Mechanisms Of Drug Resistance In Enterobacteriaceae And Pathogenicity Of UPEC To HeLa Cell

ObjectiveTo investigate the mechanisms of carbapenems resistance and/or reduced susceptibility to carbapenems in clinical isolates of Klebsiella oxytoca and Enterobacter cloacae.MethodsA carbapenem-resistant K.oxytoca ZC101 strain and a carbapenem-non-susceptible E. cloacae ZY106 strain isolated in our hospital were investigated.Minimum inhibitory concentration(MIC) values of various antibiotics were determined by agar dilution method.Conjugation experiment was carried out in mixed broth cultures,and Escherichia coli EC600 or C600 strain was used as recipient.The crudeβ-lactamase extracts of ZC101,ZY106 and their E.coli transconjugants were subjected to analytical isoelectric focusing(IEF).PCRs and DNA sequence analysis were preformed to confirm the genotype of drug resistance genes.Plasmid currying experiment was performed to confirm the attribution of reduced susceptibility to carbapenems.Outer membrane proteins(OMPs) of K.oxytoca ZC101 were isolated and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and the ompK35 and ompK36 genes were amplified by using PCR and were sequenced.OMPs of E.cloacae ZY106 were isolated and examined by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis(Urea-SDS-PAGE).ResultsMICs of imipenem,meropenem and ertapenem for ZC101 were 16μg/ml,16μg/ml, and 128μg/ml,respectively,and those for ZY106 were 2μg/ml,4μg/ml,and 16μg/ml, respectively.Conjugation study with E.coli EC600 resulted in the transfer of significant reduced carbapenem susceptibility from ZC101(MICs increased at least four-fold).The IMP-4-plasmid eliminated strain of ZC101 still has high resistance against the carbapenems.Conjugation experiments with E coli C600 resulted in the transfer of drug resistance gene from ZY106,and MICs of carbapenems were increased from 0.0625μg/ml to a range of 0.25 to 0.5μg/ml.IEF,PCRs and DNA sequence analysis confirmed that ZC101 produced IMP-4 metallo-β-lactamase and CTX-M-14 extended-spectrumβ-lactamase,while E.coli transconjugant produced a single IMP-4.Amplification of integron revealed that blaIMP-4 gene located within a class I integron that was carried on a plasmid with a size of approximately 55 kb.Meanwhile,the results of IEF,PCRs and DNA sequence analysis showed that ZY106 produced IMP-1 metallo-β-lactamase and CTX-M-3 extended-spectrumβ-lactamase,but E.coli transconjugant produced a single IMP-1.A plasmid-mediated quinolone resistance determinant qnrS1 was detected in ZY106. Transfer of the qnrS1-encoding-plasmid into E.coli EC600 by conjugation resulted in intermediate resistance to ciprofloxacin(MIC,2μg/ml) in E.coli transconjugant. Plasmid analysis indicated that the bla_IMP-1 was located in a plasmid with a size of approximately 50 kb while the size of qnrS1-encoding-plasmid was approximately 60 kb. SDS-PAGE analysis of outer membrane proteins of K.oxytoca ZC101 indicated its lack of OmpK36.Sequence analysis of ompK36 gene of K.oxytoca ZC101 showed disruption by an insertion sequence IS5.Urea-SDS-PAGE analysis of OMPs showed that ZY106 lacked an OMP of approximately 38 kDa.Conclusions1.Carbapenem resistance in K.oxytoca ZC101 is mainly due to production of IMP-4 metallo-β-lactamase combined with the loss of the OmpK36.2.Reduced susceptibility to carbapenems in E.cloacae ZY106 is mainly due to production of IMP-1 metallo-β-lactamase combined with the loss of an OMP.A plasmid-mediated quinolone resistance determinant qnrS1 is detected in the same isolate. Objectives To investigate the bacteria flora composiotn of the urinary tract(UT) in the UT infected patients;To analyze the drug resistance profile and extended spectrumβ-lactamases(ESBLs) detection rate of the Escherichia coli that caused the urinary tract infection,and to study its drug resistance for cephalosporin and quinolones;To understand the distribution of pathogenic genes in uropathogenic E.coli(UPEC);To screen the high virulent UPEC strains through HeLa cell model and to analyze the pathogenicity of UPEC to HeLa cell.Methods We retrospectively studied the data of bacteria isolating,ESBLs detection rate from different regions and drug resistant profile from 59 hospitals of Zhejiang Province in 2007.Meanwhile,we investigated the statistical data of bacteria isolated from urine specimen and other clinical specimen,drug resistance and ESBLs detection rate in the past nine years ranging from 2000 to 2008 in our hospital.We have compared the drug