导　　师： 栗绍文

学科专业： 090602

授予学位： 硕士

作　　者： ;

机构地区： 华中农业大学

摘　　要： 沙门菌是一种常见的重要人兽共患病原,由于抗生素的广泛使用,沙门菌的耐药性已非常严重。而研究表明,抗生素不仅仅是杀灭细菌的武器,还可以作为信号分子,引起细菌生物膜形成、运动性和毒力等表型的改变。c-di-GMP是一种广泛存在于细菌中的新型第二信使,可调控细菌生物膜的形成。抗生素对沙门菌的生物膜形成是否有影响,以及c-di-GMP通路是否参与其影响,目前国内外尚未见报道。本论文对109株猪源沙门菌进行耐药性和生物膜形成能力鉴定,进而研究亚抑菌浓度抗生素对沙门菌生物膜形成的影响及c-di-GMP通路在其中发挥的作用。结果如下： 1.猪源沙门菌的耐药性分析 109株猪源沙门菌分离株经血清型鉴定和生化分析,包括101株猪霍乱沙门菌和7株鼠伤寒沙门菌,另外1株未定型。采用K-B法检测沙门菌对15种抗生素的敏感性,结果显示,109株沙门菌均表现为多重耐药,以同时耐9种/(28.4/%/)和10种/(27.5/%/)抗生素的菌株最多。分离株对磺胺类药物的耐药率最高,磺胺甲基异恶唑和复方新诺明分别为99.1/%和97.2/%,对氯霉素和四环素的的耐药率也均在90/%以上,对喹诺酮类药物环丙沙星和诺氟沙星的耐药率较低。 进而采用PCR对沙门菌的耐药基因进行了检测,检出率结果表明：β-内酰胺类耐药基因blaTEM为45.0/%,氨基糖苷类耐药基因aadA1、aadA2、aacC2、aac/(3/)-Ⅳ和aph/(3'/)-Ⅱ分别为92.7/%、99.1/%、31.2/%、47.7/%和33.9/%,喹诺酮类耐药基因aac/(6'/)-Ib-cr为58.7/%,四环素类耐药基因tetA和tetB分别为54.1/%和42.2/%,氯霉素耐药基因catA1、cmlA和floR分别为33.0/%、42.2/%和69.7/%,磺胺类药物耐药基因sul Ⅱ、sul Ⅰ和sul Ⅲ别为99.1/%、60.6/%和55.0/%。而blaPSE、aadB、armA、rmtB、 qnrA、qnrB、gnrS、gepA、tetC和tetG等耐药基因均未检出。91株沙门菌/(83.5/%/)携带Ⅰ类整合子,其中33株/(36.3/%/)携带dfrA12-orfF-aadA2耐药基因盒。 2.亚抑菌浓度抗生素对沙门菌生物膜形成的影响 经微孔板法检测109株沙门菌的生物膜形成能力,筛选出5株强生物膜形成株,且均为鼠伤寒沙门菌。在亚抑菌浓度四环素、链霉素、阿米卡星和环丙沙星作用下,检测5株沙门菌株生物膜形成量的变化,结果显示,仅环丙沙星可显著增强其中3株沙门菌的生物膜形成量,并存在量效关系,在1//8MIC环丙沙星作用时,沙门菌3764株的生物膜形成量达到最大。此外,亚抑菌浓度的四环素和阿米卡星可明显抑制沙门菌3764株的swimming和swarming运动性,环丙沙星可抑制菌株的swimming,但对swarming运动无影响,链霉素则对菌株的2种运动性均无影响。 细菌第二信使c-di-GMP调控生物膜的形成,试验选择c-di-GMP的合成基因adrA、stm4511,降解基因yhjH,调控基因csgD、csrA以及运动性相关基因fliC,作为目的基因。在亚抑菌浓度的环丙沙星作用下,检测沙门菌3764株所选基因的相对转录水平变化。结果显示,与对照组相比,药物组基因yhjH、csgD、fliC、csrA和stm4511的转录水平均显著下降,adrA的转录水平无明显变化。c-di-GMP在亚抑菌浓度抗生素对沙门菌生物膜形成影响中的作用尚有待进一步研究。 Salmonella is a major zoonosis pathogen. At present antimicrobial resistance is extremely severe in Salmonella isolates due to the widespread use of antibiotics. Researches showed that antibiotics are not only weapons that can kill bacteria, and as a signal molecule it can cause the change of bacterial phenotype, such as biofilm formation, motility, virulence. C-di-GMP as a novel second messenger which exists in bacteria extensively can regulate biofilm formation in bacteria. Until now it has not been reported whether antibiotic produces an effect on biofilm formation in Salmonella and whether the c-di-GMP pathway is involved in biofilm formation. In this study, the antimicrobial resistances of109Salmonella isolates of porcine origin was evaluated, some strains with biofilm formation were screened. Influence of subinhibitory concentrations of antibiotics on biofilm formation in Salmonella isolates and whether the effect was modulated by c-di-GMP were researched. The results were showed as follows: 1. Evaluation of the antimicrobial resistance of Salmonella isolates of porcine origin Two serovars were identified among the109Salmonella isolates, among which101isolates were S. Choleraesuis,7isolates were S. Typhimurium and1isolate was unknown. The antimicrobial susceptibility was detected by Kirby-Bauer disk diffusion method. Multidrug resistance was existed in all109Salmonella isolates,31/(28.4/%/) isolates