High ward-level and within-sample diversity of Klebsiella pneumoniae on a Malawian neonatal unit revealed by single colony whole genome sequencing and post-enrichment metagenomics

Klebsiella pneumoniae is a frequent cause of antimicrobial resistant healthcare associated infections in neonates across sub-Saharan Africa, with multiple lineages associated with neonatal sepsis. However, the full diversity of circulating strains and key reservoirs facilitating transmission within hospitals is unknown. We investigated the population structure and within- sample diversity of K. pneumoniae in a Malawian neonatal unit. We recruited 94 mother- neonate pairs and collected regular stool samples, hand swabs, cot swabs and swaddling cloth samples. Additionally, we collected ward surface-swab samples and staff hand swabs weekly. To establish within sample diversity we employed a dual sequencing approach; (i) single colony picks from Extended-Spectrum Beta-Lactamase selective chromogenic agar for short-read whole genome sequencing; and (ii) post-enrichment metagenomics using plate sweeps from a non-selective agar. In total, we analysed 552 single-colony picks and 772 plate-sweeps from neonate, maternal and environmental samples. Comparing sequence types, surface antigens, antimicrobial resistance and virulence genes, and plasmid replicons, between sample types and sequencing approaches, we identified key advantages and limitations of post-enrichment metagenomics. Our approach revealed high diversity at both the ward and individual level, with a high proportion of the overall diversity likely due to Extended-Spectrum Beta-Lactamase negative organisms. ST15 and ST307 were found in high numbers using both methodologies, whilst ST14 was identified primarily from the non-selective post-enrichment metagenomic samples. Isolates and samples from ward surface swabs had more antimicrobial resistance genes and plasmid replicons than those isolated from human stool. This approach demonstrates the value of combining colony-based and metagenomic sequencing approaches, as a cost-effective alternative to shotgun metagenomics to study health care associated infections. Impact statementKlebsiella pneumoniae is an important cause of healthcare-associated infections (HAIs) in neonates, particularly in sub-Saharan Africa. While multiple strains can colonise a single person or surface, the full diversity of K. pneumoniae in the hospital environment is unknown. This study investigates ward-level and within-sample diversity of K. pneumoniae in a Malawian neonatal unit. We collected stool samples from babies and their mothers, as well as swabs from ward surfaces. To characterize bacterial diversity, we compared two approaches: (i) single- colony whole-genome sequencing of isolates selected under antimicrobial pressure and (ii) post-enrichment metagenomics, where entire microbial communities were sequenced from non- selective culture plates in a single run. We found high diversity of K. pneumoniae, with considerable diversity revealed by post-enrichment metagenomics. Samples frequently had multiple strains of K. pneumoniae within them and this varied by sample type. These data highlight the need to utilize methods that account for within-sample diversity when investigating K. pneumoniae, indicate that single colony WGS is inadequate and a combination of post- enrichment metagenomics and single colony WGS is preferable.