Drug Resistance And Genotype Analysis Of Carbapenem-resistant Klebsiella Pneumoniae Strains Isolated In Qingdao Municipal Hospital

ObjectiveThe study aims at investigate the clinical distribution,characteristicse of drug sensitivity,resistance mechanism and molecular epidemiology of carbapenem resistant Klebsiella pneumoniae?CRKP?,in order to provid evidence-based medical evidence for antibiotic treatment as well as help nosocomial infection control.MethodsThirty-two CRKPs from Qingdao municipal hospital affiliated Qingdao University from November 2015 to April 2017 was collected,susceptibility of clinical isolated strains to imipenem and?or?meropenem by BD phoenix^TM-100 automatic indentification/drug sensitivity systems and Disk diffusion test.Carbapenem inactivation method was conducted to determine the drug resistant phenotypes.Agar dilution method was conducted to determine minimum inhibitory concentrations?MICs?of antimicrobial agents.Carbapenemase genes,beta-actamase genes,quinolone genes and aminoglycoside genes was detected by polymerase chain reaction and the carbapenemase genes positive products was directly sequenced,then sequence alignment by Blastn was used to identify the genotype.The Klebsiella pneumoniae strains clonal dissemination was detected by pulsed field gel electrophoresis?PFGE?and multilocus sequence typing?MLST?.The mobility of plasmids was confirmed by conjugation tests and electrotransformation technology,the size of plasmids was determined by S1-PFGE.,carbapenemase genes was located by Southern blot.Results?1?The isolation rate of CRKP in Qingdao municipal hospital affiliated Qingdao University was 4.59%?32/697?.Drug-resistant strains was separated from 14differentdepartments,andICU?31.3%?,day-careward?12.5%?,departmentof respiratory?9.4%?are three leading department.The main clinical specimens was sputum?53.1%?and blood?28.1%?.?2?Most of these strains were extensively drug resistant?XDR?bacteria.Antibiotic susceptible rate in vitro of the strains showed that polymyxin is the most sensitive with being100%,followed by tigecycline,being90.63%,sulfamethoxazole,being 28.12%,doxycycline,being 25%,amikacin,being 18.75%,gentamycin 15.63%,levofloxacin 15.63%,.The resistance rate of the third and fourth generation Cephalosporin the resistance rate>90%.?3?32strains all carried carbapenemase genes,among them 27 strains carried KPC?84.3%?,3 strains carried NDM?9.4%?,2 strains carried BIC?6.3%?and the two genes?KPC and NDM?of carbapenemase co-existed in a single strain?CRKP23?.?4?11 strains clonal dissemination were detected by multilocus sequence typing?MLST?,3 strains?CRKP27?CRKP28?CRKP29?were ST23,3strains?CRKP20?CRKP21?CRKP28?were ST2250,2 strains?CRKP2?CRKP5?were ST15,1 strain?CRKP23?was ST1083,1 strain?CRKP22?was ST2193,1 strain?CRKP32?was ST11.The results of pulsed field gel electrophoresis showed that CRKP8 and CRKP9were very closely-related,CRKP17 and CRKP18 were very closely-related,which is possibly related to CRKP19,CRKP27 and CRKP28 were the same subtype.?5?Plasmids of 8 strains were transferred by conjugation tests has succeeded into host strain,being25%?8/32?and plasmids of 3 strains was transferred by electrotransformation technology has succeeded into host strain,being 12.5%?3/24?.S1-PFGE and Southern blot showed that carbapenemase genes was located in plasmid,the size of plasmids was 75kb¹60kb.Conclusion?1?The main clinical distribution departments of CRKP was ICU,and the main specimentypes were sputum and blood.?2?The molecular epidemiology of CRKP showed that the main resistance mechanism of CRKP was enzyme production,especially KPC-producing strains,carbapenemase genes was located in plasmids.?3?ST23 and ST2250 was predominant sequence type among the strains.?4?The same subtype of CRKP isolates in Qingdao municipal hospital affiliated Qingdao University,so it is vitally important to monitor the resistant bacteria.