Investigation Of Molecular Epidemiology Of Carbapenem Resistant Gram-negative Bacilli And Rapid Detection Of Carbapenem Resistance Determinants By MALDI-TOF MS

Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa occupied for the top four of the clinical commonly isolated gram-negative bacilli. Emergence and widespread of carbapenem-resistant gram-negative bacilli in our hospital brings in great difficulties for clinical anti-infection treatment. For the development of scientific and effective prevention and control measures, molecular classification technologies were performed to investigate the molecular epidemiology of carbapenem-resistant gram-negative bacilli. Meanwhile, a rapid detection method of carbapenem-resistance determinants was established according to the primary carbapenem-resistance mechanisms and this will earn the precious time for the clinical treatment.In part one of this study, the prevalence, carbapenem resistance mechanism and changes of the strains were investigated among the4species of carbapenem-resistant gram-negative bacilli isolated from our hospital. Carbapenem-resistant non-duplicated strains of16E. coli,87K. pneumoniae,80A. baumannii and67P. aeruginosa were collected from2nd Affiliated Hospital of Zhejiang University during the year2009to 2012. Antimicrobial susceptibility testing revealed that polymyxin B and colistin showed the best activity against gram-negative bacilli, followed by amikacin. Carbapenemase production is the predominant mechanism of carbapenem-resistant gram-negative bacteria. KPC-2is the main carbapenemase detected in E. coli, K. pneumoniae and P. aeruginosa isolates, while OXA-23is the main carbapenemase detected in A. baumannii. PFGE showed that clonal spread is serious in our hospital. The dominant KP-A type K. pneumonia spread vertically since2009, and PA-A type P. aeruginosa spread within our hospital since2010. The clonal spread of A. baumannii is serious within the year, while unlike K. pneumonia and P. aeruginosa isolates, none of the dominant clone was found through2009to2012. MLST indicated that ST131, ST11and ST463are the predominant STs of carbapenem-resistant E. coli, K. pneumoniae and P. aeruginosa, respectively. As for carbapenem-resistant A. baumannii, no predominant ST was found, while several STs belonged to the same clonal complex208(CC208), a worldwide spreading clonal complex. Besides production of carbapenemases, combination of extended-spectrum β-lactamase/AmpC and porin deficiency is another main reason for carbapenem resistance or reduced susceptibility against Enterobacteriaceae.On the basis of the above results, we established a quickly and accurately detection method of carbapenem-resistant determinants among the gram-negative bacilli by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Fifty strains of carbapenemase-producing gram-negative bacilli were selected to detect the carbapenemases by MALDI-TOF MS. The entire process takes only2hours. It is easy to operate, with low cost and suitable for large sample operation. Meanwhile, MALDI-TOF MS was performed to detect the porins of10strains of E.coli and8strains of K. pneumonia. Peaks with approximately37,000-and38,000-m/z disappeared among the porin-deficiency E.coli and K. pneumonia. Peaks with37,000-and38,000-m/z in E.coli isolates correspond to the40kDa and41kDa bands in SDS-PAGE, respectively; Peaks with38,000-m/z in K. pneumonia isolates correspond to the42kDa band, while the37,000-m/z peak detected by MALDI-TOF MS was not found in the SDS-PAGE. Tryptic in gel-digestion, MALDI-TOF/TOF MS analysis and database searching were performed to verify the corresponding relation of peaks in MALDI-TOF MS with protein bands in SDS-PAGE. The results proved that peaks with37,000-and38,000-m/z in E.coli isolates correspond to OmpF and OmpC, respectively; Peaks with38,600-m/z in K. pneumonia isolates correspond to OmpK36. A wider range of concentrations can be detected by MALDI-TOF MS with much more convenient operation, shorter time-consuming, higher resolution and fewer affecting factors comparing with SDS-PAGE.In summary, clone spread of carbapenem-resistant gram-negative bacilli is severe in hour hospital. We established a fast, accurate, low-cost, high-throughput detection method of carbapenem determinants of gram-negative bacteria and it can be used to detect the expression of carbapenemase and outer membrane proteins.