Epidemiology and Genomic Characteristics of Bloodstream Infection Caused by Carbapenem-Resistant Klebsiella pneumoniae With Decreased Susceptibility to Aztreonam/Avibactam in China
Wei Yu
Ping Shen1† 
Yunbo Chen
Kai Zhou
Xiaohui Chi
Yonghong Xiao- 1State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
- 2Shenzhen Institute of Respiratory Diseases, Second Clinical Medical College (Shenzhen People’s Hospital), Jinan University; First Affiliated Hospital (Shenzhen People’s Hospital), Southern University of Science and Technology, Shenzhen, China
Aztreonam/avibactam (AZA), as one of the novel β-lactamases and β-lactamase inhibitor combinations, is considered to be a promising option for bloodstream infection (BSI) of carbapenem-resistant Klebsiella pneumoniae (CR-Kp). However, decreased susceptibility of AZA activity in Enterobacterales has been reported. The aim of this study was to identify the mechanisms of BSI CR-Kp with decreased susceptibility of AZA (minimal inhibitory concentration above 16/4 mg/L) (AZAH-Kp). Nine BSI AZAH-Kp isolates were screened from 317 CR-Kp isolates in Blood Bacterial Resistant Investigation Collaborative System (BRICS) program. Whole genome sequencing, bioinformatics analysis, and the relative expression of blaKPC, ompK35, and ompK37 were explored for CR-Kp with decreased susceptibility to AZA. The results revealed that elevated inhibitory concentration of AZA has emerged in CR-Kp before previous clinical exposure. In addition, decreased AZA susceptibility was associated with higher KPC expression and changes in OmpK35-37.
Introduction
Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) infections, especially for bloodstream infection (BSI), remain a serious threat to public health. The non-β-lactam therapeutic options, such as colistin and tigecycline, with unsatisfactory pharmacokinetics–pharmacodynamics characteristics are less desirable for clinicians (Durante-Mangoni et al., 2019). Fortunately, several novel β-lactam–β-lactamase inhibitor combinations including aztreonam/avibactam (AZA), ceftazidime/avibactam, meropenem/vaborbactam, and imipenem-cilastatin/relebactam have been developed or under development against CR-Kp. However, except AZA, neither agent showed activity against metallo-β-lactamase (MBL) (Biagi et al., 2019). In China, the prevalence of blaNDM-1-positive CR-Kp was 13.2% in children and 4.2% in adults (Han et al., 2020). Therefore, AZA remains as a promising agent against CR-Kp.
The Food and Drug Administration (FDA) granted AZA as Qualified Infectious Disease Product (QIDP) qualification for carbapenem-resistant Enterobacteriaceae infections in November 2019. Recently, AZA is undergoing clinical trials to assess the efficacy against MBL and serine carbapenemases producing Gram-negative organisms (Cornely et al., 2020). In vitro studies have demonstrated that avibactam restored aztreonam susceptibility in 98% of aztreonam-resistant isolates (Biagi et al., 2020). However, clinical breakpoint of AZA has not been approved. Data on molecular mechanism of decreased AZA susceptibility are limited. Hence, the aim of this study is to explore the mechanisms of BSI CR-Kp with higher AZA inhibitory concentration (>16/4 mg/L) (AZAH-Kp).
Methods
Bacterial Strains
A total of nine non-duplicate BSI AZAH-Kp isolates were collected from Blood Bacterial Resistant Investigation Collaborative System (BRICS) program in 2019. Five of AZAH-Kp were collected from Hangzhou, two from Shenyang, one from Jiangsu, and one from Luoyang. Carbapenemase-producing isolates were further identified using modified Hodge test according to Clinical and Laboratory Standards Institute (CLSI) guidelines (Clinical and Laboratory Standards Institute, 2016).
Antibiotic Susceptibility Test and Molecular Identification of Carbapenemase
The minimal inhibitory concentration (MIC) of 23 antibiotics against AZAH-Kp isolates were determined by agar dilution method, while polymyxin B was used in broth dilution method. Amoxicillin, clavulanic acid, piperacillin, tazobactam, cefazolin, cefuroxime, ceftriaxone, ceftazidime, cefepime, cefoxitin, cefoperazone, sulbactam, moxalactam, aztreonam, ertapenem, meropenem, imipenem, gentamicin, amikacin, ciprofloxacin, levofloxacin, fosfomycin, tigecycline, trimethoprim, sulfamethoxazole, and avibactam were purchased from Dalian Meilun Biotech (Dalian, China). Polymyxin B and glucose-6-phosphate (G-6-P) were obtained from Sigma-Aldrich (St Louis, MO). The MIC of aztreonam combined with avibactam (8 or 16 mg/L) were further tested for AZAH-Kp isolates. Carbapenemase genes of AZAH-Kp isolates were confirmed by PCR and sequencing (Poirel et al., 2011).
