Transmission ofKlebsiellastrains and plasmids within and between Grey-headed flying fox colonies

The Grey-headed flying fox ( Pteropus poliocephalus ) is an endemic Australian fruit bat, known to carry pathogens with zoonotic potential. We recently showed these bats harbour the bacterial pathogens Klebsiella pneumoniae and closely related species in the K. pneumoniae species complex ( Kp SC). However, the dynamics of Klebsiella transmission and gene flow within flying fox colonies were not explored and remain poorly understood. Here we report a high-resolution genomic comparison of 39 Kp SC isolates from Greyheaded flying foxes. Illumina whole genome sequences (n=39) were assembled de novo and the Kleborate genotyping tool was used to infer sequence types (STs). Oxford Nanopore sequences were generated for 13 isolates (one for each distinct ST) in order to generate high-quality completed reference genomes. Read mapping and variant calling was used to identify single nucleotide variants (SNVs) within each ST, using the relevant reference genome. In silico genome-scale metabolic models were generated to predict and compare substrate usage to 59 previously published Kp SC models for isolates from human and environmental sources, which indicated no distinction on the basis of metabolic capabilities. High-resolution genome comparisons identified five putative strain transmission clusters (four intra- and one inter-colony, n=2-15 isolates each, ≤25 pairwise SNVs). Inter-colony transmission of Klebsiella africana was found between two flying fox populations located within flying distance. The 13 completed genomes harboured 11 plasmids, all of which showed 37-98% coverage (mean 73%) and ≥95% identity to those previously reported from human-associated Kp SC. Comparison of plasmids from different flying fox associated Kp SC indicated an interspecies horizontal plasmid transmission between K. pneumoniae and K. africana for a 98 kbp plasmid, pFF1003. These data indicate that Kp SC are able to transmit directly via flying fox populations or indirectly via a common source, and that these isolates can harbour plasmids with similarity to those found in human derived Kp SC, indicating gene flow is occurring between isolates from Grey-headed flying fox Kp SC and human clinical isolates.