bioRxiv preprint doi: https://doi.org/10.1101/761924; this version posted September 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Preterm infants harbour diverse Klebsiella populations, including atypical species that encode 2 and produce an array of antimicrobial resistance- and virulence-associated factors 3 4 Yuhao Chen1*, Thomas C. Brook2*, Cho Zin Soe3, Ian O’Neill3, Cristina Alcon-Giner3, Onnicha 5 Leelastwattanagul4, Sarah Phillips3, Shabhonam Caim3, Paul Clarke5,6, Lindsay J. Hall3†, Lesley 6 Hoyles1,7† 7 8 1Department of Surgery & Cancer, Faculty of Medicine, Imperial College London, London, United 9 Kingdom 10 2Department of Biomedical Sciences, Faculty of Science and Technology, University of Westminster, 11 London, United Kingdom 12 3Gut Microbes & Health, Quadram Institute Bioscience, Norwich Research Park, Norwich, United 13 Kingdom 14 4Bioinformatics & Systems Biology Program, School of Bioresources and Technology, King 15 Mongkut's University of Technology Thonburi (Bang Khun Thian Campus), Bangkok, Thailand 16 5Neonatal Intensive Care Unit, Norfolk and Norwich University Hospitals NHS Foundation Trust, 17 Norwich, United Kingdom 18 6Norwich Medical School, University of East Anglia, Norwich, United Kingdom 19 7Department of Biosciences, School of Science and Technology, Nottingham Trent University, 20 Nottingham, United Kingdom 21 22 *These authors contributed equally to this work. 23 †Correspondence: Lindsay J. Hall, [email protected]; Lesley Hoyles, 24 [email protected] 25 Abbreviations: AMR, antimicrobial resistance; ANI, average nucleotide identity; CARD, Comprehensive Antibiotic Resistance Database; 26 CC, clonal complex; CDS, coding sequence; CFU, colony-forming unit; EUCAST, European Committee on Antimicrobial Susceptibility 27 Testing; GI, gastrointestinal; LOS, late-onset sepsis; LPE, lactose-positive Enterobacteriaceae; MAG, metagenome-assembled genome; 28 MIC, minimal inhibitory concentration; MLST, multi-locus sequence typing; NEC, necrotizing enterocolitis; NICU, neonatal intensive care 29 unit; NNUH, Norfolk and Norwich University Hospital; ORF, open reading frame; OTU, operational taxonomic unit; P:C:IA, 30 phenol:chloroform:isoamyl alcohol; SNP, single nucleotide polymorphism; ST, sequence type; UEA, University of East Anglia; VFDB, 31 Virulence Factor of Pathogenic Bacteria Database. 32 1 bioRxiv preprint doi: https://doi.org/10.1101/761924; this version posted September 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 33 ABSTRACT 34 Klebsiella spp. are frequently enriched in the gut microbiota of preterm neonates, and overgrowth is 35 associated with necrotizing enterocolitis, nosocomial infections and late-onset sepsis. Little is known 36 about the genomic and phenotypic characteristics of preterm-associated Klebsiella as previous studies 37 have focussed on recovery of antimicrobial-resistant isolates or culture-independent molecular 38 analyses. Faecal samples from a UK cohort of healthy and sick preterm neonates (n=109) were 39 screened on MacConkey agar to isolate lactose-positive Enterobacteriaceae. Whole-genome 40 sequences were generated for isolates. Approximately one-tenth of faecal samples harboured 41 Klebsiella spp. (Klebsiella pneumoniae, 7.3 %; Klebsiella quasipneumoniae, 0.9 %; Klebsiella 42 grimontii, 2.8 %; Klebsiella michiganensis, 1.8 %). Isolates recovered from NEC- and sepsis-affected 43 infants and those showing no signs of clinical infection (i.e. ‘healthy’) encoded multiple β-lactamases, 44 which may prove problematic when defining treatment regimens for NEC or sepsis, and suggest 45 ‘healthy’ preterm infants contribute to the resistome. No difference was observed between isolates 46 recovered from ‘healthy’ and sick infants with respect to in vitro siderophore production (all encoded 47 enterobactin in their genomes). All K. pneumoniae, K. quasipneumoniae, K. grimontii and K. 48 michiganensis faecal isolates tested were able to reside and persist in macrophages, indicating their 49 immune evasion abilities. Using a curated dataset of Klebsiella oxytoca, K. grimontii and K. 50 michiganensis whole-genome sequences, metapangenome analyses of published metagenomic data 51 confirmed our findings regarding the presence of K. michiganensis in the preterm gut, and highlight 52 the importance of refined analyses with curated sequence databases when