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Background <p>This study aimed to investigate the molecular basis of polymyxin B resistance in clinical carbapenem-resistant <i>Klebsiella pneumoniae</i> (CRKP) isolates.</p> Methods <p>A total of 52 polymyxin B-resistant CRKP clinical isolates were collected at West China Hospital between 2018 and 2021. Polymyxin B susceptibility testing was performed using the broth microdilution method. Whole-genome sequencing was conducted to determine sequence types (STs), capsular loci, resistance genes, virulence genes and mutations in genes associated with polymyxin B resistance. Transcriptome sequencing was used to compare changes in gene expression between selected high-minimum inhibitory concentration (MIC) isolates after polymyxin B exposure and controls cultured without polymyxin B.</p> Results <p>The majority (98.08%, 51/52) of CRKP isolates produced carbapenemases, predominantly KPC-2 (92.16%, 47/51). ST11 (88.46%, 46/52) and capsular type KL64 (71.15%, 37/52) were the most prevalent. Eleven isolates (21.15%) were found to carry genetic markers associated with hypervirulence, including <i>rmpA</i>, <i>rmpA2</i>, <i>iucABCD</i>,<i> iutA</i> and <i>peg-344.</i> Genetic alterations in the <i>mgrB</i> gene were identified in 80.77% (42/52) of the polymyxin B- resistant isolates. These alterations were primarily mediated by insertion sequences (88.10%, 37/42), with IS<i>5</i> family elements being the most common. Isolates with <i>mgrB</i> alterations exhibited significantly higher polymyxin B MICs (geometric mean 28.51&#xa0;mg/L, MIC₅₀ 32&#xa0;mg/L, MIC₉₀ 64&#xa0;mg/L) than those without (geometric mean 11.31&#xa0;mg/L, MIC₅₀ 12&#xa0;mg/L, MIC₉₀ 32&#xa0;mg/L) (<i>P</i><i> = 0.003</i>). Several amino acid substitutions were identified in polymyxin B resistance-related proteins, including D86E, E57G, and A41T in PmrA; G345E, T157P, and L213M in PmrB; D191A in PhoP; A63E and A20S in PhoQ; and S257L and M54I in PmrC. Notably, some mutations, such as M54I in PmrC and A20S and A63E in PhoQ, have not been previously reported. No <i>mcr</i> genes were detected. Transcriptomic analysis of isolates P40 (MIC &gt; 64&#xa0;mg/L) revealed upregulation of <i>pmrA</i> (log₂FC = 1.02, adjusted <i>P</i> &lt; 0.001), but downregulation of <i>ompA</i> (log₂FC = -2.85), <i>arnA</i> (log₂FC = -1.46), <i>ugd</i> (log₂FC = -1.01) <i>and tolC</i> (log₂FC = -1.66) (adjusted <i>P</i> &lt; 0.001 for all) after polymyxin B exposure.</p> Conclusions <p>In southwest China, polymyxin B resistance in the studied CRKP isolates was most frequently associated with chromosomal mutations, particularly insertional inactivation of the <i>mgrB</i> gene. The emergence of polymyxin B- and carbapenem-resistant ST11-KL64 hypervirulent clone is a potential threat to antiinfection treatment.</p>

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Molecular analysis of polymyxin B resistance in clinical carbapenem-resistant Klebsiella pneumoniae isolates from a large tertiary care teaching hospital in Southwest China

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  • 发表日期：

    2026年5月20日

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  • Siying Wu,
  • Quanfeng Liao,
  • Ya Liu,
  • Jin Deng,
  • Weili Zhang,
  • Mei Kang

摘要

Background

This study aimed to investigate the molecular basis of polymyxin B resistance in clinical carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates.

Methods

A total of 52 polymyxin B-resistant CRKP clinical isolates were collected at West China Hospital between 2018 and 2021. Polymyxin B susceptibility testing was performed using the broth microdilution method. Whole-genome sequencing was conducted to determine sequence types (STs), capsular loci, resistance genes, virulence genes and mutations in genes associated with polymyxin B resistance. Transcriptome sequencing was used to compare changes in gene expression between selected high-minimum inhibitory concentration (MIC) isolates after polymyxin B exposure and controls cultured without polymyxin B.

Results

The majority (98.08%, 51/52) of CRKP isolates produced carbapenemases, predominantly KPC-2 (92.16%, 47/51). ST11 (88.46%, 46/52) and capsular type KL64 (71.15%, 37/52) were the most prevalent. Eleven isolates (21.15%) were found to carry genetic markers associated with hypervirulence, including rmpA, rmpA2, iucABCD, iutA and peg-344. Genetic alterations in the mgrB gene were identified in 80.77% (42/52) of the polymyxin B- resistant isolates. These alterations were primarily mediated by insertion sequences (88.10%, 37/42), with IS5 family elements being the most common. Isolates with mgrB alterations exhibited significantly higher polymyxin B MICs (geometric mean 28.51 mg/L, MIC₅₀ 32 mg/L, MIC₉₀ 64 mg/L) than those without (geometric mean 11.31 mg/L, MIC₅₀ 12 mg/L, MIC₉₀ 32 mg/L) (P = 0.003). Several amino acid substitutions were identified in polymyxin B resistance-related proteins, including D86E, E57G, and A41T in PmrA; G345E, T157P, and L213M in PmrB; D191A in PhoP; A63E and A20S in PhoQ; and S257L and M54I in PmrC. Notably, some mutations, such as M54I in PmrC and A20S and A63E in PhoQ, have not been previously reported. No mcr genes were detected. Transcriptomic analysis of isolates P40 (MIC > 64 mg/L) revealed upregulation of pmrA (log₂FC = 1.02, adjusted P < 0.001), but downregulation of ompA (log₂FC = -2.85), arnA (log₂FC = -1.46), ugd (log₂FC = -1.01) and tolC (log₂FC = -1.66) (adjusted P < 0.001 for all) after polymyxin B exposure.

Conclusions

In southwest China, polymyxin B resistance in the studied CRKP isolates was most frequently associated with chromosomal mutations, particularly insertional inactivation of the mgrB gene. The emergence of polymyxin B- and carbapenem-resistant ST11-KL64 hypervirulent clone is a potential threat to antiinfection treatment.