resistance of E coli strains isolated from urine specimens with those from other specimens.Drug resistant genes of the resistant strains were detected by specific PCR and the products were sequenced by PCR.Genotypes and mutations of those genes were confirmed by blasting with the known genes in GenBank.Genetic relationship of isolated strains was examined by pulsed field gel electrophoresis(PFGE).Molecular typing of E coli strains isolated from urinary tract infected patients was determined by a PCR method.Virulence genes of those E coli strains were determined by using PCR method to analyze the distribution of pathogenic genes in those E coli strains.The E coli strains with high pathogenicity isolated from urinary tract infected patients were initially screened by using adhesion experiments and Trypan-blue staining.HeLa cell early apoptosis induced by the high pathogenic strains was examined by flow cytometry and the morphology changes were confirmed by using transmission electron microscopy.Results The results from 59 hospitals of Zhejiang province in 2007 showed that E coli is the most common cause of infection.ESBLs detection rate of E coli from different regions of Zhejiang province is above 50%.The resistance rate against cephalosporins is above 45%,while the resistance rate of quinolones is the highest,above 60%. Retrospective study of data from our hospital in the past nine years indicated that E coli is the most frequently isolated strain from urine specimen for the past nine years.The ESBLs positive E coli strain detection increased from 7.2%in 2000 to 53.7%in 2008. The resistant rate of cephalosporins also increased from 20%to 50%.However, resistance to ciprofloxacin and gentamicin was maintained at high levels since nine years ago,around 70%and 60%respectively.The drug resistant rate against cephalosporins in E coli isolated from urinaray tract specimens is significantly lower than that in the strains isolated from other sources,while the resistant rate against quinolones and aminoglycoside is similar in the strains from different specimen sources.Through case inquiring,we selected 28 cases of hospitalized patients,who showed a leukocyte level at above ++ by routine urianalysis,and had symptoms of urinary tract infection.Among the 28 E coli strains isolated from the urinary tract infected patients, 64.29%of the isolates were resistant to cefotaxime and 62.49%was resistant to ciprofloxacin.Results of detection of drug resistance by PCR and DNA sequencing indicated that two isolates harbored both CTX-M-15 and CTX-M-14 genes,and 16 isolates only had CTX-M-14 gene.DNA sequence analysis of gyrA and parC in the quinolones resistant strains found that there were several mutations in the QRDRs.The qnr gene in two isolates was detected as qnrA1 and qnrS1,respectively.PFGE analysis showed that the E coli strains isolated from urine specimen had high genetic heterogeneity,with only two strains belonging to the same clone according to PFGE profile.PCR molecular typing indicated that those isolates mainly belong to type D (60.71%),following type B2(35.71%).TypeⅠpilus gene was the most common virulence gene with a positive rate at about 96.43%,while only six strains were positive for usp gene.We obtained two high virulent isolates through the pathogenic screening experiment(strain 6N and strain 27N). Both strains had various virulence genes.Strain 27N and 6N contained very similar virulence gene profile except that strain 27N did not contain usp gene.PCR molecular typing showed that strain 6N and 27N belonged to group B2 and group D,respectively. Both strains can destroy HeLa cell within 3 hours causing cell death.Results of apoptosis detected by flow cytometer revealed that strain 6N induced 20.75%of HeLa cells to an early stage apoptosis within 1.5 hours.On the other hand,strain 27N induced only 1.55%of HeLa cells to the early stage apoptosis.Morphology changes observed by transmission electron microscopy confirmed that the strain 6N can induce HeLa cells early apoptosisConclusions1.E.coli has an important role in bacterial infection,especially in urinary tract bacterial infection.2.Resistance of E coli to cephalosporin is mainly caused by the ESBLs production.The primary genotype of ESBLs in E coli is CTX-M-14 type.3.Resistance to quinolones is mainly caused by the mutations in gyrA and parC gene, along with qnr plasmid mediated drug resistance.4.High virulent UPEC strain carrying usp gene can induce HeLa cell rapid early apoptosis.