Abstract
The number of multidrug-resistant strains of Riemerella anatipestifer continues to increase, and new strategies for the treatment of associated infections are necessary. Recently, numerous studies have shown that efflux pumps (EPs) play key roles in universal bacterial mechanisms that contribute to antibiotic resistance. In addition, studies have shown that the effects of antibiotics that are subjected to efflux can be reinforced by their combined use with efflux pump inhibitors (EPIs). Unfortunately, the role of the efflux system in R. anatipestifer remains barely understood. In this study, we evaluated the role of EPs and resistance genes in the resistance generated by clinical strains of R. anatipestifer to antibiotics. A set of 10 R. anatipestifer strains were characterized by drug resistance, associated resistance genes, and antibiotic profiles in the presence and absence of EPIs. Efflux activity was studied on a real time basis through a fluorometric method. Quantification of the levels of mRNA transcription of efflux pump genes (EPGs) was determined by RT-qPCR. Several approaches (detection of resistance genes, drug susceptibility testing, and growth kinetics analysis) were used to assess the correlation between the effect of the EPIs and the resistance levels. Analysis of the R. anatipestifer growth inhibition tests showed that the antibiotic activity was enhanced by the synergy of EPIs. Among the various resistance genes that confer antibiotic resistance, different minimum inhibitory concentrations (MICs) were observed. The different levels of resistance were reduced by EPIs. Real time fluorometry showed that all the R. anatipestifer strains presented inherent efflux activity, conferring varying levels of inhibition in the presence of EPIs. Moreover, 15 EPGs were overexpressed in the presence of antibiotics. The addition of EPIs to antibiotics led to downregulation in the expression of some EPGs and a simultaneous increase in drug resistance and sensitivity. These results demonstrated the contribution of these EPs in the resistant phenotype of the clinical strains of R. anatipestifer that are under investigation, independently of the resistant genotype of the respective strains. Intrinsic efflux activity was possibly linked to the evolution of resistance in multidrug-resistant isolates of R. anatipestifer. Furthermore, the inhibition of EPs by EPIs could enhance the clinical effects of antibiotics.
Highlights
Duck serositis, caused by Riemerella anatipestifer, is an important communicable disease (Zheng et al, 2012; Guo et al, 2017)
Microorganisms possess remarkable capacities to counteract the action of antimicrobial agents, thereby, conferring resistance
The mechanisms of resistance mainly entail the production of drug inactivating enzymes, alteration of drug targets, mobile resistant genetic elements, and prevention of drug access; this last mechanism refers to the functions of drug efflux and influx (Du Toit, 2017)
Summary
Duck serositis, caused by Riemerella anatipestifer, is an important communicable disease (Zheng et al, 2012; Guo et al, 2017). R. anatipestifer is a gram-negative, rod-shaped, non-sporeforming, non-motile bacterium that can primarily infect ducks that are l−5 weeks of age, via the respiratory or digestive tracts (Chikuba et al, 2016). Infection with this pathogen leads to great economic losses owing to the significant weight loss observed in ducklings and high mortality rates of up to 75% (Wang Q. et al, 2017). At least 21 different serotypes of R. anatipestifer have been identified, and crossimmunoprotection among different serotypes has been barely reported (Zhai et al, 2013; Gyuris et al, 2017) All these factors complicate the prevention of this disease.
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