AUTHOR=Zhu Lanlan , Li Ping , Zhang Guangyi , He Zhiyong , Tao Xingyu , Ji Yicheng , Yang Wenjing , Zhu Xiaofang , Luo Wanying , Liao Wenjian , Chen Chuanhui , Liu Yang , Zhang Wei 

TITLE=Role of the ISKpn element in mediating mgrB gene mutations in ST11 hypervirulent colistin-resistant Klebsiella pneumoniae

JOURNAL=Frontiers in Microbiology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2023.1277320

DOI=10.3389/fmicb.2023.1277320

ISSN=1664-302X

ABSTRACT=<sec id="sec1"><title>Background</title><p>Colistin has emerged as a last-resort therapeutic against antibiotic-resistant bacterial infections, particularly those attributed to carbapenem-resistant Enterobacteriaceae (CRE) like CRKP. Yet, alarmingly, approximately 45% of multidrug-resistant <italic>Klebsiella pneumoniae</italic> strains now manifest resistance to colistin. Through our study, we discerned that the synergy between carbapenemase and IS elements amplifies resistance in <italic>Klebsiella pneumoniae</italic>, thereby narrowing the existing therapeutic avenues. This underscores the instrumental role of IS elements in enhancing colistin resistance through mgrB disruption.</p></sec><sec id="sec2"><title>Methods</title><p>From 2021 to 2023, 127 colistin-resistant <italic>Klebsiella pneumoniae</italic> isolates underwent meticulous examination. We embarked on an exhaustive genetic probe, targeting genes associated with both plasmid-mediated mobile resistance-encompassing <italic>bla</italic>KPC, <italic>bla</italic>NDM, <italic>bla</italic>IMP, <italic>bla</italic>VIM, <italic>bla</italic>OXA-48-like, and <italic>mcr-1</italic> to <italic>mcr-8</italic>-and chromosome-mediated resistance systems, including <italic>PhoP/Q</italic>, <italic>PmrA/B</italic>, and <italic>mgrB</italic>. PCR amplification revealed the presence of virulence-associated genes from the <italic>pLVPK</italic> plasmid, such as <italic>rmpA</italic>, <italic>rmpA2</italic>, <italic>iucA</italic>, <italic>iroB</italic>, and <italic>peg344</italic>. <italic>mgrB</italic> sequencing was delegated to Sangon Biotech, Shanghai, and the sequences procured were validated using BLAST. Our search for IS elements was navigated through the IS finder portal. Phenotypically, we harnessed broth microdilution (BMD) to ascertain the MICs of colistin. To sketch the clonal lineage of mgrB-mutated CoR-Kp isolates, sophisticated methodologies like MLST and PFGE were deployed. S1-PFGE unraveled the intrinsic plasmids in these isolates. Our battery of virulence assessment techniques ranged from the string test and capsular serotyping to the serum killing assay and the <italic>Galleria mellonella</italic> larval infection model.</p></sec><sec id="sec3"><title>Results</title><p>Among the 127 analyzed isolates, 20 showed an enlarged <italic>mgrB</italic> PCR amplicon compared to wild-type strains. These emerged over a three-year period: three in 2021, thirteen in 2022, and four in 2023. Antimicrobial susceptibility tests revealed that these isolates consistently resisted several drugs, notably TCC, TZP, CAZ, and COL. Additionally, 85% resisted both DOX and TOB. The MICs for colistin across these strains ranged between 16 to 64 mg/L, with a median of 40 mg/L. From a genetic perspective, MLST unanimously categorized these <italic>mgrB</italic>-mutated CoR-hvKp isolates as ST11. PFGE further delineated them into six distinct clusters, with clusters A and D being predominant. This distribution suggests potential horizontal and clonal genetic transmission. Intriguingly, every mgrB-mutated CoR-hvKP isolate possessed at least two virulence genes akin to the <italic>pLVPK-like</italic> virulence plasmid, with <italic>iroB</italic> and <italic>rmpA2</italic> standing out. Their virulence was empirically validated both <italic>in vitro</italic> and <italic>in vivo</italic>. A pivotal discovery was the identification of three distinct insertion sequence (IS) elements within or near the mgrB gene. These were:ISKpn26 in eleven isolates, mainly in cluster A, with various insertion sites including +74, +125, and an upstream −35.ISKpn14 in four isolates with insertions at +93, −35, and two upstream at −60.IS903B present in five isolates, marking positions like +74, +125, +116, and −35 in the promoter region. These diverse insertions, spanning six unique locations in or near the mgrB gene, underscore its remarkable adaptability.</p></sec><sec id="sec4"><title>Conclusion</title><p>Our exploration spotlights the ISKpn element’s paramount role in fostering <italic>mgrB</italic> gene mutations in ST11 hypervirulent colistin-resistant <italic>Klebsiella pneumoniae</italic>. Employing MLST and PFGE, we unearthed two primary genetic conduits: clonal and horizontal. A striking observation was the ubiquitous presence of the <italic>KPC</italic> carbapenemase gene in all the evaluated ST11 hypervirulent colistin-resistant <italic>Klebsiella pneumoniae</italic> strains, with a majority also harboring the <italic>NDM</italic> gene. The myriad mgrB gene insertion locales accentuate its flexibility and the overarching influence of IS elements, notably the pervasive IS5-like variants ISKpn26 and IS903B. Our revelations illuminate the escalating role of IS elements in antibiotic resistance within ST11 hypervirulent colistin-resistant <italic>Klebsiella pneumoniae</italic>, advocating for innovative interventions to counteract these burgeoning resistance paradigms given their profound ramifications for prevailing treatment modalities.</p></sec>