Application of a novel multi-screening signature-tagged mutagenesis assay for identification of Klebsiella pneumoniae genes essential in colonization and infection.

Klebsiella pneumoniae is a common cause of urinary tract infections (UTIs) and pneumonia, especially in immunocompromised individuals. Epidemiological studies have revealed that K. pneumoniae infections are frequently preceded by gastrointestinal colonization and the gastrointestinal tract is believed to be the most important reservoir for transmission of the bacteria. To identify genes involved in the ability of K. pneumoniae to colonize the intestine and infect the urinary tract, a novel multi-screening signature-tagged mutagenesis (MS-STM) assay was implemented. In the MS-STM assay, PCR-amplified tags present in the inoculum as well as recovered pools from each infection model are simultaneously subjected to hybridization using each specific tag as a probe. Therefore, screenings of a mutant library in more than one infection model is significantly eased compared to the traditional signature-tagged mutagenesis methodology. From a total of 1,440 K. pneumoniae transposon mutants screened, 13 mutants were identified as attenuated in intestinal colonization as well as the UTI model. In addition, six mutants attenuated only in the UTI model were identified. Transposon insertion sites in attenuated mutants were, among others, in genes encoding well-known K. pneumoniae virulence factors such as lipopolysaccharide and capsule, as well as in genes of unknown function.