studying closely related 53 species present in metagenomic data. 54 2 bioRxiv preprint doi: https://doi.org/10.1101/761924; this version posted September 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 55 INTRODUCTION 56 The gut microbiota encompasses bacteria, archaea, lower eukaryotes and viruses, with these 57 microbes contributing to host gastrointestinal (GI) and systemic health. Host–microbiome interactions 58 within the intestine are particularly important in neonates, contributing to development of the immune 59 response, establishment of the gut microbiome and protection from infections [1,2]. Term infants (i.e. 60 gestation 37 weeks) are rapidly colonised after exposure to the mother’s microbiota and the 61 environment, with streptococci and Enterobacteriaceae dominating in the initial phases [1], and 62 Bifidobacterium spp. becoming prominent as the infant grows [1]. 63 In contrast, colonization of preterm infants (i.e. <37 weeks’ gestation) occurs in neonatal 64 intensive care units (NICUs) and is shaped by the significant number of antibiotics (‘covering’ (i.e. to 65 cover possible early onset infection from birth) and treatment) these infants receive in the first days 66 and weeks post birth. The microbiota in preterm infants is enriched for bacteria such as 67 Enterobacteriaceae, Enterococcus and Staphylococcus [3,4]. 68 Critically, colonization of these at-risk infants with potentially pathogenic taxa, in concert with 69 an unstable microbiome, and immaturity of their GI tract and immune system, is thought to contribute 70 to nosocomial infections such as late-onset sepsis (LOS) or necrotizing enterocolitis (NEC) [5–10]. 71 The family Enterobacteriaceae is a large group of Gram-negative bacteria encompassing many 72 pathogens [e.g. Escherichia (Esc.) coli, Klebsiella pneumoniae, Shigella (Shi.) dysenteriae, 73 Enterobacter (Ent.) cloacae, Serratia (Ser.) marcescens and Citrobacter spp.]. While coagulase- 74 negative staphylococci are the most common cause of LOS in preterm infants, Enterobacteriaceae 75 that translocate from the preterm gut to the bloodstream also cause this condition [8,9]. In addition, 76 Enterobacteriaceae are associated with higher morbidity than the staphylococci, and blooms in 77 Proteobacteria – thought to be linked to impaired mucosal barrier integrity – have been reported 78 immediately prior to the diagnosis of LOS [8,9,11]. Predictions made from shotgun metagenomic data 79 show replication rates of all bacteria – and especially the Enterobacteriaceae and Klebsiella – are 80 significantly increased immediately prior to NEC diagnosis [12]. This altered gut microbiome 81 influences intestinal homeostasis and contributes to NEC [13], in tandem with the immature preterm 82 immune system contributing to intestinal pathology in response to blooms of Proteobacteria. 3 bioRxiv preprint doi: https://doi.org/10.1101/761924; this version posted September 16, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 83 Associations between Klebsiella-related operational taxonomic units (OTUs) and the 84 development of NEC have been noted, suggesting members of this genus contribute to the aetiology 85 of NEC in a subset of patients [14,15]. Although Sim et al. [14] found one of their two distinct groups 86 of NEC infants had an overabundance of a Klebsiella OTU, these researchers failed to identify a 87 single predominant species of Klebsiella, recovering representatives of several genera (K. 88 pneumoniae, Klebsiella oxytoca, Ent. cloacae, Ent. aerogenes, Esc. coli and Ser. marcescens) from 89 samples. Klebsiella spp. and their fimbriae-encoding genes were significantly enriched in faeces 90 collected immediately prior to the onset of NEC in a US infant cohort. These fimbriae may contribute 91 to the overexpression of TLR4 receptors observed in preterm infants [12]. Confirming the role of 92 these bacteria in NEC will require reproducing certain aspects of the disease in model systems, using 93 well-characterized bacteria recovered from preterm infants [11,14]. 94 To date, there is limited information on the genomic and phenotypic features of preterm- 95 associated Klebsiella spp. Thus, to characterise these important opportunistic pathogens, and to build 96 a collection of preterm-associated Klebsiella strains for use in future mechanistic studies relevant to 97 preterm-infant health,