It is to be expected that other still unknown factors are required for K. pneumoniae to colonize and reside in the GI tract. An increased knowledge of such factors is an important step in the search for new strategies to prevent colonisation and subsequent infection of susceptible patients with K. pneumoniae. One approach to identify novel pathogenic virulence mechanisms is to employ screening of genomic libraries. Such libraries are constructed by digesting genomic DNA, cloning it into vectors

and transforming them into cells that can be screened for a desired phenotype [16–19]. In a previous study, we constructed a library of K. pneumoniae DNA expressed in Escherichia coli and successfully AZD8055 nmr used it Romidepsin cell line to screen for K. pneumoniae genes involved in biofilm formation in vitro[18]. The objective of this study was to identify genes involved in K. pneumoniae intestinal colonisation by screening of the K. pneumoniae genomic library in a well-established

mouse model of GI colonisation. To our knowledge, this is the first use of a genomic library as a positive-selection-based in vivo screening model. We demonstrate successful in vivo selection of clones containing GI colonisation promoting K. pneumoniae genes, thus validating this novel screening approach. Results Clones containing colonisation promoting genes are selected in the mouse GI colonisation model We initially assessed the colonisation abilities of K. pneumoniae clinical isolate C3091 and E. coli laboratory strain EPI100 in the mouse model of GI colonisation. We found that while both strains persistently colonized the intestines of the infected mice, the bacterial counts in faeces were more than 100-fold

higher for C3091 than for EPI100 (Figure 1). Thus K. pneumoniae C3091 is a superior coloniser of the intestinal tract likely via possession of genes not present in the E. coli strain and which promote enhanced colonisation ability. Figure 1 Colonisation of the intestine by K. pneumoniae C3091 and E. coli EPI100. The two Methamphetamine strains were fed individually to sets of three mice. Colonisation was quantified from plating of faeces on selective media. Symbols for day 0 represent the size of inoculum. The results are presented as the mean log (CFU/g faeces) ± sum of means (SEM). To identify GI colonisation promoting genes, a library consisting of 1,152 fosmids, each containing approximately 40 kb random K. pneumoniae C3091 DNA, expressed in E. coli EPI100 was screened in the mouse GI colonisation model. The library was arrayed in 12 pools each containing 96 fosmid clones. The 12 pools were fed individually to a set of two mice, and following 17 days of colonisation, fosmids were purified from colonies picked from platings of faecal samples and characterised. The 17-day colonisation period was chosen to ensure enough time for detectable selection of clones containing colonisation promoting genes.