Tn 4661-mediated transfer of bla CTX-M-15 from Klebsiella michiganensis to an outbreak clone of Pseudomonas aeruginosa

Abstract

Carriage of CTX-M-type extended-spectrum β-lactamase (ESBL) is rare in Pseudomonas aeruginosa. During routine surveillance of an endemic ST-621 P. aeruginosa at a large hospital, isolate MRSN 100690 carrying bla _CTX-M-15 was cultured from a patient (P2). This was the first detection of this ESBL in the endemic ST-621 lineage. All 1 488 bacterial isolates collected from the same facility in the 12 months prior to the incidence of 100 690 were screened for the presence of bla _CTX-M-15. A set of 183 isolates was identified, in which corresponding patient metadata was evaluated for spatiotemporal overlaps with P2. The resulting three isolates, along with 100 690, were long-read sequenced using the Oxford Nanopore MinION platform to determine a potential donor of bla _CTX-M-15. The screen revealed a single Klebsiella michiganensis isolate, MRSN 895358, which carried an IncA/C2 plasmid harbouring bla _CTX-M-15. Notably, the patient harbouring 895358, P1, occupied the same hospital room as P2 9 months prior. Genomic alignment revealed that both isolates shared an identical 80.8 kb region containing the IncA/C2 plasmid replicon and bla _CTX-M-15. This region was plasmid bound in 895 358, but chromosomally bound in 100 690 due to Tn4661-mediated transposition. ESBL bla _CTX-M-15 was acquired and subsequently integrated into the chromosome of a ST-621 P. aeruginosa, likely initiated by plasmid transfer from a K. michiganensis strain.

Keywords: CTX-M; ESBL; Pseudomonas aeruginosa; Tn4661; nosocomial infection.

Fig. 1.. Spatiotemporal distribution of 100 690 and 895 358. A schematic following the movement of P1 (light blue) and P2 (dark blue) across the hospital ward following their initial intake. Hospital buildings are indicated as B1 or B2 and hospital rooms are indicated by black squares, labelled as R1, R2, or R3. Movement across rooms is indicated by a solid black line and continued admission in the same room by a dashed line. Culturing of 895358, 100 690, and 103 016 is indicated by orange, red, or green shading, respectively.

Fig. 2.. Proposed pathway of ESBL acquisition by 100 690. (a) Initial acquisition of Kmi895358_P1(CTX) by 100 690 (likely via conjugation) from 895 358. (b) Rearrangement of Kmi895358_P1(CTX) resulting in an 80.8 kb homologous region and a 126.6 kb non-homologous region. (c) A copy of Tn4661 inserts into the 80.8 kb homologous region. (d) This Tn4661 homologous region then integrates into the 100 690 chromosome via Tn4661-mediated recombination, (e) resulting in the observed structure containing bla_CTX-M-15 within the 100 690 chromosome.

Fig. 3.. Mauve and pairwise alignment of (a) Kmi895358_P1(CTX) with CP026281.1 K. oxytoca and CP097701.1 K. pneumoniae segment; and (b) Kmi895358_P1(CTX) with the 100 690 chromosome. Plasmid sequence colour is identical to Fig. 2; 80.8 kb homologous region (dark blue), 126.6 kb non-homologous region (light blue), or Tn4661 (orange). Percent shared identity between aligned sequences is depicted; aligned sequences with <95% shared identity are unshaded.