Genome Sequencing and Data Analysis
Whole genome sequencing (WGS) for AZAH-Kp isolates was performed using Illumina HiSeq PE150 platform (Novogene Bioinformatics Technology Co., Ltd., Beijing, China). The resistance genes, plasmid replicons, multilocus sequence type (MLST) and virulence genes were identified by ResFinder v3.0 web server (http://www.genomicepidemiology.org). The single-nucleotide polymorphisms (SNPs) phylogeny was performed using kSNP3.
The sequencing data for AZAH-Kp isolates had been deposited at GenBank under accession number PRJNA737207.
Quantitative Real-Time PCR
Relative quantification of blaKPC, ompK35, and ompK37 genes in comparison to 16S rRNA gene was performed in triplicate by quantitative real-time PCR (qPCR) from log-phase cultures of AZAH-Kp and 41 AZA susceptible (0.125/4–2/4 mg/L) isolates with same MLST and blaKPC gene. CR-Kp ATCC BAA-1705 was used as the reference isolate.
Results
Determination of MIC
All isolates were positive for modified Hodge test. A summary of 24 antibiotics MIC against AZAH-Kp is shown in Table 1. Except 98690 and 109096, other AZAH-Kp isolates were resistant to ceftazidime/avibactam as well. It is of note that the MIC of AZA decreased with the increase in avibactam concentration.
Table 1 Susceptibility testing and genotypic characteristics of 9 AZAH-Kp isolates.
Resistance Genes
Nine AZAH-Kp isolates belonged to ST11 and harbored wild-type blakpc-2. Other different β-lactamse genes were identified and shown in Table 1. Genetic environment of blakpc-2 gene demonstrated all AZAH-Kp isolates harbored ISKpn27 (Supplementary Figure S1). In addition, all isolates carried ColRNAI and IncFII plasmid replicon types. However, IncHI1B and RepB were not identified in 108728 and 108738. SNPs phylogeny showed same regional source clustered closely (Supplementary Figure S2).
Outer Membrane Porin
Among outer membrane porin genes, ompK36 was not identified, while ompK35 and ompK37 had mutations. In addition, all isolates had truncated ompK37. Three under development mutations (I70M, N230G, and I128M) relating to carbapenem resistance were found in OmpK37. Furthermore, other undefined class mutations were found as well in OmpK35 and OmpK37 (Supplementary Table S1).
qPCR
Their relative expression of blaKPC gene in AZAH-Kp isolates was more than twofold higher than that in AZA susceptible isolates (p=0.0032) (Figure 1A). The relative expression of ompK35 in AZA susceptible isolates (MIC=0.125/4 mg/L, 3.1 ± 1.6) was 9.3-fold higher than that in AZAH-Kp isolates (0.3 ± 0.6, p=0.002) (Figure 1B). However, there was no statistical significance of the relative expression of ompK37 between AZA susceptible (MIC=0.125/4 mg/L) and AZAH-Kp isolates (p=0.606) (Supplementary Figure S3).
Figure 1 Relative blaKPC and ompK35 expression level in selected isolates. (A) blaKPC; (B) ompK35. p < 0.05 was consider significant different.
Discussion
Recent surveillance data reported the lower MIC of AZA (MIC90 = 0.5/4 mg/L) against CRE, especially for metallo-β-lactamase (MBL)-producing isolates (Sader et al., 2021). Our previous data also showed that the MIC90 of AZA was 1/4 mg/L, extending the observations for BSI CR-Kp (Yu et al., 2021). In the present study, we found the decreased susceptibility mechanisms of AZAH-Kp strains isolated from patients without history of previous AZA exposure. Our results indicated that AZAH-Kp needs sustained attention, although AZA remains potent against BSI CR-Kp. Overexpression of blaKPC and changes in outer membrane porin may be responsible for reduced susceptibility to AZA in AZAH-Kp isolates. Increasing the avibactam concentration to AZA could improve the sensitivity.
