Study On Antimicrobial Resistance Mechanisms In Campylobacter Isolates From Livestock And Poultry

Campylobacter,especially Campylobacter jejuni and Campylobacter coli,are important foodborne pathogens that can be transmitted to humans through food chain,causing gastroenteritis as well as other disease.The overuse of antimicrobial agents in both food animals and clinical settings promote the emergence and dissemination of multidrug resistance in Campylobacter.The prevalence of multidrug resistance Campylobacter in China is much higher than that in other countries and the characterized antimicrobial resistance mechanisms could not explain this situation.In this study,we aim to investigate and identify novel resistance mechanisms based on high prevalence and high level of Campylobacter resistance to multiple antimicrobials.The efflux pump CmeABC is an important intrinsic mechanism conferring multidrug resistance to various antimicrobial agents and toxins and also plays an important role in its physiology in Campylobacter.Our study found that those Campylobacter isolates showed exceedingly high-level resistance to ciprofloxacin contains the CmeABC variant.Sequence analysis of the CmeABC variant indicated that CmeA and CmeC were homologous?>98%amino acid identities?to CmeA and CmeC in NCTC 11168,while CmeB in these strains showed only 81%amino acid sequence identity to CmeB in NCTC 11168.Further study confirmed that this efflux pump CmeABC variant could confer Campylobacter isolates increased resistance to multiple antimicrobials,thus we designated this CmeABC variant as RE-CmeABC.RE-CmeABC promoted the emergence of fluoroquinolone-resistant Campylobacter mutants and expanded the mutant selection window for fluoroquinolones.The extremely high-level resistance to ciprofloxacin was conferred by RE-CmeABC together with the C257T mutation.Accumulation assays suggested that RE-CmeABC has an enhanced efflux function than wild type CmeABC.Subsequently,we performed structural modeling of CmeB to predict the interaction between substrates and their structures.The results revealed that RE-CmeB tends to use the mutated residues to bind drugs,which suggested that the amino acid changes enhance drug binding in RE-CmeB.Furthermore,a total of 2002 Campylobacter isolates of various regions in China was detected for the presence of RE-cmeABC gene by PCR,the result suggested that rising prevalence of RE-cmeABC in Campylobacter isolates was observed during 2012-2014 and the detection rate of RE-cmeABC in C.jejuni was significantly higher than in C.coli.RE-cmeyABC-carrying isolates showed both overall genetic diversity and regional clonality by PFGE.Natural transformation verified that horizontal transmission involved in dissemination of this variant,which explained that RE-cmeABC-positive Campylobacter isolates increased so quickly to some extent.These findings reveal a previously undescribed mechanism for enhanced multidrug resistance and open a new direction for us to understand how bacteria adapt to antibiotic treatment.In China,the gentamicin resistance rate in Campylobacter has been increased in recent years.However,the cause of resistance mechanisms is largely unknown.Analysis of 607 Campylobacter strains of chicken and swine origins collected in the year of 2014 showed that 15.6%?25/160?of C.jejuni isolates and 79.9%?357/447?of C.coli isolates were resistant to gentamicin.We examined the gentamicin resistance determinants by PCR.The aph?2'?-If was identified in 10.0%?16/160?of C.jejuni and 57.7%?258/447?of C.coli isolates,which was significantly higher than aacA/aphD with 2.5%?4/160?in C.jejuni and 15.2%?68/447?in C.coli,respectively.The aph?2"?-If gene could transfer to susceptible strain,NCTC 11168 by natural transformation.Whole genome sequencing indicated that aph?2'?-If gene was found in a novel resistant genomic island,which was inserted between conserved genes,cj0299 and panB.This novel genomic island included multiple resistance genes,aph?2'?-If,aac,aadA,aph2,aad9,aphA3,aphA7,and cat,which could confer Campylobacter resistance to gentamicin,kanamycin,neomycin,amikacin and chloramphenicol.Cloning of aph?2"?-If into gentamicin susceptible C.jejuni NCTC 11168 confirmed its function in conferring high-level resistance to gentamicin and kanamycin.The aph?2"?-If gene was firstly reported in China,and was firstly identified on chromosome in Campylobacter.Furthermore,molecular typing by PFGE suggested that both horizontal transmission and clonal expansion were involved in dissemination of aph?2"?-If gene in China.Fosfomycin can effectively treat enteritis caused by Campylobacter,and is alternative antimicrobial agent for the treatment of campylobacteriosis.We found a C.coli DZB4 from swine origin,which was highly resistant to fosfomycin?>512?g/mL?.The fosfomycin resistance in DZB4 could be transferred to C.jejuni NCTC 11168 by natural transformation.Whole genome sequence of the transformant showed that a 9 Kb,11 ORFs,segment was inserted between conserved genes cadF and CC01582 on the chromosome of NCTC 11168.Interestingly,an ORF encodes a 136 amino acid protein that exhibits 26.9%,34.2%and 63.9%identity to fosfomycin resistance determinants FosA,FosB and FosX,respectively.Functional cloning of this ORF revealed that it could confer Campylobacter and E.coli resistance to fosfo4ycin,designated fosX^CC,and could disseminate between C.coli and C.jejuni.In conclusion,we identified several novel antimicrobial resistance mechanisms in Campylobacter of food animal origin in China.For the first time,we described the emergence of an efflux pump variant,named RE-CmeABC,which is much more potent in conferring Campylobacter resistance to multiple antibiotics and involved in extremely high level resistance to ciprofloxacin.Moreover,the characterization of aph?2"?-If and fosX^CC gene mediating aminoglycosides and fosfomycin resistance,respectively,are also firstly described in this study.These findings not only demonstrated the novel antimicrobial resistance mechanisms in Campylobacter but also provided new insights for research on drug resistance in Campylobacter.