| Background and objectivesSince the invention of antibiotics and its continuous application in clinical treatment,the resistance of bacteria has also increase,which has reduced the effectiveness of antibiotics and increased the difficulty of treating diseases,which has become a major problem affecting global public health.Extended-spectrum?-lactamase is a group of enzymes that hydrolyze penicillin,cephalosporin,and aztreonam,and is the main drug resistance that causes Gram-negative bacteria to develop resistance to broad-spectrum ?-lactam antibiotics.Carbapenems can be used to treat infections caused by a wide range of beta-lactamase-producing enterobacteriaceae isolates.However,due to the unreasonable use of carbapenem drugs,anti-carbapenem-resistant Enterobacteriaceae bacteria continue to appear,which brings difficulties to clinical work.KPC enzyme is the most important enzyme in class A carbapenemase.It can hydrolyze carbapenem drugs.The infection mortality caused by KPC enzyme-producing bacteria is high,which poses a huge challenge to clinical diagnosis and treatment.Klebsiella pneumoniae is a gram-negative pathogen and is commonly associated with hospital-acquired and community-acquired infections such as pneumonia,septicemia and urinary tract infections.Selection pressure due to the widespread use of antibiotics,K.pneumoniae strains in hospital often have multiple drug resistance,and health care related infections caused by these strains treatment is usually very difficult,in particular K.pneumoniae produced on carbon penicillium alkene drug resistance,makes this a difficult even more significant.K.pneumoniae has a high ability to acquire mobile genetic components,such as plasmids containing antibiotic resistance genes.The widespread spread of carbapenase-resistant K.pneumoniae is reported to be caused by horizontal transfer of moving elements such as plasmids and insertion sequences.In view of its high ability to spread,enterobacteriaceae bacteria carrying KPC-2 pose a substantial threat to clinical antibacterial therapy in China.The occurrence of CRKP infection is generally considered to be related to the patient's physical condition,whether the patient has complications,the length of time the patient is admitted to the hospital,whether the patient is treated with invasive equipment,and the patient's recently used antibiotics,etc.The importance of bacteria and the mechanism of intestinal colonization and transmission of drug resistance are not yet known.Previous studies have shown that CRKP colonization is an important risk factor for nosocomial infection in hospitals.This subject intends to continuously observe the intestinal colonization of CRKP in a specific critically ill patient,use bacterial tests,whole genome sequencing and other methods to obtain the virulence and drug resistance of the infected strain,and try to clarify the resistance of intestinal colonization CRKP in this specific patient drug and molecular characteristics.The research results will provide new ideas for the occurrence,diagnosis and treatment of intestinal colonization CRKP,and have significant scientific significance and important clinical value for the early prevention and control of infection.MethodsA total of 65 clinical stool samples of a patient were collected from the ICU ward of the First Affiliated Hospital of Zhejiang University School of Medicine in this study.Bacteria were isolated and identified from the collected stool samples.A total of 88 carbapenemase-resistant Gram-negative strains were isolated.Drug susceptibility test was performed on the isolated bacteria to obtain the drug-resistant phenotype of the bacteria.Pulse-field gel electrophoresis(S1-PFGE)and DNA blotting(Southern blot)of the plasmid were performed to determine whether the KPC gene was present in the plasmid and the plasmid size was determined..Genomic sequencing,splicing and analysis of isolated bacteria were performed to determine the drug-resistant genes,virulence genes,MLST typing,genetic environment of KPC and phylogenetic analysis.Results1.In this study,88 CRKP strains of gram-negative enterobacter carbapenase-resistant were isolated from 65 clinical fecal samples collected.PCR amplification was used to identify carbapenem resistance genes in 88 strains of CRKP isolated.The identification results showed that all 88 CRKP strains were positive for blaKPC gene.2.Drug susceptibility results showed that 86 of 88 strains of K.pneumoniae carrying blaKPC-2 were resistant to carbapenems,and most of the bacteria were resistant to piperacillin/tazobactam and cefotaxime,ceftazidime,cefpirome,aztreonam,ciprofloxacin showed resistance,sensitive to gentamicin,tobramycin,amikacin,and fosfomycin.3.Southern blot hybridization results of S1PFGE and blaKPC-2 gene showed that the blaKPC-2 gene carried by bacteria was plasmid-mediated.There are not less than 6 positive plasmids carrying blaKPC-2 gene in these clinical strains.The plasmid carrying blaKPC-2exists in the isolate,the plasmid size is in the range of 28.8kb?173.4kb,the common plasmid size is?54.7kb,?78.2kb and?138.9kb3.MLST results showed that 60 of 88 CRKP strains were ST11 type and 28 strains were ST37.And 88 strains of CRKP carried multiple resistance genes.These include quinolones,sulfonamides,aminoglycosides,fosfomycins,beta-lactams,macrolides,and tigecyclines.Not only that,bacteria also carry many virulence genes,of which 88 strains of CRKP carry fim and mrk operons encoding type 1 and type 3 pili,and ecp pili protein.4.There were 6 different blaKPC-2 genetic environments in 88 CRKP,and blaKPC-2 had a conserved sequence:klcA-hp-hp-hp-blaKPC-2-ISKpn27-Tn3.Phylogenetic analysis showed that CRKP were roughly divided into two branches,one branch was ST11 type CRKP and the other branch was ST37 type CRKP.In addition,some bacteria showed closer kinship,and some bacteria showed distant kinship,indicating that the CRKP in the patient was diverse.Conclusions1.A total of 88 CRKP strains were collected in this study.All 88 strains carried the blaKPC gene and almost all were resistant to carbapenems,indicating that KPC-producing K.pneumoniae was stably colonized in the intestine of this patient.KPC-2 pneumoniae produced by intestinal colonization has extensive multidrug resistance,carries multiple drug resistance genes and virulence genes,and poses a huge challenge to clinical diagnosis and treatment.2.The size of the plasmid carrying blaKPC-2 varies greatly among different strains.The blaKPC-2 gene is mostly located in the plasmid and there are moving elements around it,which has the risk of transmission.KlcA-hp-hp-hp-blaKPC-2-ISKpn27-Tn3 is a conserved sequence.3.The bacteria isolated from this patient have SNP differences,indicating that during the hospitalization of the patient during the use of antibacterial drugs,the bacteria have undergone evolutionary mutation. |
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