AZA-resistant breakpoint has not been assigned. High-level MIC of AZA is rarely observed among clinical isolates, and the molecular mechanism of decreased AZA susceptibility is limited. CMY-16 mutants (Tyr150Ser and Asn346His) were primarily responsible for the decreased susceptibility during inoculation with AZA, whereas wild AZAH-Kp without blaKPC had multiple resistance mechanisms (Niu et al., 2020; Mendes et al., 2021). In this study, we identified nine AZAH-Kp strains isolated from patients without treatment history of AZA. However, no mutations were found in KPC. Recent studies have indicated that isolates harboring mutated blaKPC gene was the leading cause of resistance to ceftazidime/avibactam (Giddins et al., 2018). In addition, blaKPC overexpression also played an important role in ceftazidime/avibactam resistance (Shen et al., 2017). Our findings are similar with the above result that high-level AZA resistance was closely related to blaKPC expression. Moreover, increased avibactam concentration could overcome the high-level inhibitory concentration of AZA.
Notably, chromosomal modification of outer membrane porins, such as OmpK35-37, could effectively abrogate bactericidal effect of antibiotics in CR-Kp (Wong et al., 2019). Our results align with previous publications (Shen et al., 2017; Venditti et al., 2020). Mutations of ompK35 and ompK37 were observed in all AZAH-Kp isolates. In addition, ompK36 deficiency was identified in all isolates. A recent study also reported production of DHA-1 combined with drug efflux, and porin deficiencies exhibited elevated MIC of AZA in three AZAH-Kp (Mendes et al., 2021). Hence, outer membrane porins act in concert to effectively lower active AZA concentration in CR-Kp.
To our knowledge, this work revealed the molecular mechanisms of decreased susceptibility to AZA against BSI CR-Kp. However, there were several limitations in our study. First, we did not assess the entire carbapenemase in CR-Kp isolates. Moreover, this study only provided data for BSI. Further studies of CR-Kp with different carbapenemase and infection sites are warranted.
Conclusions
In conclusion, high-level inhibitory concentration of AZA against BSI CR-Kp has emerged independently from clinical use. Excessive expression of KPC and changes in OmpK35-37 give rise to AZA high-level inhibitory concentration.
Data Availability Statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.
Author Contributions
The work presented here was carried out in collaboration between all authors. WY and YX developed the concept and designed the study. WY, PS, and KZ carried out genome sequencing and data analysis. YC and XC co-worked on associated data collection. The manuscript was written by WY and corrected by YX. All authors contributed to the article and approved the submitted version.
Funding
This study was funded by Key Research and Development Program of Zhejiang Province (No. 2021C03068) and Independent Task of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (No. 2022zz01). The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s Note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Acknowledgments
We thank all cooperative hospitals of Blood Bacterial Resistant Investigation Collaborative System (BRICS) program for their collection of the isolates.
Supplementary Material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb.2022.926209/full#supplementary-material
Supplementary Figure 1 | Schematic diagram of the genetic environment of the blakpc-2 gene in 9 AZAH-Kp isolates.
Supplementary Figure 2 | The phylogenetic tree of 9 AZAH-Kp isolates.
Supplementary Figure 3 | Relative ompK37 expression level in selected isolates.
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Keywords: carbapenem-resistant Klebsiella pneumoniae, aztreonam/avibactam, blaKPC, membrane porin, mutation
Citation: Yu W, Shen P, Chen Y, Zhou K, Chi X and Xiao Y (2022) Epidemiology and Genomic Characteristics of Bloodstream Infection Caused by Carbapenem-Resistant Klebsiella pneumoniae With Decreased Susceptibility to Aztreonam/Avibactam in China. Front. Cell. Infect. Microbiol. 12:926209. doi: 10.3389/fcimb.2022.926209
Received: 22 April 2022; Accepted: 23 May 2022;
Published: 22 June 2022.
Edited by:
Krisztina M. Papp-Wallace, United States Department of Veterans Affairs, United StatesReviewed by:
Fupin Hu, Fudan University, ChinaSiqiang Niu, The First Affiliated Hospital of Chongqing Medical University, China
Marios Karvouniaris, University General Hospital of Thessaloniki AHEPA, Greece
Copyright © 2022 Yu, Shen, Chen, Zhou, Chi and Xiao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Yonghong Xiao, xiaoyonghong@zju.edu.cn
†These authors have contributed equally to this work