Background

Infections caused by Gram-negative organisms resistant to third-generation cephalosporins and carbapenems are a major global health problem with few treatment options. Resistance is mediated by extended-spectrum beta-lactamases (ESBLs) or carbapenemases that are frequently located on large plasmids capable of transfer between different bacterial species. Detecting plasmid transmission in hospitals remains difficult with no gold-standard methods.We therefore aimed to develop a novel methodology for detecting plasmid transmission using Oxford Nanopore long-read whole genome sequencing.

Methods

We conducted a 6-month prospective study in the haematology/bone marrow transplant ward of our hospital to detect plasmid transmission between patients. Patients were screened for colonisation on admission, weekly, then on discharge. All clinical isolates causing infectionwere collected. Our protocol for determining likely plasmid transmission combined plasmid replicon information (from MobSuite), plasmid distances (with pling), single nucleotide variants, and patient movement data.

Results

We collected 171 ESBL and/or carbapenemase isolates from 253 patients. Of these 171 isolates, only 11 came from clinical infections, the remaining 160 were colonisation. Isolateswere long-read sequenced and assembled with Hybracter. Antimicrobial resistance geneswere detected with AMRFinderPlus. Most isolates were E. coli (n=89), followed by Klebsiella (n=42), Enterobacter (n=30) and Citrobacter (n=7). Acquired ESBL/carbapenemase genes were found on plasmids in 68% of genomes.

We extracted 107 ESBL/carbapenemase plasmids for further analysis. Using our systematic plasmid comparison protocol, we detected only three instances of likely plasmid transmissionbetween patients, compared to eight instances of strain transmission.

Conclusions

Whilst plasmid transmission was rare in our dataset, we found that considering multiple metrics as well as patient metadata is important when clustering plasmids. Usage of a single tool was often insufficient when attempting to tease apart complex plasmid communities. This study demonstrated that there is significant AMR transmission occurring between patients on this ward, which can then lead to infections by these resistant colonising isolates.