Danièle Meunier

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.

Background: Ceftazidime/avibactam, launched in the United Kingdom (UK) in March 2017, is an antibiotic against multidrug-resistant Gram-negative pathogens. It was selected for the government’s subscription model pilot, for incentivising new antibiotic development, which began in December 2020. Aim: Ahead of the pilot, we assessed ceftazidime/avibactam testing, resistance (2016−2020) and usage (2017−2020) levels in England, as baselines for future surveillance. Methods: From routine surveillance samples, we retrieved reported ceftazidime/avibactam resistance categorisation. From reference laboratory samples, we reviewed minimum inhibitory concentration (MICs) and molecular data. Among surveillance samples, per cent resistance was estimated. Referred samples’ MICs, by carbapenemase gene presence, were investigated. Ceftazidime/avibactam hospital use was measured in defined daily doses (DDDs). Results: Overall, 6.3% (4,200/66,914; 95% confidence interval (95%CI): 6.1–6.4%) of surveillance-reported ceftazidime/avibactam-tested Gram-negative bacteria were resistant. Percentage resistance per bacterial species varied over time, somewhat stabilising as testing was established, with between April 2019 and March 2020, 1.3% Escherichia coli (288/22,736; 95%CI: 1.1−1.4%), 12.6% Pseudomonas aeruginosa (690/5,495; 95%CI: 11.7−13.5%) and 6.1% of Klebsiella pneumoniae (314/5,179; 95%CI: 5.4−6.7%) being resistant. For 8,437 referred Enterobacterales, MIC determination found 11.5% (968/8,437; 95%CI: 10.8–12.2%) resistant. Among resistant isolates, 89.3% (864/968; 95%CI: 87.1–91.1%) had metallo-β-lactamase (MBL) genes. Of 908 MBL-negative isolates, producing ≥ 1 non-metallo-carbapenemase(s), 2.1% (19/908; 95%CI: 1.3–3.2%) were resistant. Since March 2017, 69.5% (105/151) of English National Health Service Trusts used ceftazidime/avibactam. Monthly usage progressed from 21 to 744 DDDs in March 2020. Conclusion: For appropriate treatment, carbapenemase gene detection and variant identification in ceftazidime/avibactam surveillance matters. Detecting emerging resistant pathogens and preventing spread within healthcare settings requires vigilance.

Introduction. Combination of PCR and Elek testing to identify toxigenic corynebacteria has revealed organisms described as non-toxigenic toxin-gene bearing (NTTB) Corynebacterium diphtheriae or C. ulcerans (i.e. PCR tox positive; Elek negative). These organisms carry part or all of tox, but are unable to express diphtheria toxin (DT) and present a challenge to clinical and public health case management. Gap analysis/Hypothesis. There are few data on the theoretical risk of NTTB reversion to toxigenicity. This unique cluster and subsequent epidemiologically linked isolates allowed the opportunity to determine any change in DT expression status. Aim. To characterize a cluster of infections due to NTTB in a skin clinic and subsequent cases in two household contacts. Methodology. Epidemiological and microbiological investigations were carried out according to existing national guidance at the time. Susceptibility testing used gradient strips. The tox operon analysis and multi-locus sequence typing (MLST) was derived from whole-genome sequencing. Alignment of the tox operon and phylogenetic analyses were performed using clustalW, mega, the public core-genome MLST (cgMLST) scheme and an in-house bioinformatic single nucleotide polymorphism (SNP) typing pipeline. Results. Isolates of NTTB C. diphtheriae were recovered from four cases (cases 1 to 4) with epidermolysis bullosa attending the clinic. Two further isolates were subsequently recovered from case 4, >18 months later, and from two household contacts (cases 5 and 6) after a further 18 months and 3.5 years, respectively. All eight strains were NTTB C. diphtheriae biovar mitis, belonged to the same sequence type (ST-336) with the same deletion in tox. Phylogenetic analysis showed relatively high diversity between the eight strains with 7–199 SNP and 3–109 cgMLST loci differences between them. The number of SNPs between the three isolates from case 4 and two household contacts (cases 5 and 6) was 44–70 with 28–38 cgMLST loci differences. Conclusions. We report a cluster of NTTB C. diphtheriae cases in a skin clinic and evidence of onward household transmission. We conclude the deletion in the tox was responsible for the non-expression of DT. There was no evidence of reversion to DT expression over the 6.5 year period studied. These data informed revision to guidance in the management of NTTB cases and their contacts in the UK.

