| Background and objectiveCarbapenem-resistant Klebsiella pneumoniae（CRKP）is currently the main focus of drug-resistant bacterial infections in clinical settings,which poses a significant threat to human health due to limited treatment options for its resistance to multiple antimicrobial agents.According to China Antimicrobial Resistance Surveillance System（CARSS）,the resistance rate to carbapenems of clinical Klebsiella pneumoniae（KP）strains isolated from Chinese hospitals increased from 6.4 percent in 2014 to 11.3 percent in 2021.The production of carbapenase,especially Klebsiella pneumoniae carbapenemase（KPC）,remains the most common resistance mechanism to carbapenem for CRKP in China.Ceftazidime/abvibatam（CAZ/AVI）,a novelβ-lactam/β-lactamase inhibitor,was approved for marketing in China in 2019,bringing hope for the treatment of CRKP（Especially for the KPC-producing KP:KPC-KP）.Imipenem/relebactam（IMI/REL）is also a novelβ-lactam/β-lactamase inhibitor under clinical development,having been approved by the US Food and Drug Administration（FDA）in 2019 and European Medicines Agency（EMA）in 2020 for the clinical treatment.However,it is worrying that the resistance of CRKP to these two new compound preparations has been reported increasingly.And the mutations in KPC enzyme,porin Omp K35/36,penicillin-binding protein PBP2/3,and the overexpression of bla KPChave been reported to be the main cause of the resistance to CAZ/AVI in KPC-KP.Meanwhile,the mutation or deletion of Omp K35/36 and the overexpression of bla KPCwere also reported to be associated with the production of resistance to IMI/REL in KPC-KP.However,the epidemiology and related mechanisms of cross-resistance or co-resistance of two compound preparations are rarely reported.This study aims to clarify the in vitro drug sensitivity of KPC-producing CRKP strains for the two new compound preparations（CAZ/AVI and IMI/REL）,and to further explore the resistance mechanisms.Thus,we can evaluate the in vitro antimicrobial activity of the two new compound preparations that can treat KPC-KP in this region and make an antimicrobial-resistance warning.MethodsCRKP isolates were collected from three tertiary hospitals in Guangdong Province from 2015 to 2022.Polymerase Chain Reaction（PCR）and Sanger sequencing were used to identify the carbapenemase（KPC,NDM,VIM,IMP,OXA）and capsular serotypes of the isolates.The KPC-KP strains were included as the study subjects,according to the recommendation of Clinical and Laboratory Standards Institute（CLSI）-2021 M100 standard,the Minimal inhibitory concentration（MIC）of Imipenem（IMI）,Meropenem（MEM）,Ertapenem（ETP）,Polymyxin B（PB）,Tigecycline（TGC）,CAZ/AVI,and IMI/REL were tested for the isolates using microbroth dilution method in vitro.The susceptibility results of MEM、IMI、ETP、CAZ/AVI and IMI/REL can be interpreted according to the breakpoint of CLSI-2021M100 standard.The susceptibility results of PB can be interpreted by the breakpoint of European Committee for Antimicrobial Susceptibility Testing（EUCAST）.The susceptibility results of TGC can be interpreted by both the breakpoint of the US FDA and the EUCAST.In addition,the clinical data were collected to analyze the molecular epidemiological characteristics of the strains.KPC-producing CRKP strains resistant to at least one of CAZ/AVI and IMI/REL were screened.Then multilocus sequence typing（MLST）and resistance genes bla ESBLand bla Amp Cwere determined by Whole Genome Sequencing（WGS）for these strains.To explore the resistance mechanisms of KPC-KP to CAZ/AVI and IMI/REL,mutations in KPC,Omp K35/36 and PBP2/3 were also determined by WGS.Meanwhile,the expression level of bla KPCwas detected in both isolates susceptible and non-susceptible to CAZ/AVI and IMI/REL using Quantitative Real-time PCR（q PCR）.ResultA total of 876 CRKP strains isolated from different patients were identified,of which,780 strains were KPC-KP.And 469 strains had complete clinical records.Of the 780 KPC-KP strains,79.5%and 20.5%were isolated from male and female patients,respectively.The proportion of KPC-KP strains increased with patients’increasing age and mainly concentrated on the population over 60 years old（61.2%）.Intensive care unit（ICU）,respiratory department and neurosurgery department were three departments where KPC-KP mostly isolated,accounted for 45.4%,14.9%and8.3%,respectively.Sputum,urine and blood were the top three sample sources of KPC-KP strains,accounting for 50.3%,17.5%and 10.4%,respectively.In addition,KPC-KP strains had the highest proportion in autumn（33.7%）,followed by that in summer（27.1%）.Among the 780 KPC-KP strains,98.3%of which produced KPC-2carbapenemase,and the remaining subtypes of KPC were KPC-26（0.5%）,KPC-33（0.4%）,KPC-117（0.4%）,KPC-12（0.1%）,KPC-29（0.1%）,and KPC-85（0.1%）.A total of 16 capsular serotypes were identified,among which KL64 was the most common type,accounting for 57.5%and then was KL47,accounting for 32.8%.The overall resistant rates of 780 KPC-KP strains for MEM、IMI and ETP were all higher than 98.0%.The susceptible rate of TGC was 87.8%and 21.0%,respectively when using FDA