were resistant to nine antimicrobials, and30/(27.5/%/) isolates were resistant to ten antimicrobials. The most prevalent resistance ratio were sulfamethoxazol /(99.1/%/) and selectrin /(97.2/%/) respectively, the resistance rate of isolates to chloramphenicol /(95.4/%/) and tetracycline /(94.5/%/) is also over90/%. However, most isolates were sensitive to ciprofloxacin and norfloxacin. The prevalence of antimicrobial resistant genes was detected. The results showed that the percentage of β-lactam resistance gene blaTEM was45.0/%; aminoglycoside resistance gene aadAl, aadA2, aacC2, aac/(3/)-Ⅳ and aph/(3'/)-Ⅱwas92.7/%,99.1/%,31.2/%,47.7/%and33.9/%, respectively; fluoroquinolone resistance gene aac/(6'/)-Ib-cr was58.7/%; tetracycline resistance gene tetA was54.1/%and tetB was42.2/%; chloramphenicol resistance gene catAl, cmlA and floR was33.0/%,42.2/%and69.7/%; sulfonamide resistance gene suⅢ, sul Ⅰ and sulⅢ was99.1/%,60.6/%and55.0/%. However, blaPSE, aadB, armA, rmtB, qnrA, qnrB, qnrS, qepA, tetC and tetG genes were not detected. Class1integrons was common present /(83.5/%/) in Salmonella isolates,33isolates carried resistance gene cassettes of dfrA12-orfF-aadA2. 2. Effect of subinhibitory concentrations of antibiotics on biofilm formation in Salmonella isolates The biofilm-forming ability of109Salmonella isolates was detected by microtiter plate assay, and five isolates were identified as strong biofilm formation strains which all were S. Typhimurium. Influences of specific antibiotics /(tetracycline, streptomycin, amikacin and ciprofloxacin/) at subinhibitory concentrations on the biofilm formation in Salmonella isolates were researched. Only ciprofloxacin significantly enhanced biofilm formation with a dose-dependent effect in three Salmonella isolates. The amount of biofilm formation reached the maximum in Salmonella isolate3764in1//8MIC of ciprofloxacin. In addition, tetracycline and amikacin at subinhibitory concentrations inhibited swimming and swarming of Salmonella isolate3764. Ciprofloxacin can inhibit swimming, but it had no effect on swarming of isolate. However, streptomycin had no effect on swimming and swarming of isolate. C-di-GMP as the bacterial second messenger regulated biofilm formation. C-di-GMP synthesis gene/(adrA, stm4511/), degradation gene /(yhjH/), regulatory gene /(csgD, csrA/) and motility related gene /(fliC/) were selected as the target genes. Relative transcription level of these genes was detected at subinhibitory concentrations of ciprofloxacin in Salmonella isolate3764. Compared with the control group, the transcription level of yhjH, csgD, fliC, csrA and stm4511were decreased significantly; however, there is no apparently change in transcription level of adrA. Subinhibitory concentrations of ciprofloxacin enhanced biofilm formation in Salmonella isolates, but the mechanism remains to be further researched.

关 键 词： 沙门菌 耐药性 亚抑菌浓度 抗生素 生物膜

分 类 号： [S852.61]

领　　域： [农业科学] [农业科学] [农业科学]