Introduction. Increasing numbers of carbapenemase-producing Enterobacterales (CPE), which can be challenging to treat, have been referred to the national reference laboratory in England since the early 2000s. Gap Statement/Aim. Previous studies on CPE in the UK have focussed on localized outbreaks. We applied whole-genome sequencing (WGS) to isolates referred to the national reference laboratory over 30 months to inform our understanding of CPE epidemiology in England. Methodology. The first confirmed CPE from each new patient referred by an English diagnostic laboratory between 1 January 2014 and 30 June 2016 was sequenced on an Illumina HiSeq 2500. Multiple isolates from the same patient were included from either different species or the same species with different carbapenemase genes. The data were analysed using an in-house bioinformatics pipeline that determines species identification, multi-locus sequence typing (MLST) profile and antimicrobial resistance gene content. Results. A total of 2658 non-duplicate CPE were sequenced amongst which three host organisms belonging to diverse sequence types (STs) predominated: Klebsiella pneumoniae (1380/2658, 51.9 %; 177 STs), Escherichia coli (723/2658, 27.2 %; 133 STs) and Enterobacter cloacae (294/2658, 11.1 %; 88 STs). Thirty different carbapenemase gene variants were identified, although bla _OXA-48-like (1122/2658, 42.2%), bla _NDM (692/2658, 26.0 %), bla _KPC (571/2658, 21.5 %), bla _VIM (100/2658, 3.8 %) and bla _IMP (33/2658, 1.2 %) predominated. ST/carbapenemase gene pairings represented widely distributed high-risk clones or clusters at a regional or hospital level. Conclusion. CPE referred to the national reference laboratory are diverse, suggesting multiple introductions to England and a role for horizontal transfer of carbapenemase genes in English CPE epidemiology.

Transferable linezolid resistance due to optrA, poxtA, cfr and cfr-like genes is increasingly detected in enterococci associated with animals and humans globally. We aimed to characterize the genetic environment of optrA in linezolid-resistant Enterococcus faecalis isolates from Scotland. Six linezolid-resistant E. faecalis isolated from urogenital samples were confirmed to carry the optrA gene by PCR. Short read (Illumina) sequencing showed the isolates were genetically distinct (>13900 core SNPs) and belonged to different MLST sequence types. Plasmid contents were examined using hybrid assembly of short and long read (Oxford Nanopore MinION) sequencing technologies. The optrA gene was located on distinct plasmids in each isolate, suggesting that transfer of a single plasmid did not contribute to optrA dissemination in this collection. pTM6294-2, BX5936-1 and pWE0438-1 were similar to optrA-positive plasmids from China and Japan, while the remaining three plasmids had limited similarity to other published examples. We identified the novel Tn6993 transposon in pWE0254-1 carrying linezolid (optrA), macrolide (ermB) and spectinomycin [ANT(9)-Ia] resistance genes. OptrA amino acid sequences differed by 0–20 residues. We report multiple variants of optrA on distinct plasmids in diverse strains of E. faecalis . It is important to identify the selection pressures driving the emergence and maintenance of resistance against linezolid to retain the clinical utility of this antibiotic.

