Signals and genes driving mixed community biofilm formation

• Main Author: Muhammad Zulfadhly Mohammad Muzaki
• Other Authors: Staffan Kjelleberg
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2022
• Subjects: Science::Biological sciences::Microbiology::Microbial ecology; Science::Biological sciences::Microbiology::Bacteria; Science::Biological sciences::Molecular biology; Science::Biological sciences::Genetics
• Online Access: https://hdl.handle.net/10356/161578
• Institution: Nanyang Technological University

Most studies on the signals and mechanisms of biofilm formation to date have focused on single-species biofilms. However, biofilms in natural environments such as dental plaques, cystic fibrosis lungs and streams often exist as mixed-species biofilm communities. Due to the adverse impacts of biofilms in chronic infections and the potential industrial applications of biofilms such for bioremediation, it is important to understand the interspecies interactions and signals that shape these mixed-species biofilm communities. Here, an experimental mixed-species biofilm community consisting of Pseudomonas aeruginosa PAO1, Pseudomonas protegens Pf5 and Klebsiella pneumoniae KP-1/MGH78578 was studied to determine how the signals and interspecies interactions within the community affect the structure, spatial organization, composition and antimicrobial resistance of the biofilm community. The P. aeruginosa acylated homoserine lactone (AHL) based quorum sensing (QS) was found to modulate the composition of the mixed-species biofilm community. The loss of P. aeruginosa QS was associated with a significant decrease in the proportion of K. pneumoniae and a concomitant increase in P. protegens Pf5. Inactivation of QS genes did not significantly change growth rates or biofilm formation, suggesting that the QS regulated functions were responsible for the observed changes in composition. Continuous-culture dual-species biofilm experiments revealed that the effect of QS on P. aeruginosa and K. pneumoniae was neutral, while QS modulates competition between P. aeruginosa and P. protegens, with P. aeruginosa QS mutants being less competitive against P. protegens than the wild-type. Dual-species biofilm experiments showed that P. aeruginosa QS effector mutants (e.g., hcnB, rhlA, pvdR) were similarly less competitive compared to its wild-type when cultivated with P. protegens. