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Antimicrobial Resistance Profiles and Genotypes of Campylobacter Spp Isolates and Subtyping of Them Based on Matrix Assisted Laser Desorption Ionization Time of Flight Mass

Author: ZouYuHan
Tutor: JiaoXinAn；HuangJinLin
School: Yangzhou University
Course: Genetics
Keywords: Campylobacter jejuni Campylobacter Antibiotic resistance Disk diffusion method Broth microdilution method Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
CLC: R446.5
Type: Master's thesis
Year: 2010
Downloads: 102
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Abstract


Campylobacter is one of the important pathogens cause of human bacterial diarrhea worldwide, one of the most common is Campylobacter jejuni and Campylobacter infection. Both in developing and developed countries, Campylobacter infection rates are up in the trend; serious rising infection rates caused by the rapid emergence of drug-resistant Campylobacter. While studies have shown that the clinical use of antibiotics, especially quinolones is to speed up bending the main reason for the emergence of bacteria resistant. Generally, antibiotic susceptibility testing standard method of operation of most micro-organisms are applicable, which is widely accepted and applications provided by the CLSI (Clinical Laboratory Standards Association) guidelines. Campylobacter is a micro-aerobic, microaerobic conditions to grow, the experimental operating standards of its antibiotic susceptibility testing is not available, there is no the drug breakthrough point clear criteria of. Therefore, in the clinical microbiology laboratory testing appear in many different ways, for example: the disk diffusion method and the broth dilution method. The purpose of this study is to (1) compare disk diffusion method and broth dilution method to determine the disk diffusion method Campylobacter susceptibility testing can become a reliable alternative method (2) analysis of drug-resistant genes in Campylobacter jejuni and distribution of Campylobacter jejuni molecular epidemiological surveillance, research and development of new antibacterial drugs and guide clinical provide a scientific basis for the proper use of antibiotics. (3) the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) Detection and typing of Campylobacter. In this study, disk diffusion method and broth microdilution method were measured 203 Campylobacter jejuni and 19 Campylobacter resistance to 11 antibiotics. 203 Campylobacter resistance to 11 antibiotics, according to the broth microdilution method were: of Erythromycin (2.46%), streptomycin (11.82%), ciprofloxacin (91.13%), amoxicillin (14.78%), clindamycin (4.93%)), the Cefotaxime (5.91%), tetracycline (84.73%), levofloxacin (83.74%), norfloxacin (88.67%), enrofloxacin (88.67 %), and nalidixic acid (88.18%). The results of the disk diffusion method and broth microdilution method, two methods ciprofloxacin, norfloxacin, enrofloxacin, levofloxacin, nalidixic acid and streptomycin results with higher consistency, several other antibiotics coincidence rate is relatively low. According to the statistical analysis of the results, we suggest the following: erythromycin sensitive the breakthrough point by in ≤ 0.5μg/mL to ≤ 1μg/mL; the the tetracycline sensitive breaking point from the ≤ 4μg/mL changed 2μg/mL; Brooklyn the neomycin resistance inhibition zone diameter ≤ 18mm to ≤ 14mm; amoxicillin sensitive breaking point by change ≤ 8μg/mL to ≤ 4μg/mL drug breakthrough point ≥ 32μg/mL instead ≥ 64μg / mL, which will significantly improve the coincidence rate of the two methods. The test results showed that Campylobacter resistance gene, six streptomycin resistant isolates carrying the aadA gene, accounting for 2.96% of the total, 12 streptomycin resistant to bending the bacteria carrying aadE, accounting for 5.91% of the total. 84.73% of the isolates carrying tetO gene, these isolates were highly resistant to tetracycline. Erythromycin-resistant Campylobacter gene sequence analysis of the results showed that erythromycin resistant Campylobacter presence of a A2075G point mutation in the 23S rRNA gene. However, on 23 quinolone-resistant Campylobacter gyrA gene sequence analysis showed that, does not appear typical of the C257T mutation. Using semi-quantitative RT-PCR was used to detect the the 23 quinolone resistance of Campylobacter cmeB gene expression. The results showed that the 16 Campylobacter cmeB overexpression. This result show that the The gene overexpression cmeB and Campylobacter related to quinolone antibiotic resistance. MALDI-TOF MS genotyping method for the study of the relationship between Campylobacter strains genotyping study of nine isolates cultured by the same conditions, the application of MALDI-TOF MS detection. The results show that the mass spectrum of Campylobacter in 2000-9551 within the range of mass-to-charge ratio, identification showed No. 8 Campylobacter database MB 5 195 0 5 THL Campylobacter is highly homologous to the the NCTC11168 and numbered the MB 7 240 0 5 THL Campylobacter highly homologous. MALDI-TOF MS genotyping of Campylobacter to certain typing ability, can become an important tool for the identification of Campylobacter analysis.

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