Capsular locus deep sequencing to study Klebsiella populations - Klebsiella species are a leading cause of healthcare-associated infections, and gastrointestinal colonization
often precedes infection. Klebsiella colonize ~20% of intensive care patients, and 4% of these patients will
develop pneumonia, bloodstream or urinary tract infections from this pathogen. In 80% of these cases, the
infecting isolate is detectable by culture at the time of admission. These colonizing and infecting Klebsiella
strains are highly diverse, as measured by typing of the capsule locus gene wzi and their gene content. In a
case-control study of colonizing strains that progressed to infection or remained asymptomatic, we identified
147 wzi types across 245 isolates and found that some patients are colonized by more than one Klebsiella
strain simultaneously. Over 22,000 genes varied between these isolates, suggesting that genetic diversity
could lead to large differences in fitness and pathogenic potential. The diversity of colonizing strains between
and within colonized patients raises fundamental questions about how the complex dynamics of colonization
affect infection risk. This high diversity of wzi types also provides a novel tool to answer these questions: a
barcoding scheme that can enable granular characterization of Klebsiella populations and large-scale
competition experiments between non-isogenic strains. The long-term goal of our research is to identify
markers of infection risk that can be used to prevent Klebsiella infections in colonized patients. The goal of this
exploratory study is to develop deep sequencing of the wzi capsular locus as a tool to study Klebsiella
population dynamics and enable rapid and large-scale competition experiments. Our hypothesis is that the wzi
locus is a naturally-occurring barcode that, analogous to 16S sequencing, can be used to measure the relative
abundance of Klebsiella strains in large, diverse populations. Our approach is to develop a wzi amplicon-
sequencing method, apply it to measure population dynamics in patient samples and competitive fitness, and
integrate these data with clinical modeling and comparative genomics. We will leverage an existing set of rectal
swab samples from cases of infection and matched controls, and corresponding fully sequenced Klebsiella
isolates, to complete the following Specific Aims: 1) Validate wzi deep sequencing and measure population
dynamics during colonization. We will develop a wzi sequencing pipeline and validate it on contrived and
archived rectal swab samples. Then we will pilot this technique on rectal swabs to measure diversity of
Klebsiella during human colonization and its impact on infection risk. 2) Apply wzi deep sequencing to non-
isogenic competition experiments in ex vivo colonization and infection models. We will measure relative fitness
from >100 strains simultaneously in human urine, serum, bronchoalveolar fluid, and stool, and against a
potential de-colonization intervention. We will correlate fitness with clinical case status, discover and validate
novel fitness genes in each condition, and test them in animal models. The outcome of this study will be a
powerful approach to accelerate progress in understanding how Klebsiella colonization progresses to infection.