Genetic Diversity of Polymyxin-Resistance Mechanisms in Clinical Isolates of Carbapenem-Resistant Klebsiella pneumoniae: a Multicenter Study in China

Ziyao Li; Xinmeng Liu; Zichen Lei; Chen Li; Feilong Zhang; Yongli Wu; Xinrui Yang; Jiankang Zhao; Yulin Zhang; Yanning Hu; Fangfang Shen; Pingbang Wang; Junwen Yang; Yulei Liu; Binghuai Lu

doi:10.1128/spectrum.05231-22

Genetic Diversity of Polymyxin-Resistance Mechanisms in Clinical Isolates of Carbapenem-Resistant Klebsiella pneumoniae: a Multicenter Study in China

Microbiol Spectr. 2023 Feb 27;11(2):e0523122. doi: 10.1128/spectrum.05231-22. Online ahead of print.

Authors

Ziyao Li^#^{1

2

3

4}, Xinmeng Liu^#^{2

3

5}, Zichen Lei^#^{1

2

3

4}, Chen Li^{2

3

5

6}, Feilong Zhang^{2

3

5}, Yongli Wu^{2

3

4}, Xinrui Yang^{2

3

4}, Jiankang Zhao^{2

3}, Yulin Zhang^{2

3}, Yanning Hu^{2

3

5}, Fangfang Shen⁷, Pingbang Wang⁸, Junwen Yang⁹, Yulei Liu¹⁰, Binghuai Lu^{1

2

3

4

5}

Affiliations

¹ China-Japan Friendship Institute of Clinical Medical Sciences, Beijing, China.
² Laboratory of Clinical Microbiology and Infectious Diseases, Department of Pulmonary and Critical Care Medicine, Center of Respiratory Medicine, National Clinical Research Center for Respiratory Diseases, National Center for Respiratory Medicine, China-Japan Friendship Hospital, Beijing, China.
³ Institute of Respiratory Medicine, Chinese Academy of Medical Sciences, Beijing, China.
⁴ Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China.
⁵ Peking University China-Japan Friendship School of Clinical Medicine, Beijing, China.
⁶ Liuyang Traditional Chinese Medicine Hospital, Changsha, Hunan, China.
⁷ Heping Hospital affiliated with Changzhi Medical College, Changzhi, Shanxi, China.
⁸ The People's Hospital of Liuyang, Changsha, Hunan, China.
⁹ Department of Laboratory Medicine, Zhengzhou Key Laboratory of Children's Infection and Immunity, Children's Hospital Affiliated with Zhengzhou University, Zhengzhou, Henan, China.
¹⁰ Department of Laboratory Medicine, Beijing Anzhen Hospital, Beijing, China.

^# Contributed equally.

Abstract

Polymyxin has been the last resort to treat multidrug-resistant Klebsiella pneumonia. However, recent studies have revealed that polymyxin-resistant carbapenem-resistant Klebsiella pneumonia (PR-CRKP) emerged due to the mutations in chromosomal genes or the plasmid-harboring mcr gene, leading to lipopolysaccharide modification or efflux of polymyxin through pumps. Further surveillance was required. In the present study we collected PR-CRKP strains from 8 hospitals in 6 provinces/cities across China to identify the carbapenemase and polymyxin resistance genes and epidemiological features by whole-genome sequencing (WGS). The broth microdilution method (BMD) was performed to determine the MIC of polymyxin. Of 662 nonduplicate CRKP strains, 15.26% (101/662) were defined as PR-CRKP; 10 (9.90%) were confirmed as Klebsiella quasipneumoniae by WGS. The strains were further classified into 21 individual sequence types (STs) by using multilocus sequence typing (MLST), with ST11 being prevalent (68/101, 67.33%). Five carbapenemase types were identified among 92 CR-PRKP, bla_KPC-2 (66.67%), bla_NDM-1 (16.83%), bla_NDM-5 (0.99%), bla_IMP-4 (4.95%), and bla_IMP-38 (0.99%). Notably, 2 PR-CRKP strains harbored both bla_KPC-2 and bla_NDM-1. The inactivation of mgrB, associated significantly with high-level polymyxin resistance, was mainly caused by the insertion sequence (IS) insertion (62.96%, 17/27). Furthermore, acrR was inserted coincidently by ISkpn26 (67/101, 66.33%). The deletion or splicing mutations of crrCAB were significantly associated with ST11 and KL47 (capsule locus types), and diverse mutations of the ramR gene were identified. Only one strain carried the mcr gene. In summary, the high IS-inserted mgrB inactivation, the close relationship between ST11 and the deletion or splicing mutations of the crrCAB, and the specific features of PR-K. quasipneumoniae constituted notable features of our PR-CRKP strains in China. IMPORTANCE Polymyxin-resistant CRKP is a serious public health threat whose resistance mechanisms should be under continuous surveillance. Here, we collected 662 nonduplicate CRKP strains across China to identify the carbapenemase and polymyxin resistance genes and epidemiological features. Polymyxin resistance mechanism in 101 PR-CRKP strains in China were also investigated, 9.8% of which (10/101) were K. quasipneumoniae, as determined via WGS, and inactivation of mgrB remained the most crucial polymyxin resistance mechanism, significantly related to high-level resistance. Deletion or splicing mutations of crrCAB were significantly associated with ST11 and KL47. Diverse mutations of the ramR gene were identified. The plasmid complementation experiment and mRNA expression analysis further confirmed that the mgrB promoter and ramR played a critical role in polymyxin resistance. This multicenter study contributed to the understanding of antibiotic resistance forms in China.

Keywords: K. quasipneumoniae; antibiotic resistance; carbapenemase-resistant Klebsiella pneumoniae; genetic diversity; mgrB inactivation; multicenter study; polymyxin resistance mechanism; polymyxin-resistant Klebsiella pneumoniae.