Research On Drug Resistance And Resistance Genes Among Carbapenem-resistant Pseudomonas Aeruginosa From Clinical Intectious Patients

Objective The study was conducted to determine the resistance of multiple antibiotics, distribution of carbapenemases and integrons, the lack of outer membrane porin among the clinical isolates of carbapenem-resistant Pseudomonas aeruginosa from hospitalized patients in Tianjin Medical University General Hospital. To analyze the molecular epidemiological characteristics of nosocomial multi-drug resistant isolates, guide clinical to control spread of resistant isolates in the hospital effectively.Methods A total of60carbapenem-resistant Pseudomonas aeruginosa were isolated from2009to2012in the Tianjin Medical University General Hospital. Remove the duplicate isolates from the same site of the same patient. The identification of bacteria and antimicrobial susceptibility test was performed by using VITEK-2compact automatic system and ancillary drug sensitive card. Cefoperazone/sulbactam susceptibility test was performed by using disk diffusion method (K-B). Isolates of carbapenem-resistant Pseudomonas aeruginosa were screened for carbapenemases by EDTA double disk synergy method. PCR was conducted to detect carbapenemases gene (Metallo-(3-lactamase gene, OXA gene), outer-membrane porin gene (OprD2gene) and the integrase gene (intll, intI2and int13). Isolates harboring class I or class II integrase genes were further amplified integrons variable region by PCR and sequencing analysis of resistance gene cassette carried in the integron variable region.Results The resistance status of60isolates of carbapenem-resistant Pseudomonas aeruginosa to15kinds of antibiotics was severe. All the isolates exhibited resistant to imipenem and meropenem at the same time. The resistant rate to Cefoperazone-sulbactam was the lowest (38.3%), the second for levofloxacin (60.0%). The rest of the antimicrobial resistant rates are higher than the70.0%. Among the60isolates,24isolates (40.0%) demonstrated Metallo-P-lactamase positive in the EDTA double disk synergy method. PCR gene amplification indicated that12isolates (20.0%) were positive for IMP.6isolates (10.0%) were positive for VIM.4isolates (6.7%) were positive for SPM.10isolates (16.7%) were positive for OXA-10.21isolates (35%) were positive for class Ⅰ integron, which no GIM, SIM, OXA-1, OXA-2gene and class Ⅱ, class Ⅲ integron were detected.36isolates (60.0%) showed the outer-membrane porin(OprD2) gene deletion.Among positive isolates for class I integron,16isolates (76.2%) amplified class I integron variable region.2kinds of integron variable region were totally found. A700bp and a1200bp integron variable region were obtained from13isolates and3isolates, respectively.The sequenced confirmed that700bp integron variable region carried the resistance gene cassette array with aacA4and1200bp integron variable region carried the resistance gene cassette array with aadB-aacA4.Conclusion These findings show that the group of carbapenem-resistant Pseudomonas aeruginosa distribution has wards of central tendency, and resistant to multiple antibiotics is serious. The most antimicrobial resistant rate is more than85.0%. EDTA double disk synergy method could be used as a screening test for rapid detection of Metallo-β-lactamase, which was simple and feasible, but it still need to be confirmed by PCR assays designed for the detection of genes encoding carbapenemases. Metallo-β-lactamase and OXA enzyme production as well as the lack of outer-membrane porin is one of the main resistance mechanisms of carbapenem-resistant Pseudomonas aeruginosa. Class I integron mainly confer resistance to aminoglycoside. Class I integron is an important cause for Pseudomonas aeruginosa to multi-drug resistance and resistant gene dissemination.