antibiotics were tested by E-test method in vitro according to the clinical and Laboratory Standards Institute.The resistance to azithromycin,rifampin and cotrimoxazole was 100%(85/85),1.0%(1/85)and 7.0%(6/85),respectively.All of the tested isolates were susceptible to minocycline,sparfloxacin,doxycycline,tetracycline,gentamicin,ciprofloxacin and levofloxacin.Minocycline and sparfloxacin had the lowest MIC50 value(0.032?g/ml).The polymorphism of 2632 T/C in the 23 SrRNA of Brucella was a possible reason of B.melitensis resistant to azithromycin.RpoB gene mutations have not been observed in the isolates resistant to rifampin.Minocycline and sparfloxacin may be served as the first line drug for Brucellosis treatment.The sensitivity of rifampicin and cotrimoxazole to 149 B.melitensis strains from 24 regions in China was tested.The results showed that the resistance rate of B.melitensis to cotrimoxazole was 25.5%(38/149),and the resistant strains were distributed in 18 provinces,and all 149 strains were sensitive to rifampin.