Molecular Epidemiology And Genetic Characteristics Of Fluoroquinolone-Resistant Escherichia Coil And Klebsiella Pneumoniae Isolated From Primary Healthcare Setting

Fluoroquinolones are synthetic antimicrobial agents, one of the most widely used antibiotics in clinical. The high frequency (64.5%-71.8%) of fluoroquinolone resistance in Escherichia coli was present since1990s in China. Up to now, the studies of molecular epidemiology and mechanism of fluoroquinolones resistance was focus on tertiary hospitals in central city, However, a shortfall exists in study on county hospitals and community infections. It is particularly important to understand the prevalence and characteristics of fluoroquinolone resistance in county hospitals for guiding the clinical application of these antibiotics.In the first section, a total of590consecutive nonduplicate clinical E. coli and569Klebsiella pneumoniae isolates from community infections were collected from30county hospitals distributed in seven geographic regions of China. Antimicrobial susceptibilities of isolates to17antimicrobial agents were determined; plasmid-mediated quinolone resistance (PMQR) genes qnrA, qnrB, qnrC, qnrD, qnrS, aac(6’)-Ib-cr, qepA and oqxAB were detected by PCR, and mutations in quinolone resistance-determining regions (QRDRs) of gyrA and gyrB subunit of DNA gyrase, parC and parE subunit of topoisomerase IV were screened by PCR; then sequenced by Sanger dideoxy-mediated chain termination and analyzed using DNASTAR Lasergene v7.1software, amino acid mutations were determined using the control strain E. coli K-12and Klebsiella pneumoniae MGH78578as a reference; Efflux pump inhibition test was performed on fluoroquinolones resistance isolates without QRDR mutations.51.2%of590E. coli isolates were resistant to ciprofloxacin, PMQR genes were detected in220(37.3%) strians, qnr genes were detected in22isolates (3.7%) and the prevalence of aac(6’)Ib-cr, qepA and oqxAB were19.7%,14.1%and3.9%respectively.24isolates contained more than2PMQR genes. All the fluoroquinolones resistant isolates had QRDR mutations and93.7%of them harbored at least3mutated points; In fluoroquinolones sensitive strains,66.7%were identified QRDR mutations. For569K. pneumoniae, only8.3%isolates resistant to ciprofloxacin, PMQR genes were detected in398(70.0%), qnr genes were detected in208isolates (36.6%) and the prevalence of aac(6’)Ib-cr and qepA were9.1%and43.8%. In fluoroquinolones resistant strains,73.9%of them were present QRDR mutations and19.6%harbored at least3point mutations; In fluoroquinolones sensitive strains,4%were identified QRDR mutations.12fluoroquinolones resistance isolates of K. pneumonia without QRDR mutations could be converted to sensitive to fluoroquinolones by efflux pump inhibitors l-naphthylmethyl-piperazine(NMP).In this section, we found great disparity in fluoroquinolones resistance between E. coli and K. pneumoniae (51.2%versus8.3%) in isolatesd from community-acquired infections from county hospitals. One of the reason was that the lower fluoroquinolones resistance state have existed in China for a long term, another was24.5%isolate of K. pneumoniae were from children (0-14olds), with resistance rate of4.2%. Incidence of QRDR mutations in E. coli was high, both in resistant group (100%) and sensitive group (66.7%). Accumulation effect of QRDR mutations in E. coli was significantly stronger than K. pneumoniae (resistance group73.9%, sensitive group4%) to the drug resistance.In the second section, Multilocus Sequence Typing (MLST) was performed on302ciprofloxacin-resistant E. coli and46ciprofloxacin-resistant K. pneumoniae. The primers and protocols of E. coli referred to UCC database (http://mlst.ucc.ie/mlst/dbs/Ecoli) and K. pneumoniae referred to pasteur database (http://bigsdb.web.pasteur.fr/klebsiella/klebsiella.html). Sequence data were analyzed using the MLST website. The distribution of sequence types (STs) across the regions was analyzed using eBURST V3and BioNumerics. Identification of plasmids replicon was performed by PCR-based replicon typing (PBRT) and another two replicons ColE and IncR. This PBRT+ColE+IncR method can identify20kinds of incompatible replicon of Enterobacteriaceae. Then conjugation experiment was carried out on those isolates containing more than2kinds of PMQR genes.A total of60ST types were identified in302ciprofloxacin-resistant E. coli isolates. The most prevalent was ST131(14.6%), followed by ST1193(7.9%), ST405(6.6%), ST69(6.3%), and ST648(6.3%).15,26,28,11,17,22and13kinds of STs were distributed in east China, central southern China, north China, northeast China, northwest China, south China and southwest China, respectively.23ST types were identified in46ciprofloxacin-resistant K. pneumoniae isolates. Clonal complex CC11(ST11, ST340, ST258) was the most prevalent with15.2%, followed by ST1(13.0%), ST15(10.9%), ST147(10.9%) and ST875(8.7%).3,2,5,4,8,5and7kinds of STs were distributed in east China, central southern China, north China, northeast China, northwest China, south China and southwest China, respectively. IncF was the most common (83.3%) replicon type in E. coli and always coexisting with other replicons,45.8%of them could conjugated with recipents. ColE-like was the most common (70%) replicon type in K. pneumoniae,75%of them could conjugation.In this section, we found the sequence types of ciprofloxacin-resistant E. coli was various, and scattered in seven regions of China, have none outbreak clone type. Although only46ciprofloxacin-resistant K. pneumoniae were found, the sequence types were abundant, more than23kinds, which were also scattered in different areas.In conclusion,, with the analysis E. coli and K. pneumoniae isolated from community-acquired infections in30county hospitals distributed in seven geographic regions of China, we found that fluoroquinolones resistance in E. coli was high, significantly difference of accumulation effect on QRDR mutations between E. coli and K. pneumoniae mighr be the important factors contributing to for the great disparity in fluoroquinolones resistance between them. Fluoroquinolones resistance isolates were multiple origins and scattered in area distribution. High fluoroquinolone resistance could be the result of long-term accumulation in multiple resistant mechanisms, rather then dominant clonal spread.