Genetic and molecular determinants of Pseudomonas aeruginosa mono- and dual-species biofilm formation

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen, and it became one of the greatest threats to human health worldwide due to their adaptation ability for various environments and resistance against multiple classes of antibiotics. These characteristics of the bacterium contributed to them to become a major causative agent of healthcare-associated infections (HAIs). They are, also, known to develop robust biofilms. Biofilm is a community of microbes that inhabits on various surfaces and typically surrounded by extracellular matrices (ECM). Chapter I is a literature review that includes brief history of biofilms, background information of P. aeruginosa and its biofilm, biofilm infections and multi-species biofilms. In chapter II, the investigation was initiated to identify genes that affect the enhancement of the P. aeruginosa biofilm by the sub-minimal inhibitory concentrations (MICs) treatment of antibiotics. We screened a transposon (Tn) mutant library of PAO1, a prototype P. aeruginosa strain. Among ~5,000 mutants, a fiuA gene mutant was verified to form very defective biofilms in the presence of sub-MIC carbenicillin. The fiuA gene encodes ferrichrome receptor A, involved in the iron acquisition process. Of note, biofilm formation was not decreased in the ΔpchΔpvd mutant defective in the production of pyochelin and pyoverdine, two well-characterized P. aeruginosa siderophore molecules. Moreover, the ΔfiuA, a non-polar fiuA deletion mutant, produced a significantly decreased level of elastase, a major virulence determinant. Mouse airway infection experiments revealed that the mutant expressed significantly less pathogenicity. In chapter III, we investigate pathogenic factors of the Pseudomonas aeruginosa polymicrobial biofilm. Since the introduction of biofilm infection model in medical field, it has discovered that biofilms are responsible for majority of chronic infections. However, the treatments of the chronic biofilm infections are still very limited to surgical removal of the infected sites. Most of the chronic biofilm infections are known to be polymicrobial infections. Pseudomonas aeruginosa and Enterococcus faecalis are two of the most spotted bacterial species in biofilm infections but the study of the interactions between these bacteria was very limited. In this investigation, we observed phenotypic changes in the dual-species biofilm of P. aeruginosa and E. faecalis, such as dramatic enhancement in elasticity of the biofilm, and distinct spatial distribution of each bacterial species in the biofilm. We have found that these phenotypic characteristics were associated with exopolysaccharides (EPS), especially Pel and Psl. Together, our results suggest that fiuA gene has pleiotropic functions that affect P. aeruginosa biofilm development and virulence. The targeting of FiuA could enable the attenuation of P. aeruginosa virulence and may be suitable for the development of a drug that specifically controls the virulence of this important pathogen. Also, Psl is more associated to the bacteria-surface adhesion and the interaction between P. aeruginosa cells to form a structured biofilm, and Pel is more related to interspecies interaction in the polymicrobial biofilm. Therefore, these two EPS can be targets for polymicrobial biofilm infection eradication.

녹농균는 기회감염균으로서 다양한 환경에 대한 높은 적응력과 여러 항생제에 대한 저항성으로 인해 인류의 건강에 큰 위협으로 급부상하고 있다. 이러한 균의 특성들은 이 균이 원내감염을 일으키는 주요 원인이 되는데 큰 역할을 하였다. 또한 이 균은 바이오필름을 매우 잘 형성하는 균으로 알려져 있다. 바이오필름은 미생물들의 공동체 집단으로서 다양한 물질의 표면에 존재하며 일반적으로 세포외기질 등에 의해 둘러싸여 있다. Chapter I 은 바이오필름에 대한 간단한 역사와 녹농균과 녹농균의 바이오필름에 대한 기본 정보, 바이오필름 감염과 multi-species 바이오필름에 대한 literature review로 구성되어 있다. Chapter II에서는 녹농균 바이오필름의 형성이 최소생장억제농도 이하의 항생제 처리 시, 오히려 더 증가하는 현상의 원인이 되는 유전자를 찾기 위해 연구가 시작되었다. 녹농균의 protoptye인 PAO1균주의 transposon (Tn) mutant library의 돌연변이를 약 5,000개 정도 검사한 결과, fiuA 유전자에 transposon이 삽입된 균주에서 carbenecillin이 최소생장억제농도 이하로 처리되었을 경우, 바이오필름의 형성이 결여됨이 확인되었다. fiuA 유전자는 녹농균의 철 이온 획득에 관여하는 ferrich...