Next generation sequencing: The key to understand Klebsiella pneumoniae biofilms?

Background: The incidence of healthcare-associated infections (HAI) is determined by underlying disease conditions and exposure to high risk medical interventions. In Portugal since 1980s K. pneumoniae is a recognized etiological agent of epidemic and endemic infections in healthcare units. An increasing rate of K. pneumoniae strains resistant either to extended cephalosporins or carbapenems has been observed and one of the mechanisms responsible for the emergence of drug resistance could be the biofilm assembly. The capacity of K. pneumoniae to form biofilm was first described in the 1980s for abiotic surfaces and ten years later on biotic surfaces. The antibiotic failure to penetrate through the biofilm layers, the emergence of mutations which might be easily transferred horizontally, and quorum sensing have been pointed as responsible for the increased antibiotic resistance of bacteria within biofilms. The main objective was to study the biofilm structure and the kinetic assembly associated to antibiotic resistance profile in K. pneumoniae strains capsulate or not, and identify the genes involved in biofilm assembly on full genome sequencing of studied strains. Material and Methods: Twoo K. pneumoniae isolates collected in 1980 (Kp45, Kp703) and one (Kp2948) in 2011 were studied. Kp703 was encapsulated and the remaining had capsular type K:2. The bacterial ability to assemble biofilms on cell culture plates was evaluated. For SEM analysis, biofilms were allowed to form on six wells cell culture plates (Nunc) for 12h at 37ºC. DNA was extracted using QiAamp DNA mini kit following the manufactures instructions. Full genome sequence was performed using next-generation sequencing platform MiSeq (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s instructions. The RAST platform was used for annotation and MAUVE platform for multiple alignments. Results: The three isolates were able to assemble biofilms although following different kinetics. K. pneumoniae strains Kp703 and Kp45 followed similar kinetics with identical biomass increase nevertheless these bacteria differ in capsule expression. Full-sequencing and annotation of genomes of isolates was performed in order to explain the differences found in biofilm assembly. Preliminary data already revealed that the K. pneumoniae strains displaying enhanced biofilm-forming ability is genetically different from the others, and, in particular, present some specific features enrolling genomic regions believed to be biofilm-related in other bacteria (an intact prophage, genes coding for a filamentous haemoagglutinin, a haemolysin expression modulating protein and an YdeA protein). Conclusion: K. pneumoniae lacking capsule, regarded as less virulent, have a better performance as biofilm assembler and exhibited the highest increase in antibiotic resistance when organized within biofilms. The analysis of the full genome sequence will allow reachingon K.pneumoniaebiofilms and provide novel opportunities to exploit the overall fitness ofK. pneumoniaeunder antibiotic stress