Characterization Of Carbapenem-resistant Klebsiella Pneumoniae Strains Obtained From Clinical Bloodstream-infections

Objective: Klebsiella pneumoniae(KPN)is a Gram-negative Enterobacteriaceae bacteria strain.In recent years,bloodstream infection caused by KPN has become a significant problem in hospitals.Furthermore,the transfer of various resistance genes between closely related strains of bacteria is a phenomenon that has been observed frequently.Carbapenem antibiotics belong to the wide antibacterial spectrum and are characterized by strong antibacterial activity and stability against β-lactamase.They are frequently used for the treatment of Gram-negative bacterial infections,as the last line of defense.However,under the pressure of selection and even abuse of clinical antibiotics,especially third-generation cephalosporin and carbapenem drugs,the drug resistance of KPN is developing into a major challenge.The recurrence of carbapenem-resistant Klebsiella pneumoniae(CRKP)and the drug resistance rate is continuously increasing every year.CRKP are capable of causing bloodstream infections,which are widely prevalent.It is difficult to control these infections,which leads to a high mortality rate,which in turn has led to great challenges in clinical treatment.Most KPN strains isolated from clinical specimens are concentrated in the sputum;therefore,in this study,the characteristics of CRKP strains isolated from clinical bloodstream infections were analyzed.We believe that this study provides a theoretical basis for future studies on clinical infection control of CRKP.Methods: We collected 32 CRKP strains from the blood samples obtained from different ward areas between January,2017 and June,2018.The bacteria were identified by routine bacterial isolation and culture methods.We used the French Mérieux VITEK2-compact automatic bacteria analyzer for bacterial identification and drug susceptibility testing.The antibiotics tested included ampicillin,ampicillin/sulbactam,piperacillin/tazobactam,ceftezole,ceftriaxone,cefotetan,ceftazidime,cefepime,aztreonam,ciprofloxacin,levofloxacin,amikacin,gentamicin,tobramycin,imipenem,ertapenem,and trimethoprim/sulfamethoxazole.The minimum inhibitory concentration(MIC)of tigecycline was determined using the micro-broth dilution method for the 32 strains,and the carbapenemase phenotype was detected by the modified Hodge test(MHT)using real-time PCR.The drug resistance gene was detected,and multi-locus sequence analysis(MLSA)was used for bacterial homology analysis.Results: The 32 CRKP strains isolated in this study were highly resistant to most antibiotics.The resistance rate toward amikacin was 43.8%,and the tobramycin resistance rate was 53.1%.The resistance rates for gentamicin,the compound sulfamethoxazole,aztreonam,ciprofloxacin,and levofloxacin were 59.4%,68.8%,93.8%,96.9%,and 96.9%,respectively.The resistance rates for ceftriaxone,ceftazidime,cefotetan,ampicillin/sulbactam,ertapenem,and imipenem were 100%.The MIC value of tigecycline was determined using the micro-broth dilution method.One tigecycline-resistant Klebsiella pneumoniae(TRKP)strain was obtained,accounting for 3.13% of all the strains isolated,whereas the remaining 31(96.88%)strains were sensitive to tigecycline,suggesting that KPN remains highly sensitive to tigecycline.Among the 32 strains of CRKP,27(84.38%)provided positive results for the modified Hodge test(MHT);the PCR result was treated as control,the sensitivity of the MHT was 95.83%(23/24),and the paired data chi-square test was used for statistical analyses.The difference was not statistically significant(P = 0.375,ie P > 0.05).The strains carrying the drug resistance genes KPC-2 24(75%),CTX-M9 22(68.75%),CTX-M1 3(9.38%),and the membrane pore protein encoding gene Ompk36 deletion 29(90.63%)were detected by PCR.The 22 strains harboring the CTX-M9 resistance gene showed Ompk36 deletion,two strains showed Ompk37 deletion(6.25%),while none of the strains showed Ompk35 deletion.None of the strains were found to harbor the OXA48,IMP-4,and NDM-1 genes,while most of the strains were found to belong to ST11 through MLST analyses(78.13%).The strains ST2894,ST1,ST2235,ST2230,ST1517,ST147,and ST550 accounted for one isolate each.Twenty-four strains carrying the Blakpc-2 gene were confirmed to belong to the ST11 strain.Conclusion: We concluded that the presence of the drug resistance genes KPC-2 and CTX-M9 accompanied by the deletion of the membrane pore protein encoding gene Ompk36 contributed to the main mechanism underlying carbapenem resistance of the KPN strains isolated from the clinical bloodstream infections in our hospital.The ST11 strain is the most prevalent in the country.The CRKP strain isolated from the clinical bloodstream infection cases in our hospital is highly resistant and exhibits a prevalent trend that requires immediate attention.