Background: ESBL- and carbapenemase-producing Pseudomonas aeruginosa are prevalent in, for example, the Middle East, Eastern Europe and Latin America, though rarer elsewhere. Because P. aeruginosa readily mutate to become carbapenem resistant via loss of OprD, isolates producing ESBLs are often as broadly resistant as those producing carbapenemases. We hypothesized that: (i) relebactam might overcome class A carbapenemases directly in P. aeruginosa; and (ii) relebactam’s inhibition of AmpC, which gives a generalized potentiation of imipenem against the species, might restore imipenem susceptibility in OprD-deficient ESBL producers. Methods: MICs were determined using CLSI agar dilution for P. aeruginosa isolates producing ESBLs, principally VEB types, and for those producing GES-5, KPC and other carbapenemases. Results: Relebactam potentiated imipenem by around 4–8-fold for most P. aeruginosa isolates producing VEB and other ESBLs; however, MICs were typically only reduced to 4–16 mg/L, thus mostly remaining above EUCAST’s susceptible range and only partly overlapping CLSI’s intermediate range. Strong (approx. 64-fold) potentiation was seen for isolates producing KPC carbapenemases, but only 2-fold synergy for those with GES-5. Predictably, potentiation was not seen for isolates with class B or D carbapenemase activity. Conclusions: Relebactam did potentiate imipenem against ESBL-producing P. aeruginosa, which are mostly imipenem resistant via OprD loss, but this potentiation was generally insufficient to reduce imipenem MICs to the clinical range. Imipenem resistance owing to KPC carbapenemases was reversed by relebactam in P. aeruginosa, just as for Enterobacterales.

Objectives: To characterise the genetic environment of optrA in linezolid-resistant Enterococcus faecalis isolates from Scotland. Methods: Linezolid-resistant E. faecalis were identified in three Scottish Health Boards and confirmed to carry the optrA gene at the national reference laboratory. WGS was performed with short read (Illumina MiSeq) and long read (Oxford Nanopore MinION) technologies to generate complete genome assemblies. Illumina reads for 94 E. faecalis bloodstream isolates were used to place the optrA -positive isolates in a larger UK phylogeny. Results: Six optrA -positive linezolid-resistant E. faecalis were isolated from urogenital samples in three Scottish Health Boards (2014-2017). No epidemiological links were identified between the patients, four were community-based, and only one had recent linezolid exposure. Reference-based mapping confirmed the isolates were genetically distinct (>13,900 core SNPs). optrA was located on a plasmid in each isolate and these plasmids showed limited nucleotide similarity. There was variable presence of transposable elements surrounding optrA , (including IS 1216 , IS 3 , and Tn 3 ) and not always as a recognisable gene cassette. OptrA amino acid sequences were also divergent, resulting in four protein variants differing in 1-20 residues. One isolate belonged to ST16 and clustered with three other isolates in the UK collection (76-182 SNPs), otherwise the optrA- positive isolates were genetically distinct from the bloodstream isolates (>6,000 SNPs). Conclusions: We report multiple variants of the linezolid resistance gene optrA in diverse E. faecalis strain and plasmid backgrounds, suggesting multiple introductions of the gene into the E. faecalis population and selection driving recent emergence.

Carbapenem resistance in Gram-negative bacteria is a public health concern. Numerous government and agency reports consequently discuss ‘CRE’ and ‘CROs’, meaning ‘Carbapenem-Resistant Enterobacterales’ or ‘Carbapenem-resistant organisms’. Unfortunately these terms are fuzzy. Do they include (i) Proteeae with inherent imipenem resistance, (ii) porin-deficient Enterobacterales resistant to ertapenem but not other carbapenems, (iii) Enterobacterales with OXA-48-like enzymes that remain ‘carbapenem susceptible’ at breakpoint, and (iv) Pseudomonas aeruginosa that merely lack OprD? Counting carbapenemase-producing Enterobacterales/organisms (‘CPE’ or ‘CPOs’) is better but still insufficient, because different carbapenemases have differing treatment implications, particularly for new β-lactam/β-lactamase inhibitor combinations. At the least it is essential for authors, journals, and regulatory agencies to specify the carbapenemases meant. The future may demand even greater precision, for mutations can alter activity, and the ability to confer resistance, within carbapenemase families.