Study On The Molecular Basis Of ISEcp1Involved In Drug Resistance In Shigella

Shigellosis remains a public health concern throughout the world and has become an actual threat. Owing to the wide use of antibiotics, the emergence of resistant strains of Shigella spp. is of great interest both in developing and industrialised countries.β-Lactamases, enzymes that can hydrolyse β-lactams, have attracted much attention in β-lactam resistance research. Extended-spectrum β-lactamases (ESBLs), which can hydrolyse expanded-spectrum cephalosporins (such as cefotaxime, ceftriaxone, ceftazidime or cefepime) and monobactams (aztreonam), represent a public health concern. Among them, the CTX-M family has the highest number of variants.It has been shown that different insertion sequences are associated with blaCTX-M genes. ISEcpl-like insertion sequences have been frequently reported. They have been associated with four of the six blaCTX-M gene clusters.ObjectiveThe aim of this study was to describe the distribution and location of ISEcpl and blaCTX-M, investigate the mechanisms of drug resistance and specifically to determine the role of ISEcpl transposition upstream of the blaCTX-M gene in Shigella treated with cephalothin. Moreover, the activity of ISEcpl in promoting blaCTX-M translation was also examined. Methods1. Selecting bacteria from the suspicious shigella system by microbial culture and biochemical identification;2. Susceptibility test was performed by Kirby-Bauer:Isolates of Shigella spp. were determined their susceptibility to antimicrobial agents by the disk diffusion method on Mueller-Hinton agar according to the recommendations of the Clinical and Laboratory Standards Institute. The minimal inhibitory concentration were also conducted. Escherichia coli ATCC25922was used as a quality control strain;3. In vitro, Shigella spp. resistant to cephalothin was inducted for20passages by1/2MIC of Cephalothin;4. Sequencing whole genome of sensitive strain and resistant strain by inducted and comparing the sequences;5. Comparing the location of ISEcpl and blaCTX-M sensitive strain and resistant inducted strain by PCR, cloning, sequencing;6. Investigating the activity of ISEcpl in promoting blaCTX-M translation by RT-PCR and Extended-spectrum β-lactamase detection.7. PCR ISEcpl and blaCTX-M in Shigella and describe the location of them;8. Analyzing the relationship of the drug resistance and the purpose genes.Results1. The strain generated after cephalothin treatment was named S. flexneri YDC, which exhibited a32-to16384-fold increase in its MIC compared with the basal MICof the indicator to β-lactams.2. The genome of S. flexneri YDC was different to the S. flexneri mel-sf1998023/zz including SNV, delete and insert. Besides, the copys of IS are different between mel-sf1998023/zz and YDC.3. S. flexneri YDC had one KEGG metabolic pathway over the S. flexneri mel-sf1998023/zz. The pathway was ko00930:Caprolactam degradation.4. ISEcpl was inserted upstream of the blaCTX-M gene in S. flexneri YDC but not in5. flexneri mel-sf1998023/zz.5. This blaCTX-M-55gene was preceded by promoter sequences [-35(TTGAAA) and -10(TACAAT)], which may drive the expression of β-lactam resistance.6. Electrophoresis analysis showed that expression levels of blaCTX-M-55were elevated in E. coli DH5a(pISECTX).7. The susceptibility of E. coli DH5α(pISECTX) to β-lactams was2-2048-fold higher than that of E. coli DH5α(pCTX).8. ESBL was generated only by E. coli DH5α(pISECTX) among DH5α(T), DH5α(pCTX) and DH5α(pISECTX).9.39.48%Shigella strains were resistant to β-lactams. The resistant rates for233Shigella spp. to CF was35.2%, to CZ was28.3%, to CXM was13.7%, to CTX was12.9%, to CAZ was9.0%, to FEP was5.2%.The most common was resistant to cfalothin and cefazolin.10. The drug resistant rates for CXM and CTX have tendencies to rise year by year.In the same time, the frequencies of occurrence for ISEcp1and blaCTX-M have tendencies to rise year by year too.11. The drug resistant rate for CF, CZ, CXM, CTX of ISEcpl positive group were higher than ISEcp1negative group.12. The drug resistant rate for CXM and CTX of blaCTX-M positive group were higher than blaCTX-M negative group.13. The drug resistant rate for CF, CZ, CXM, CTX of ISECTX positive group were higher than ISECTX negative group.Conclusions1. The susceptibility to β-lactams in Shigella spp.could decrease under low dose of β-lactams.2. S. flexneri YDC had one KEGG metabolic pathway over the S. flexneri mel-sf998023/zz. The pathway was ko00930:Caprolactam degradation. The KEGG metabolic pathway may be relevant to drug resistance by inducted.3. ISEcpl element could translocate upstream of blaCTX-M in S. flexneri following induction by cephalothin. After ISEcpl element could translocate upstream of blaCTX-M, the ISEcpl element appears to act as a potent activator for blaCTX-M gene expression. The ISEcpl sequence may initiate higher-level expression of the blaCTX-M gene. The promoter element of ISEcp1is necessary for the higher-level expression of blaCTX-M, leading to bacterial resistance to extended-spectrum cephalosporins.4. The drug resistant rates for β-lactams have tendencies to rise year by year with the rise of frequencies of occurrence for ISEcpl and blaCTX-M.5. ISEcpl and blaCTX-M are related to the β-lactams resistance in Shigella.