| Carbapenem-resistant Klebsiella pneumoniae（CRKP）infection has become a serious problem in hospitals around the world.Hospitals are an important source of multidrug resistant bacteria.In order to study the drug resistance status of CRKP in hospital sewage,this study analyzed the drug resistance mechanism and virulence characteristics of CRKP and the potential gene level transmission mechanism.A three-level hospital in Changchun,Jilin Province,China was monitored.Through the cultivation of imipenem resistance（4μg/m L）,vancomycin（2μg/m L）Mac Conkey agar medium,PCR and BD PhoenixTM-100 system automatic microbial identification/drug sensitivity system,carbapenem-resistant isolates were isolated and identified.It was found that CRKP was the main carbapenem-resistant strain in hospital sewage;The drug sensitivity and the resistance to chlorine-containing disinfectants were tested by the automatic microbial identification/drug sensitivity system and micro broth dilution method,and the biofilm forming ability of CRKP isolates was also measured.According to the drug sensitivity test results,further detection of carbapenem resistance genesβ-Lactamase resistance gene,plasmid-mediated quinolone resistance gene（PMQR）,16sr RNA methyltransferase,tetracycline,sulfonamide and other antibiotic resistance genes,integrase gene and virulence gene;The CRKP isolates were genotyped by using PBRT plasmid typing kit;The diffusion,transmission level and mechanism of carbapenem resistance gene were determined by conjugation transfer experiment.Subsequently,the isolated CRKP isolates were sequenced by bacterial framework map,and the sequencing results were analyzed by bioinformatics to deeply analyze the drug resistance gene,virulence gene,plasmid type and MLST type of CRKP isolates,and comprehensively analyze the drug resistance,pathogenicity and molecular epidemiological relationship between the two.The results show that:A total of 93 CRKPs were isolated from four hospital sewage samples from March to September 2021,accounting for 76.86%（93/121）of all carbapenem-resistant isolates.The resistance rates of 93 CRKP strains to imipenem and meropenem were 100%and 98.92%respectively.The resistance rates to cefazolin,cefotaxime,ceftazidime,cefepime,atranam,and piperacillin tazobactam were 100%,81.72%,63.44%,98.92%,98.92%,and 98.92%,respectively.All isolates were resistant to ampicillin,piperacillin,amoxicillin clavulanate sodium,and ampicillin sulbactam.The drug resistance rates to trimethoprim-sulfamethoxazole,chloramphenicol,ciprofloxacin,levofloxacin,moxifloxacin and tetracycline were 77.42%,30.11%,22.58%,17.20%,21.51%and 25.81%,respectively.All isolates were sensitive to colistin.68.8%of CRKP can form biofilm,and all isolates have certain resistance to chlorine-containing disinfectants.Drug resistance genes and virulence genes of 93 CRKP strains were detected.The results of drug resistance gene detection showed that the detection rates of bla KPC,bla NDM,bla IMPgenes were 91.4%（85/93）,9.68%（9/93）,4.3%（4/93）,bla CTX-M,bla SHV,bla TEM,genes were 34.4%（32/93）,32.26%（30/93）,56.99%（53/93）,aac（6’）-Ib-cr,qnr A,qnr B,qnr S genes were 12.9%（12/93）,3.22%（3/93）,36.56%（34/93）,25.56%8%（25/93）,the detection rate of tet（A）and tet（B）genes was 25.8%（24/93）The detection rates of 3.23%（3/93）,sul1 and sul2 genes were60.22%（56/93）,39.78%（37/93）,and the detection rates of cml A and flo R genes were 9.68%（9/93）,3.22%（3/93）.The detection rates of rmt B,aac（3）-II,aac（6’）-Ib resistant genes were13.98%（13/93）,35.48%（33/93）,18.28%（17/93）,bla VIM,bla OXA–48,qnr C,qnr D,tet（C）,tet（D）,tet（X）,sul3 and arm A did not amplify positive results.Results of virulence gene detection:the detection rate of rmp A gene was 6.45%（6/93）,and the detection rate of carrying rmp A2 gene was 12.9%（12/93）.There were 10 isolates（10.75%）carrying iuc A and iro B genes at the same time,of which 6 isolates carried 4 virulence genes at the same time,accounting for 6.45%（4/93）of the isolates.Only Int I1 gene was detected in the isolates,with a detection rate of 61%（71/93）.The result of replicator detection shows that there are 12 types of replicators.The most common type of replicon is Inc FIB,with 58（62.4%）isolates,followed by In FII and Inc R,with 50（53.8%）and 34（36.6%）isolates,respectively.No replicon type was found in 7.53%（7/93）isolates.Among these isolates,the most common type of replicon combination is Inc FII and Inc R,accounting for 12.9%（12/93）,followed by the combination of Inc FIB and Inc FIA,10.8%（10/93）.The results of conjugation transfer showed that the resistance genes in CRKP isolates of four carbapenem-resistant gene combinations could be transferred to the receptor bacteria horizontally,and the conjugation transfer frequency was in the order of 10-4Through genomic analysis,the PCR amplification results of the drug resistance and virulence genes of 93 CRKP isolates of Klebsiella pneumoniae were supplemented.The strains carried a variety of aminoglycosidesβ-Detailed genotyping studies have been conducted on the resistance genes and disinfectant resistance genes of lactams,quinolones,chloramphenicols,fosfomycin,and rifamycin.Detailed genotyping studies have also been conducted on virulence factor related genes,and related gene products have been described.The results of multi site sequence typing showed that there were as many as 21 types of MLST typing,with the dominant types being ST11（12.9%）and ST195（12.9%）.In this study,93 CRKP isolates were isolated from four hospital sewage samples from March to September 2021.The results showed that antibiotic resistant strains could resist the disinfectants in hospital sewage,form biofilms and colonize in the sewage.Improper treatment of hospital wastewater may lead to the spread of drug-resistant bacteria and their genes,which poses a great threat to public health and safety.Therefore,these bacteria must be eliminated before being discharged into the municipal sewage system. |
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