Refining microbiome resolution: KoSCAPE bio identifies Klebsiella oxytoca complex species in16S rRNA amplicon data and uncovers associations with NEC

Klebsiella are early colonizers and commensals of human hosts but are also opportunistic pathogens. Research has focused on Klebsiella pneumoniae due to its global contribution to nosocomial infection and antibiotic resistance. Yet Klebsiella oxytoca and several closely related species are increasingly recognized as clinically significant bacteria underlying enteric and respiratory disorders. Inadequate interspecies discrimination inhibits advancement of our understanding of these diseases including necrotizing enterocolitis (NEC) in infants. Here we leverage the taxonomic potential of the amplified V3 and V4 regions of the K. oxytoca species complex (KoSC) 16S rRNA genes. We developed a curated database of all 16S rRNA gene sequences available for KoSC genomes and the automated pipeline KoSCAPEbio for efficient interspecies discrimination. Sequence polymorphism in the V3-V4 area revealed 41 signature sequences unique to individual species. Six amplicon variants are unique to the KoSC but shared by two to three species within the complex. Genetic linkage of the specific variants to the clinically relevant tiI gene cluster provided a predictive marker for risk of enterotoxin exposure. Reiteration of the workflow with K. aerogenes complex genomes and phylogroups of the K. pneumoniae species complex (KpSC) added K. aerogenes-specific variant typing. We then applied KoSCAPEbio to interrogate publicly available datasets. Analyses resolved and quantified coresident Klebsiella in patient samples including species belonging to KoSC, K. aerogenes and the KpSC. The population structure thus delineated in this proof of principle research differs significantly in disease versus controls. NEC cases associate positively with exclusive presence of the KoSC and negatively with cooccurrence of both KoSC and KpSC, suggesting conflict within the Klebsiella may promote pathogenesis. Thus, KoSCAPEbio has potential to contribute to a better understanding of factors involved in the etiology of NEC and other Klebsiella spp. associated diseases.