Diverse modes of ceftazidime/avibactam resistance acquisition in carbapenem-resistant Klebsiella pneumoniae and Pseudomonas aeruginosa from a Chinese intensive care unit

Abstract

Objectives: To investigate the mechanisms of ceftazidime/avibactam (CZA) resistance and the nosocomial dissemination of carbapenem-resistant Pseudomonas aeruginosa (CRPA) and carbapenem-resistant Klebsiella pneumoniae (CRKP) in an intensive care unit (ICU) in China.

Methods: Clinical CRPA and CRKP isolates were obtained from an ICU of a tertiary hospital in China from August 2020 to February 2021. Antimicrobial susceptibility was determined according to CLSI. WGS, cloning experiments and kinetic parameters were conducted to reveal resistance mechanisms, molecular characteristics and dissemination of CRPA and CRKP.

Results: We isolated 32 CZA-resistant strains, including 12 CRPA and 20 CRKP strains from an ICU between August 2020 and February 2021. CZA resistance was associated with the presence of NDM and efflux pumps in CRKP strains, whereas bla_AFM-2, bla_KPC-87, and bla_PER-1 contributed to CZA resistance in CRPA strains. Compared to KPC-2, KPC-87 exhibited a 1.5-fold elevation in k_cat/K_m for ceftazidime, a 7.5-fold increase in K_i for avibactam, and a loss of carbapenem hydrolysis. bla_KPC-87 was located in the NTE_KPC-IIa like element based on the Tn3. Insertion of 656 bp Δbla_TEM-1 upstream of bla_KPC-87 introduced an additional promoter that increased KPC-87 expression. Cluster 2 and 3 of CRKP represented two different clones of ST11 transmitted between patients. KPC-87-producing ST270 CRPA strains exhibited a small-scale dissemination and cross-regional transfer with the referral of a patient. The evolutionary pathways of AFM-2-producing ST275 CRPA strains were more complex to elucidate the transmission events.

Conclusions: In CRKP and CRPA, diverse resistance mechanisms contributed to CZA resistance. These CZA-resistant strains were transmitted among patients in the ICU and even across regions to the other healthcare unit when the patient was transferred.

Keywords: Klebsiella pneumoniae; Pseudomonas aeruginosa; Ceftazidime/avibactam; Cross-regional transmission; KPC; Non-Tn4401 elements.

Fig. 1

Isolation time and molecular characterization of CZA-resistant strains. Isolation time and source of CZA-resistant strains. Squares represent K. pneumoniae strains and circles represent P. aeruginosa strains. Different colours represent various carbapenemase-carrying characteristics. The strains in K. pneumoniae cluster 1 and SNPs among the strains were marked by blue line. The orange line and the green line marked the strains in K. pneumoniae cluster 2 and cluster 3, respectively. Only the two smallest SNPs with similar separation times are labelled

Fig. 2

Genetic context analysis of bla_KPC-87, bla_AFM-2 and bla_PER-1. A Genetic context comparison of bla_KPC-87 and bla_KPC-2. Shading denotes nucleotide identity > 99%. B Promoter region comparison of bla_KPC-87 and bla_KPC-2. Sequence of Δbla_TEM-1 were labeled in yellow. Putative promoter regions are underlined, in which the − 35 and − 10 and + 1 promoter elements are shown in red characters. RBS, ribosome binding site. C Genetic context comparison of bla_AFM-2. Shading denotes nucleotide identity > 99%. D Genetic context comparison of bla_PER-1. Shading denotes nucleotide identity > 99%

Fig. 3

The phylogenetic tree of ST270 P. aeruginosa strains. Core-genome phylogenetic tree was built for 32 ST270 P. aeruginosa strains and rooted in the midpoint. Isolation year was indicted by different shades of blue. The clade 1 was labeled in yellow shade and the clade 2 was labeled in green shade. The red branches are the branches where the ST270 P. aeruginosa strains in this study. Scale bar indicates nucleotide substitutions per site