Comparison Of Fitness Between Salmonella Typhimurium And Salmonella Enteritidis Under Ciprofloxacin Selective Pressure

Non-typhoid Salmonella are one of the most common causes of food- and water-borne infections. The emergence and increasing multi-drug resistant(MDR) strains may through food animals to humans along with the food chain and posed a serious threat to human health and public health. Fluoroquinolones(FQs), presenting unique antibacterial mechanism and broad-spectrum bactericidal activity, are used in the treatment of a variety of salmonellosis. However, resistance to FQs in Salmonella rapidly emerged and become a great burden on the clinical treatment.Our previous epidemiological studies have shown that the Salmonella typhimurium was the predominant serotype among food producing animal farms, showing multiple resistant to fluoroquinolones, cephalosporins and other drugs. So we supposed that in the development of resistance the prevalent salmonella serotype had a low compensatory fitness cost which contribute to prevalent and persist of the predominant serotype strains in the environment. To answer this question, a ciprofloxacin-susceptible S. Enterica Typhimurium and a S. Enterica Enteritidis clinical isolates were selected undergoing the subsequent experiments. Serial mutants of respect serotypes were obtained in vitro from the above parent strains under increasing ciprofloxacin concentrations, undergoing to explore the mechanisms associated with the development of resistance, to compare the growth abilities and the competitiveness, and the survival and invasion ability when exposed to different concentrations of bile.A serial mutants of two different serotypes of Salmonella were obtained under the increasing ciprofloxacin pressures cultured in different medium. MICs were measured by agar dilution method, and PCR was used to detected the mutations of quinolone target genes gyr A, gyr B, par C, par E. The results showed the two parent strains had no mutations in QRDR of four target genes. In LB medium, the mutation of S464 F in gyr B occured when the Salmonella Enteritidis showed decreased susceptibility to ciprofloxacin(MIC, 0.25mg/L), once induced to high-level resistance(MIC, 32mg/L), two additional mutations also detected, the mutations of D87 A in gyr A and V461 G in par E. In M9 medium,the mutation of S464 F in gyr B occured when the Salmonella Enteritidis showed decreased IV susceptibility to ciprofloxacin(MIC, 0.125mg/L), once induced to high-level resistance(MIC, 32mg/L), the mutation of S83 F in gyr A also detected. For the Salmonella Typhimurium induced in LB medium, when the induced strains showed reduced susceptibility to ciprofloxacin, the mutation of G81 D occurred in gyr A. When the MIC of ciprofloxacin reach 16mg/L, additional mutation of G78 D in par C also appeared, then the mutation disappeared and the MIC of ciprofloxacin decreased to 4mg/L. Even if the induced concentrations of ciprofloxacin increased, the MICs still were 4mg/L. However, in the M9 culture conditions, when the MIC of ciprofloxacin reached 1mg/L, the mutation of S464 Y occurred in gyr B,and once the MIC reached 4mg/L, a further mutation of S83 Y in gyr A also detected. But then the mutation disappered and even if the induced concentrations of ciprofloxacin increased, the MICs still were 1mg/L.RT-PCR was used to detect the Acr AB-Tol C efflux pump expression levels of each mutants and associated with its increased expression of regulatory factors. The results showed that the Acr AB-Tol C efflux activity in Salmonella Enteritidis mutants had increased and Salmonella Typhimurium mutants had no Acr AB-Tol C efflux activity. As the main regulator, Ram A has regulatory role to expression of Acr AB-Tol C efflux pump.Using the colony counting method to detect the ability to grow on LB and M9 medium, while also the growth ability on LB medium supplemented with different concentrations of bile salts to determine the both serotypes strains fitness in different environments. The results indicated that under the initial antibiotic pressure, the growth ability of Salmonella Enteritidis were significantly greater than the Salmonella Typhimurium. But with the increased and sustained antibiotic pressure, the growth ability of resistant Salmonella Typhimurium wewe more significantly greater than the Salmonella Enteritidis.We assessed competitive advantages/disadvantages of the strains which showed different resistance levels at the same selected ciprofloxacin concentrations through pyrosequencing. The results revealed that the ciprofloxacin-susceptible Salmonella Enteritidis were more competitive than the corresponding Salmonella Typhimurium, but the resistant Salmonella Typhimurium more competitive than the Salmonella Enteritidis under the same ciprofloxacin concentration.In addition, we also measured the invasion ability of the two serotypes at different concentrations of bile salts. The results showed that in the absence of bile salts or at 0.1% bile salt concentration, the Salmonella Typhimurium showed an impaired invasion ability; but at 1% bile salt concentration, it was opposite.Conclusion: The mechanims in the development of resistance to ciprofloxacin was quite different betweent Salmonella Typhimurium and Salmonella Enteritidis. The development of resistance in Salmonella Typhimurium were mainly due to the mutation in gyr A, and the the efflux activity unrealted with Acr AB-Tol C pump. While the resistance to ciprofloxacin in Salmonella Enteritidis was mainly resulted from the over-expression of Acr AB-Tol C efflux pump and additional mutations in gyr B which contributed to the high level resistance; ciprofloxacin-resistant S. Enterica Typhimurium strains have the fitness advantage over the ciprofloxacin-resistant S. Enterica Enteritidis, and presented increasing invision ability comparing with ciprofloxacin-resistant S. Enterica Enteritidis when exposured to higher bile concentration.