Antimicrobial Resistance Profiles And Genotypes Of Campylobacter Spp Isolates And Subtyping Of Them Based On Matrix Assisted Laser Desorption Ionization Time Of Flight Mass

Thermophilic Campylobacter is one of the leading causes of human bacterial diarrhoea worldwide, with Campylobacter jejuni and Campylobacter coli representing the most frequently involved species. The incidence of human Campylobacter infections has increased markedly in both developed and developing countries worldwide and, more significantly, so has the rapid emergence of antibiotic-resistant Campylobacter strains, with evidence suggesting that the use of antibiotics, in particular the fluoroquinolones, as growth promoters in food animals and the veterinary industry is accelerating this trend.Campylobacter spp. are fastidious bacteria requiring microaerophilic conditions for growth. Standardized procedures for susceptibility testing are available for a wide range of organisms, and in general, the guidelines provided by CLSI are the most widely used; however, no internationally accepted criteria for susceptibility testing of Campylobacter spp. are available and breakpoints do not exist. Consequently, a number of different diffusion and dilution methods for example the disk diffusion method and the microbroth dilution method have been used in clinical, veterinary, and food microbiology laboratories.The aim of this study were (i) to conpare the the disk diffusion method and the microbroth dilution method and determine whether the disk diffusion method could be a reliable alternative method for antimicrobial susceptibility testing of Campylobacter spp; (ii) to analyze the resistance genotypes in Campylobacter jejuni and Campylobacter coli, and provide scientific data for molecular epidemiological surveillance of Campylobacter spp, research and exploitation of novel antimicrobials and guidance on clinical applying antimicrobials; (iii) to detect and subtype Campylobacter species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) .In this study, the susceptibilities of 184 Campylobacter jejuni and 19 Campylobacter coli isolates to eleven antimicribial agents were tested and analyzed by disk diffusion method and broth microdilution method. According to the broth microdilution method, resistance to Erythromycin (2.46%), Streptomycin (11.82%), Ciprofloxacin (91.13%), Amoxicillin (14.78%), Clindamycin (4.93%), Cefotaxime (5.91%), Tetracycline (84.73%), Levofloxacin (83.74%), Norfloxacin (88.67%), Enrofloxacin (88.67%), and Nalidixic Acid (88.18%) were detected. The correlation and the level of agreement between the broth microdilution and disk diffusion methods were analyzed. A high-level agreement between the two methods was evident for Ciprofloxacin, Levofloxacin, Norfloxacin, Enrofloxacin, Nalidixic Acid , and Streptomycin; while a lower level agreement was observed for other antibiotics. Our data suggest that the breakpoint for Erythromycin-susceptible Campylobacter strains may be more appropriately set at≤1μg/mL instead of≤0.5μg/mL, the MIC breakpoints for Tetracycline are changed to≤2μg/mg/ml for susceptible isolates; the zone diameter breakpoints for Clindamycin are changed to≤14mm for resistant isolates; and the MIC breakpoints for Amoxicillin are changed to≤4μg/mg/ml for susceptible isolates and≥64μg/mL for resistant isolates. The percent agreement between the microbroth dilution and the disk diffusion methods would increase dramatically.The aadA gene was detected in 2.96% isolates resistance to Streptomycin. The aadE gene was detected in 5.91% isolates resistance to Streptomycin. The tetO gene was detected in 84.73% isolates with high-level resistance to tetracycline. Sequence analysis showed that the Erythromycin resistant isolates had a point in the 23S ribosomal RNA genes (A2075G). However, none of the 23 fluoroquinolone resistant isolates had the typical point mutation in the gyrase gene gyrA(C257T). Semi-quantitative RT-PCR was used to determine the levels of expression of cmeB in 23 fluoroquinolone resistant isolates. Results showed that 16 isoltes over expressed cmeB. These data indicate that over expression of cmeB is associated with fluoroquinolone resistance in Campylobacter spp..The MALDI-TOF MS subtyping method was used to assess the extent of genetic diversity and clonality of 9 Campylobacter. spp isolates, and the differences of the mass spectrums were analyzed. All bacteria were cultured under the same conditions. The results reveal that Campylobacter jejuni could be distinguished in mass/charge (m/z) range from 2000 to 9551. 8 isolates are probable species identification with Campylobacter jejuni MB₅195₀5 THL, NCTC11168 are probable species identification with Campylobacter jejuni MB₇240₀5 THL. MALDI-TOF MS is a rapid and direct method for detection of Campylobacter jejuni from culture.