Study On The Mobile Genome Of Multidrug-resistant Klebsiella Pneumoniae

The bacterial species Klebsiella pneumoniae is an increasingly important opportunistic pathogen of man. It is responsible for nosocomial and community-acquired infections of the urinary tract, upper respiratory tract, blood stream and other anatomical sites. Dramatic increases in the levels of multiple-antibiotic resistance associated with this species, particularly to agents of last-resort such as carbapenems, pose a major emerging global problem. Further accentuating the problem is the fact that Klebsiella serve as reservoirs for the spread of antibiotic resistance genes to many bacterial species, thus amplifying the hazard of antibiotic-recalcitrant infections both within and outside hospitals. It is now well established that the bacterial mobile genome(mobilome) not only serves as a substrate for an evolutionary game of accelerated gene shuffling but that many members directly promote this process of dynamic lateral gene flow. Across the full breadth of the bacterial domain, a vast repertoire of antibiotic resistance, virulenceassociated and other traits have spread via horizontal gene transfer of various mobile genetic elements(MGEs) including plasmids, prophages, transposons, integrons and associated gene cassettes, integrative conjugative elements(ICEs) and genomic islands(GIs).We herein studied a mobilome thoroughly using the completely sequenced K. pneumoniae HS11286, an epidemic ST11 carbapenemase-producing clinical isolate obtained from the sputum of a patient in Shanghai, China in 2011 as a model organism. The mobilome of HS11286 includes six plasmids, 49 insertion sequences(ISs), nine transposons, one integron, two ICEs and seven prophages. Many of the ISs, all of the transposons and the integron are on the plasmids, comprising complex variable regions, where complicated genetic events have occurred. Analyses indicated that IS26 might be a highly active element that has driven the evolution significantly. Over 20 important drug-resistant genes were identified, among which 17 are plasmid-borne, including the most concerning bla KPC-2. Analyses revealed that Tn3, Tn1721, Tn5393, ΔIn2, ISCR2 and ISCR3 contribute significantly to the acquisition of resistant genes, while the rest of the ISs might involve in the evolution of the genetic contexts. The gene bla KPC-2 and a 26-kb region containing 13 resistant genes were serially deleted using λ red recombination and Flp-FRT recombination. Susceptibility testing against 33 antibiotics of 19 class systemically elucidated the anti-drug mechanism of the strain. Furthermore, a newly identified element ICEKpn HS11286-1 comprises a Yersinia high-pathogenicity island(HPI) in the chromosome of HS11286 was studied. A novel ICE-deletion strategy via utilization of sac B counterselection was developed. An ICE-deleted mutant was obtained. But unexpected deletion was also observed. Possible deletion scenarios were proposed based on context analysis and one of them was confirmed, showing that sitespecific recombination occurred. The sequences of the recombination sites are identical to those of the ICE, suggesting the recombination might be mediated by the ICE integrase. Though this novel genetic event awaits investigation, we hypothesize that ICE’s integrase can contribute to the recombination of the chromosome backbone in a sitespecial manner.ICEs, abundant in both Gram-positive and Gram-negative bacteria, are defined as selftransmissible integrative elements that encode functional conjugation machineries. These multi-talented entities can promote their own mobilization and thus contribute to horizontal transfer of virulence determinants, antibiotic-resistance genes and many other bacterial traits. However, the knowing of this recently categorized elements only comprises “the tip of the iceberg”. A systemic study is wanted to elucidate ICE features and to subsequently raise an identification methodology. We organized the information of 428 ICEs in 333 strains from experimental and bioinformatics analyses data and literatures as a Postgre SQL-based online database called ICEberg(http://dbmml.sjtu.edu.cn/ICEberg/). Among the collected elements, 28 families were defined based on integrase homology and synteny of ICE backbone features. ICEs typically consist of three core genetic modules:(i) integration and excision module,(ii) conjugation module and(iii) regulation module, and have been showed to possess conserved backbones and accessory regions in several well studied families. Proteins of ICEs that have been classified in ICEberg were further analyzed. For 15 ICE families with the number of member ≥5, pairwise comparison of proteins encoded by different members was carried out by blastp. Proteins were classified based on sequence similarity. Statistics of the appearance of each feature in each ICE family were used to interpret the conservation and diversity of ICEs. Results showed that the members share conserved backbone but also accommodate various accessory genes. The accessory genes account for about half of the features on average, while the conserved genes comprise ICE backbones. ICE-specific structural patterns mapping to recombination and conjugation modules were then examined by analyzing conserved components with selected profile hidden Markov models(profile HMMs), which consequently offered a conserved landscape of ICE recombination and conjugation modules. A set of relevant profile HMMs were collected. Based on the above discoveries, we were able to propose a mathematical model for ICE prediction based on the conserved pattern of recombination and conjugation. Combined with direct repeat features, a patterns-based hits co-location pipeline was established and successfully identified ICEs when tested with several control organisms. The tool however needs future refinement to be more intelligent and accurate on boundary identification.In conclusion, we annotated the mobile genome of a sequenced microorganism and found its link with antibiotic resistance, virulence and evolution. Then we managed to interpret the conserved features of ICEs and develop an ICE prediction strategy accordingly. Collectively, our results showed the mobilome plays a key role in promoting evolution and in the acquisition and spreading of antibiotic resistance and pathogenesis-related attributes. The research presented here can allow us gain an insight into the bacterial mobilome.