Detection And Sequence Analysis Of Correlative Virulence Genes And Drug Resistance Genes Of Superiority Escherichia Coli Serotypes Isolated From Cow Mastitis

In this study, we used clinical isolates of mastitis source Escherichia coli in Ningxia, the advantages serotypes were identified by the method of plate agglutination, using the disk diffusion method to analyze its resistance phenotype, the resistance genes and virulence gene were detected by PCR methods. Furthermore, we have analyzed the relationship between the resistance and the serotype, at the same time two virulence genes sequence irp2and iucA were analyzed. The main results are as follows:1. According to GenBank reports sequences, we have designed a universal primers to identify E. coli16S rRNA, then using this method to identify the173mastitis source Enterobacteriaceae collected by our laboratory, eventually identified107strains of Escherichia coli.2. According to the relevant literature, we selected30kinds of0antigen serum to identify107clinical isolates of mastitis source Escherichia coli, the identification results show that78serotypes were identified from clinical isolated107bacterial strain cow mastitis source Escherichia coli, contain6advantage serotypes O101(15/78),0158(15/78), O53(8/78), O10(7/78),086(5/78),092(6/78), accounting for71.8%in all clinical isolated107bacterial strain, and29stereotypes strains did not stereotypes.3. We used producers diffusion method by CLSI,11kinds of drug to be used to the susceptibility testing on107clinical isolated mastitis source Escherichia coli. The results showed that isolated bacterial strain for11kinds of antimicrobial agents with different degrees of drug resistance, in which the drug resistance rate of94.4%to cotrimoxazole,tetracycline reached90.7%, gentamicin reached86.9%, ampicillin and amoxicillin reached93.5%, in addition to doxycycline and kanamycin have reached more than60%, several other drugs also reached a higher level. It appears53kinds of E. coli resistance spectrum in all107bacterial strain, does not appear1resistance,2resistance and11resistance bacterial strains. Drug resistance type0resistance is the least resistance (only one), the highest reaching10resistance.4. According to related sequences by GenBank, we designed sequences of sulfa:sull, sul2, sul3; tetracyclines:tetA, tetB, tetE; aminoglycosides:aadA1, aadA2, aadB, aadD, aacC2, aacC4, aac (3)-Ⅰa, aac (3)-IIa, aph (3)-IIa; β-lactams:CTX-M, SHV, OXA, TEM; HPI Island:irp2, fyuA; LEE Island: eaeA, ler; furthermore we also designed the specific primers PCR reaction of F41, LT1, ST1, iucA, iucB, iutA, established the corresponding PCR detection methods, the results show that the method is sensitive and reliable, so that it can be used for clinical testing. After107mastitis source Escherichia coli bacterial strains clinical isolated were detected, and14kinds of drug resistance genes were detected; seven kinds of virulence genes were detected, respectively, sull (31.8%), sul2(37.4%), sul3(2.8%), tetA (17.8%), tetB (14.0%), aacC2(32.7%), aacC4(13.1%), aadA1(9.3%), aadA2(10.3%), aadB (38.3%), aac (3)-IIa (27.1%), CTX-M (10.3%), OXA (8.4%), TEM (47.7%), irp2(49.5%), fyuA (49.5%), ler (3.7%), eaeA (3.7%), iucA (38.3%), iucB (56.1%), iutA (49.5%). But we did not detected the other genes.5. Amplified irp2and iucA genes on the dominant serotype0158,0101in30bacterial strains of E. coli, after gene sequence analysis, the results showed that the two genes sequence homology very high with GenBank reported, irp2reached more than95.9%, and iucA more than98%. In addition, the different sequences of both genes appeared regular mutations in both, which occur irp2gene sequence of six kinds, of which there are three sequences occured4bp deletion near279genetic locus and the AT mutation to TC on531genetic locus. All six kinds of sequences, there are304genetic locus point mutation A to C,343genetic locus point mutation C to A,346genetic locus A to G,376genetic locus point mutation C to A and518genetic locus point mutation T to C. The iucA gene emerged four kinds of sequences, which iucA-2, iucA-3, iucA-4occurred point mutation C to G at751genetic locus; while iucA-1, iucA-2, iucA-4occured point mutation T to C at1089genetic locus, the four sequences had all appeared point mutation as A to G at1037,1042,1047three genetic locus.