14Healthcare-acquired infections (HAIs) kill tens of thousands of people each year 15 and add significantly to healthcare costs. Multidrug resistant and epidemic strains are a 16 large proportion of HAI agents, and multidrug resistant strains of Klebsiella pneumoniae, 17 a leading HAI agent, have become an urgent public health crisis. In the healthcare 18 environment, patient colonization of K. pneumoniae precedes infection, and transmission 19 via colonization leads to outbreaks. Periodic patient screening for K. pneumoniae 20 colonization has cost-effective and life-saving potential. In this study, we describe the 21 design and validation of KlebSeq, a highly informative screening tool that detects 22Klebsiella species and identifies clinically important strains and characteristics using 23 highly multiplexed amplicon sequencing without a live culturing step. We demonstrate 24 the utility of this tool on several complex specimen types including urine, wound swabs 25 and tissue, several types of respiratory, and fecal, showing K. pneumoniae species and 26 clonal group identification and antimicrobial resistance and virulence profiling, including 27 capsule typing. Use of this amplicon sequencing tool can be used to screen patients for K. 28 pneumoniae carriage to assess risk of infection and outbreak potential, and the expansion 29 of this tool can be used for several other HAI agents or applications. 30 highly significant levels (3-6). Healthcare-and outbreak-associated strain types of K. 37 pneumoniae that appear highly transmissible and have a propensity for antimicrobial 38 resistance (AMR) or virulence gene acquisition are a growing proportion of the K. 39 pneumoniae species (7-18). ST258, the crux of the worldwide carbapenemase-producing 40 Enterobacteriaceae (CPE) threat, disseminated rapidly around the world's healthcare 41 systems despite its recent emergence (17). Its progenitor strains in clonal group (CG) 258 42 also cause outbreaks and carry many important ESBL and carbapenemase genes (9, 19-43 21). Several other strain types such as those in CG14, CG20, and CG37, also frequently 44 appear as multidrug resistant and in outbreak situations (7, 10, 12, 15). 45Host colonization is likely an important reservoir driving transmission of these 46 strains. In the healthcare environment, intestinal colonization of K. pneumoniae is risk 47 for detecting CPE or ESBL producers, which have several limitations including turn-60 around time, narrow application, lack of sensitivity and specificity, subjectivity, and 61 extensive labor for high-throughput screening (31, 38). Automated systems also require 62 up-front organism culture and isolation, with many of the same limitations (31). PCR-63 based assays are rapid, but often use DNA from culture, or if used on DNA extracted 64 from specimens, may have low sensitivity. Additionally, a limited number of tests can be 65 run simultaneously, and may miss important AMR genes not previously known to 66 circulate in a given locale (31, 39). 67In this study, we describe...