breakpoint and EUCAST breakpoint.PB and CAZ/AVI showed good antibacterial activity against KPC-KP strains,and the susceptible rates were 92.4%and 97.4%,respectively.Only 0.9%of the KPC-KP strains were susceptible to IMI,but the susceptible rate increased to 99.0%after combining with REL,and the MIC50and MIC90of IMI both were decreased by 256 folds:from64μg/m L and 128μg/m L to 0.25μg/m L and 0.5μg/m L,respectively.20 and 6 of 780 KPC-KP strains were resistant to CAZ/AVI and IMI/REL,respectively,and 4 of them were resistant to both these two antimicrobial agents.All resistant strains were ST11 clones,and the most common capsular serotypes were KL64 and KL47.All 22 KPC-KP strains resistant to CAZ/AVI and/or IMI/REL were multi-drug resistant（MDR）strains,but 20（90.9%）and 17（77.3%,FDA breakpoint）strains were sensitive to PB and TGC,respectively.However,when the EUCAST breakpoint was used,the resistance rate of TGC was 77.3%（17 strains）.Further,the resistance mechanisms of 22 strains resistant to CAZ/AVI and/or IMI/REL were analyzed.The consistent mutation in OmpK35 was detected:OmpK35has an early stop codon at amino acid 63.Except for 2 strains that were lack of Omp K36,the consistent mutations of Omp K36 were detected in the remaining 20strains resistant to CAZ/AVI and/or IMI/REL:Omp K36 has glycine and aspartic acid repeat at amino acid 134（134-135 GD）.In addition,3 strains developed additional mutations in Omp K36:one was a missense mutation（K196E）,one was a frameshift mutation（S125fs）,and another one was a premature termination（Q296*）.And 4strains had mutations in KPC-2 enzymes:3 strains had the D179Y mutation（KPC-33）and one strain had the A172V mutation（KPC-85）.And no mutations of PBP2 or PBP3 were detected in all of the strains.The relative expression level of bla KPCfor strains resistant to CAZ/AVI significantly increased compared with the sensitive strains,and the relative expression level of bla KPCfor strains intermediate and resistant to IMI/REL significantly increased compared with the sensitive strains.Of the 22 strains,4 strains had KPC-2 mutations,and they were all resistant to CAZ/AVI and showed high MIC values（≥64μg/m L）,but were all sensitive to IMI/REL.And three of them produced KPC-33,one strain showed sensitive,intermediate,and resistant to MEM（MIC value 4μg/m L）,respectively.Two strains were sensitive to Doripenem and one strain was intermediate to Doripenem.All these three strains were sensitive to IMI.The KPC-2 enzyme in 13 resistant strains had no mutations.And All these strains Omp K35 had Stop codon and Omp K36 had only 134–135 GD.All 13 strains were resistant to CAZ/AVI with MIC values 16–32μg/m L and only one of these strains which carried additional bla SHV-158was resistant to IMI/REL.Two strains were susceptible to CAZ/AVI but resistant to IMI/REL（MIC values is between 4μg/m L and 8μg/m L）.The two strains had no mutations in KPC-2 enzyme,but had Stop codon in Omp K35 and the deletion of Omp K36.Among the four strains resistant to both CAZ/AVI and IMI/REL,none of the KPC-2 enzymes had mutation,but all had Stop codon in Omp K35.And three of these strains had additional mutations in Omp K36:K196E,Q296*,and S125fs,and the MIC value for IMI/REL was 4μg/m L,8μg/m L,and 16μg/m L,respectively for these three strains.And only one of these strains which had a MIC value to IMI/REL of16μg/m L also carried both bla SHV-158and bla SHV-12.Conclusions1.Among all the 780 KPC-KP strains,98.3%of them produced KPC-2 enzymes,and the other KPC subtypes were KPC-26（0.5%）,KPC-33（0.4%）,KPC-117（0.4%）,KPC-12（0.1%）,KPC-29（0.1%）,and KPC-85（0.1%）.The capsular serotypes were mainly KL64 and KL47 for all the KPC-KP strains.2.The gradually increased susceptibility of antibacterial agents for all KPC-KP strains is:TGC（87.8%,FDA breakpoint）,PB（92.4%）,CAZ/AVI（97.4%）,and IMI/REL（99.0%）,but the susceptible rate of TGC decreased to 21.0%using the EUCAST breakpoint,which deserves clinical attention.3.There are total 22 isolates resistant to CAZ/AVI and/or IMI/REL,among which 20 isolates（90.9%）and 17 isolates（77.3%,FD breakpoint）are susceptible to PB and TGC,respectively.However,the resistant rate of TGC increased to 77.3%with the EUCAST breakpoint.4.Mutations of the KPC-2 enzyme could possibly cause the resistance to CAZ/AVI,but are less associated with resistance to IMI/REL.When bla KPChad no mutations,mutations of Omp K35 and Omp K36 accompanied by the increased expression level of bla KPCmay often result in the resistance to CAZ/AVI but not to IMI/REL.5.Different from previous studies on Omp K36-mediated IMI/REL resistance,three new amino acid mutation sites of Omp K36 were found in IMI/REL-resistant strains in our study:missense mutation（K196E）,premature termination（Q296*）,and frameshift mutation（S125fs）.In addition,SHV beta-lactamases(bla SHV-12and bla SHV-158)were found in IMI/REL-resistant strains.K196E,Q296*,S125fs or deletion of Omp K36 and relatively high expression level of bla KPCmay play an important role in mediating co-resistance to both CAZ/AVI and IMI/REL. |
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