Genomic and molecular characterisation of a Klebsiella pneumoniae clinical isolate resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam

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Abstract
This article reports an unusual Klebsiella pneumoniae clinical isolate, KpMVR1, resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam, and investigates the underlying genetic alterations using comparative genomics and molecular experiments. Resistance to carbapenems and third-generation cephalosporins is increasing in K. pneumoniae globally, restricting therapeutic options. The β-lactam/β-lactamase inhibitor combinations are widely used to circumvent β-lactamase-mediated resistance. In 2021, isolate KpMVR1 was recovered from a hospitalised patient in England. Two additional isolates with the same variable-number tandem-repeat profile—KpMVS1, collected from the same patient 42 days before KpMVR1, and KpMVS2, from another patient in the same hospital—were susceptible to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam. Illumina and nanopore whole-genome sequencing and hybrid genome assembly were conducted for these three isolates. Annotated genome assemblies were compared to identify genetic variation, and mutagenesis experiments were performed to verify predicted functional alterations. All isolates belonged to a novel clone ST8134 and carried bla KPC-2 -like alleles (KpMVR1: bla KPC-157 ; KpMVS1 and KpMVS2: bla KPC-2 ) in presumptively conjugative plasmids. IS Ec68 caused a frameshift mutation in KpMVR1’s ompK36 gene, reducing the meropenem-vaborbactam and imipenem-relebactam susceptibility. KPC-157 demonstrated decreased hydrolysis of imipenem and ceftazidime when compared with KPC-2. KpMVR1 also encoded a disrupted transcriptional repressor MarR and a destabilising mutation in AcrB, a component of the AcrAB-TolC multidrug efflux pump. In conclusion, KpMVR1 harboured complex resistance-associated genetic alterations, with evidence for in vivo emergence of antimicrobial resistance. Our study underlines routine screening for resistant pathogens in vulnerable patients to guide antimicrobial chemotherapy as well as the need to characterise underlying resistance mechanisms to help assess the potential for onward transmission. Data summary: Illumina and nanopore sequencing reads, hybrid genome assemblies, and anonymised metadata of isolates KpMVS1, KpMVR1, and KpMVS2 have been deposited in databases of the National Center for Biotechnology Information ( www.ncbi.nlm.nih.gov ) under BioProject accession PRJNA1084250, with BioSample accessions SAMN46778009 (KpMVS1), SAMN46778010 (KpMVR1), and SAMN46778011 (KpMVS2). The genome assemblies of these isolates have also been deposited in Pasteur Institute’s database for K. pneumoniae species complex ( bigsdb.pasteur.fr/klebsiella/ ) under ids 75608 (KpMVS1), 75609 (KpMVR1), and 75610 (KpMVS2). Impact statement: This is the first bla KPC -positive K. pneumoniae isolate referred to the UK’s national reference laboratory with resistance to three last-resort β-lactam/β-lactamase inhibitor combinations meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam, implicating in vivo emergence of this unusual resistance profile during prolonged antimicrobial chemotherapy. This isolate belonged to a novel clone ST8134 and harboured a plasmid-borne bla KPC-2 -like allele bla KPC-157 . We identified complex genetic alterations in this isolate: chromosomal large deletions, point mutations, and an IS Ec68 -induced loss-of-function truncation of the ompK36 porin gene. We determined the impact of KPC-2, KPC-157, and the ompK36 truncation on the susceptibility of K. pneumoniae to meropenem, meropenem-vaborbactam, imipenem, imipenem-relebactam, imipenem-avibactam, aztreonam, aztreonam-avibactam, ceftazidime, ceftazidime-avibactam, and cefiderocol. Our work underscores the need to monitor emerging resistance to beta-lactam/beta-lactamase inhibitor combinations in healthcare and to understand underlying resistance mechanisms for assessing the potential of resistance transmission.
Funding Information
  • Wellcome Trust (PSN109)
  • National Institute for Health and Care Research (NIHR200876)

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