Influence Of Subinhibitory Concentrations Of Antibiotics On Biofilm Formation In Antimicrobial-Resistant Salmonella Of Porcine Origin

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 originTwo 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 isolatesThe 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.