Epidemiology and Genomic Characteristics of Bloodstream Infection Caused by Carbapenem-Resistant Klebsiella pneumoniae With Decreased Susceptibility to Aztreonam/Avibactam in 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.

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).
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.

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.

Resistance Genes
Nine AZAH-Kp isolates belonged to ST11 and harbored wildtype bla kpc-2 . Other different b-lactamse genes were identified and shown in Table 1. Genetic environment of bla kpc-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).

DISCUSSION
Recent surveillance data reported the lower MIC of AZA (MIC 90 = 0.5/4 mg/L) against CRE, especially for metallo-b-lactamase (MBL)-producing isolates . Our previous data also showed that the MIC 90 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 bla KPC 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 bla KPC 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 bla KPC gene was the leading cause of resistance to ceftazidime/avibactam (Giddins et al., 2018). In addition, bla KPC overexpression also played an important role in ceftazidime/avibactam resistance (Shen et al., 2017). Our findings are similar with the above result that highlevel AZA resistance was closely related to bla KPC 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 . 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.

ACKNOWLEDGMENTS
We thank all cooperative hospitals of Blood Bacterial Resistant Investigation Collaborative System (BRICS) program for their collection of the isolates.