The biofilm formation and iron acquisition systems of Klebsiella pneumoniae

Abstract

Klebsiella pneumoniae is an opportunistic bacterial pathogen and a common cause of healthcare-associated infections. Due to the emergence of antimicrobial-resistant and hypervirulent strains, K. pneumoniae is recognised as a major public health threat. The ability of some K. pneumoniae strains to form biofilms increases this concern, particularly in healthcare settings where bacteria can colonise surfaces of indwelling devices. Biofilms can mediate more effective host colonisation and bacterial cells within biofilms are often more resistant to antimicrobial treatments.

Given the importance of biofilms in enhancing virulence and complicating treatment regimens, greater understanding of biofilm formation among Klebsiella isolates is required. This dissertation revealed that the K. pneumoniae clinical isolates AJ094, AJ097 and AJ218 exhibited different biofilm formation capabilities, and their biofilms had variable responses to DNase and proteinase treatment, suggesting qualitative differences. The early-stage biofilms formed by AJ094 and AJ218 could be destabilised by DNase, suggesting that the presence of extracellular DNA in the biofilm matrix was important for biofilm development. Dispersal of AJ218 biofilms by proteinase treatment indicated the importance of protein components such as fimbriae in maintaining the biofilm. Proteomic analysis revealed that cells were more metabolically active in the planktonic compared to the biofilm state, and differential expression of certain proteins suggested physiological variation between planktonic and biofilm cells. Proteomic experiments also showed that type 3 fimbrial proteins were expressed at higher levels in the biofilm state, particularly in the AJ218 strain, which is known to form biofilms via type 3 fimbriae.

Some proteins expressed in the biofilm state were involved in metal ion uptake and AJ094, AJ097 and AJ218 were all shown to require iron supplementation for optimal growth and biofilm formation in minimal media. Mutation of an enterobactin-mediated iron acquisition gene in AJ094 significantly reduced biofilms under iron limiting conditions compared to iron-replete conditions. On the other hand, the siderophore yersiniabactin was shown to be less important than enterobactin for supporting in vitro biofilm formation and growth.

Highly invasive Klebsiella strains often carry genes for yersiniabactin synthesis and, compared to enterobactin, this siderophore is less well-understood. A transposon mutagenesis approach was employed in this study to identify novel regulators and efflux systems for yersiniabactin. The study was successful in identifying known yersiniabactin-related factors, including genes located outside of the yersiniabactin operon that were directly or indirectly related to yersiniabactin metabolism and transport. During construction of the transposon mutant library, a spontaneous 70-kb chromosomal deletion occurred in the parent strain. The deletion mutant exhibited reduced siderophore activity when associated with a transposon insertion within a hypothetical gene (orf684). However, when the 70-kb region was present, inactivation of the orf684 gene no longer caused reduced siderophore activity.

This study has improved our understanding of biofilm composition and the requirement of iron and siderophores in the formation of K. pneumoniae biofilms. Further research into fundamental characteristics of iron, siderophores and matrix components in regard to bacterial growth and biofilm formation may lead to the development of novel drugs or preventive strategies to reduce the burden of bacterial infection.