K. pneumoniae is a leading cause of antimicrobial-resistant (AMR) healthcare-associated infections, neonatal sepsis and community-acquired liver abscess, and is associated with chronic intestinal diseases. Its diversity and complex population structure pose challenges for analysis and interpretation of K. pneumoniae genome data. Here we introduce Kleborate, a tool for analysing genomes of K. pneumoniae and its associated species complex, which consolidates interrogation of key features of proven clinical importance. Kleborate provides a framework to support genomic surveillance and epidemiology in research, clinical and public health settings. To demonstrate its utility we apply Kleborate to analyse publicly available Klebsiella genomes, including clinical isolates from a pan-European study of carbapenemase-producing Klebsiella, highlighting global trends in AMR and virulence as examples of what could be achieved by applying this genomic framework within more systematic genomic surveillance efforts. We also demonstrate the application of Kleborate to detect and type K. pneumoniae from gut metagenomes.

30Alcohol-based hand rubs are international pillars of hospital infection control, restricting 31 transmission of pathogens such as Staphylococcus aureus. Despite this success, health care 32 infections caused by Enterococcus faecium (Efm) -another multidrug resistant pathogen -are 33 increasing. We tested alcohol tolerance of 139 hospital Efm isolates, obtained between 1997 and 34 2015 and found Efm post-2010 were 10-fold more tolerant to alcohol killing than older isolates. 35 Using a mouse infection control model, we then showed that alcohol tolerant Efm resisted standard 36 70% isopropanol surface disinfection and led to gastrointestinal colonization significantly more 37 often than alcohol sensitive Efm. We next looked for bacterial genomic signatures of adaptation. 38 Tolerant Efm have independently accumulated mutations modifying genes involved in carbohydrate 39 uptake and metabolism. Mutagenesis confirmed their roles in isopropanol tolerance. These findings 40 suggest bacterial adaptation and complicate infection control recommendations. Additional policies 41 and procedures to prevent Efm spread are required. 42 [146 words] 43 44 3

type; QACs, quaternary 28 ammonium compounds; KL, K-locus; ybt, yersiniabactin; clb, colibactin; ICEKp, integrative conjugative 29 element K. pneumoniae; pLVPK, large virulence plasmid of K. pneumoniae; CPS, capsular 30 polysaccharides; MLST, multilocus sequence typing; YbSTs, yersiniabactin sequence types; CbSTs, 31 colibactin sequence types; CR-Kp, carbapenem-resistant K. pneumoniae; MIC, minimum inhibitory 32 concentration; ESBL, extended-spectrum beta-lactamase; HM, heavy metal; ML, maximum likelihood; 33 MDR, multidrug resistance; PDR, pandrug resistance; Inc, incompatibility; IS, insertion sequence; KPZM, 34 Zn2+/Mn2+metabolism module; QRDR, quinolone-resistance determining region; PMQR, plasmid-35 mediated quinolone resistance.36 2 Abstract 37 The emergence and dissemination of carbapenem-resistant hypervirulent Klebsiella 38 pneumoniae (CR-hvKp) is a worrisome public health issue compromising the treatment and 39 outcome of infections caused by this pathogen. We performed a detailed virulome and 40 resistome analysis of representative KPC-and/or CTX-M-producing K. pneumoniae 41 belonging to clonal group (CG) 258 (sequence types ST11, ST258, ST340, ST437), 42 circulating in Argentina, Brazil, Chile, Colombia and Peru; with further evaluation of the 43 virulence behavior using the Galleria mellonella infection model. Genomic analysis of K. 44 pneumoniae strains recovered from the human-animal-environment interface revealed a wide 45 resistome characterized by the presence of genes and mutations conferring resistance to 46 human and veterinary antibiotics, quaternary ammonium compounds (QACs) and heavy 47 metals. Plasmid Inc typing revealed the presence of a wide diversity of replicon types with 48 IncF, IncN, IncR and Col-like being frequently detected. Moreover, KPC-2-producing K. 49 pneumoniae belonging to ST11 (KL-64 andKL-105) and ST340 (KL-15) carried multiple 50 variants of distinct yersiniabactin siderophore (ybt) and/or genotoxic colibactin (clb) genes. In 51 this regard, ICEKp3, ICEKp4 and ICEKp12 were identified in strains belonging to ST11 and 52 ST340, recovered from Argentina, Brazil, Chile and Colombia; whereas ybt 17 and a novel 53 ybt sequence type (YbST346) were identified together with clb in ICEKp10 structures from 54 ST11 and ST258, from Brazil and Colombia, respectively. K. pneumoniae ST11 55 (ICEKp10/YbST346 and ICEKp4/ybt 10) strains killed 100% of wax moth larvae, in a similar 56 way to hypervirulent K1/ST23 strain (ybt-and clb-negative) carrying the pLVPK-like 57 plasmid, indicating enhanced virulence. In summary, our results indicate that yersiniabactin, 58 colibactin and an expanded resistome have contributed to enhanced virulence and persistence 59 of KPC-2-producing K. pneumoniae CG258 in South America. Therefore, active surveillance 60 of hospital-associated lineages of K. pneumoniae should not only focus on clonal origin and 61 antimicrobial resistance, but also on the virulence factors ybt and clb. 62 3 INTRODUCTION 63