Antibacterial Resistance In Clostridioides Difficile And Mechanism Of Resistance To Rifamycin

Objective: Clostridioides difficile,which is a Gram-positive,sporeforming,toxin-producing obligate anaerobic bacterium is the most commonly identified cause of healthcare-associated infection and has established itself as an important community pathogen.Antibacterial agents may promote C.difficile infection by disrupting intestinal microflora structure and allowing C.difficile to proliferate,colonize the colon,produce toxins and cause infection.Symptoms of C.difficile infection include fever,diarrhea and in severe cases pseudomembranous colitis and even death.Many epidemiological studies have shown an alarming increase in incidence of C.difficile infection worldwide over the last 20 years,with a significant financial burden on the healthcare system.The increased number of infections has been mainly associated with the emergence of highly virulent BI/NAP1/027 C.difficile strains.Resistance to multiple antibacterial agents plays a critical role in driving C.difficile dissemination and epidemiology.Genetic analysis have demonstrated that C.difficile has a versatile genome content,which is the determinant for antibacterial agents resistances.Several mechanisms responsible for antibacterial agents resistance have been identified in C.difficile,including chromosomal resistance genes,mobile genetic elements,alterations in the antibacterial agent targets or in metabolic pathways,and biofilm formation.Furthermore,recent evidences support that C.difficile resistance to some antibacterial agents may be complex and multifactorial.The object of this study was to further explore the antibacterial agents susceptibility and mechanism of resistance to rifamycin of C.difficile in Shijiazhuang district.Methods:Two hundred nonduplicated strains were selected from Hebei Provincial Bank for Medical Culture Collections verified by matrix assisted laser desorption/ionization time of flight mass spectrometry and 16 S rRNA gene sequencing.Polymerase chain reaction for toxin genes and binary toxin gene test and ribotype were used.Agar dilution method was performed to determine the minimal inhibitory concentration of 14 antibacterial agents including fidaxomicin,metronidazole,and vancomycin and so on against C.difficile.Bioinformatics methods were used to further explore the protein structural basis of reduced susceptibility of C.difficile to rifamycin.Results:All strains in this study were toxigenic,and were assigned to 46 ribotypes.RT 012,RT 001,HB 024 and RT 017 were predominant and accounted for 48.50 %.The antibacterial agents resistance rates were distinctly higher for ciprofloxacin,clindamycin,erythromycin and tetracycline which were more than 30.00 % than for rifaximin,levofloxacin,ceftriaxone,tigecycline,rifampin and chloramphenicol which were less than 20.00 %,while all strains were susceptible to meropenem,fidaxomicin,metronidazole and vancomycin.Multi-drug-resistant rate was as high as 68.50 %.Data from sequencing of rifamycin resistance determination region revealed that specific mutations,Asp492 Tyr,His502Asn,His502 Tyr,His502Asp and Arg505 Lys,were responsible for the reduced susceptibility to rifamycin.Protein structure modeling and molecular docking provided further evidence that the affinity between rifamycin and RNA polymerase beta subunit mutants was decreased.Conclusions:This study found that C.difficile was highly susceptible to fidaxomicin,metronidazole and vancomycin which are for the treatment of C.difficile infection,whereas C.difficile was highly resistant to ciprofloxacin,clindamycin,erythromycin and tetracycline which should be taken seriously.It was illustrated that the decrease in affinity of rifamycin and its target protein may explain the reduced susceptible of C.difficile to rifamycin.