Resistome and virulome accretion in an NDM-1-producing ST147 sublineage of Klebsiella pneumoniae associated with an outbreak in Tuscany, Italy: a genotypic and phenotypic characterisation

Abstract

Background Carbapenemase-producing Enterobacterales (CPE), particularly those producing metallo-β-lactamases, are among the most challenging antibiotic-resistant pathogens, causing outbreaks of difficult-to-treat nosocomial infections worldwide. Since November 2018, an outbreak of New Delhi metallo-β-lactamases-positive CPE (NDM-CPE) has emerged in Tuscany, Italy. In this study, we aimed to investigate the NDM-CPE associated with the outbreak and characterise the responsible Klebsiella pneumoniae clone.Methods We used whole-genome sequencing and bioinformatic analysis to characterise NDM-CPE isolates that caused bloodstream infections in 53 patients at 11 hospitals in Tuscany and that were collected between Jan 1, 2018, and July 5, 2019 (ie, the early phase of the outbreak and preceding months). The CPE isolates characterised in this study were isolated and identified at the species level and as NDM producers by six diagnostic microbiology laboratories that serve the 11 hospitals. We used comparative genomic analysis, antimicrobial susceptibility testing, plasmid conjugal transfer assays, evaluation of virulence potential in the Galleria mellonella infection model, and serum bactericidal assays to further characterise the clone causing the outbreak. FindingsThe outbreak was sustained by an ST147 K pneumoniae producing NDM-1, which had a complex resistome that mediated resistance to most antimicrobials (except cefiderocol, the aztreonam-avibactam combination, colistin, and fosfomycin). The clone belonged to a sublineage of probably recent evolution, occurred by the sequential acquisition of an integrative and conjugative element encoding the yersiniabactin siderophore, an FIB(pQil)-type multiresistance plasmid carrying bla NDM-1 , and a transferable chimeric plasmid, derived from virulence elements of hypervirulent K pneumoniae, carrying several resistance and virulence determinants. Infection of G mellonella larvae revealed a variable virulence potential. The behaviour in serum bactericidal assays was different from typical hypervirulent K pneumoniae strains, with variable grades of serum resistance apparently associated with mutations in specific chromosomal loci (csrD, pal, and ramR).Interpretation This description of a sublineage of ST147 K pneumoniae with a complex resistome and virulome that is capable of sustaining a large regional outbreak adds to existing research on the evolutionary trajectories within high-risk clones of K pneumoniae. Global surveillance programmes are warranted to track the dissemination of these lineages, and to prevent and control their spread.

Nosocomial outbreak by NDM-1-producing Klebsiella pneumoniae highly resistant to cefiderocol, Florence, Italy, August 2021 to June 2022

“…The resistance profile was consistent with the resistome of the isolates (see Supplementary Table S1 for the list of the acquired resistance genes revealed by the in silico analysis), which overall reflected that of the Tuscan ST147-vir clone [ 9 ], with minor inter-isolate variability.…”

“…In late 2018, an outbreak of NDM-producing Enterobacterales emerged in Tuscany in central Italy, involving several hospitals especially from the north-western area [ 7 , 8 ]. The outbreak was mostly sustained by strains of an ST147 NDM-Kp sublineage (named ST147-vir clone) which uniformly exhibited FDC susceptibility [ 9 ]. Since 2019, at our facility, the largest regional tertiary care hospital located in the central area of Tuscany, cases with NDM-Kp related to the regional outbreak have been recurrently observed following the transfer of infected or colonised patients from other hospitals or long-term-care facilities, with limited intra-hospital transmission.…”

“…Whole genome sequencing (WGS) was carried out either on the first isolate from patients who experienced only carriage of FDC-resistant NDM-Kp, or on the first isolate from the most clinically relevant infection (BSI > LRTI > UTI), when more isolates were available from the same patient (n = 9 patients) ( Figure 3 ). WGS was performed by Illumina Miseq (San Diego, United States) as previously described [ 9 ]. WGS analysis revealed that all the FDC-resistant NDM-Kp isolates belonged to ST147 and were related with the ST147-vir clone circulating in Tuscany [ 9 ] ( Figure 3 ).…”

“…KP-1PI, KP-25PA, KP-26PI, KP-8POA were included as comparators belonging to the ST-147-vir clone circulating in Tuscany [ 9 ]. ERS845991 (from Hungary), MRSN519386 (from Middle East region), SIKP004 (from Thailand), MRSN501925 (from Germany), Kpv_ST147_SE1_2_NDM (from United Kingdom) and 2020EL-00059 (from United States) strains were included in the analysis as outliner representants of ST147 K. pneumoniae.…”

Empiric treatment of patients with sepsis and septic shock and place in therapy of cefiderocol: a systematic review and expert opinion statement

“…It was characterized by a complex resistome responsible for resistance to most antimicrobials except from cefiderocol and few others. The acquisition of a chimeric plasmid, indeed, which included genes for the siderophores yersiniabactin and aerobactin, granted cefiderocol activity [ 18 ]. A cefiderocol MIC value of < 4 μg/ml was observed, in addition to the superiority in the eradication of bacteria with carbapenem resistance (meropenem and imipenem MIC > 6 μg/ml) [ 15 ].…”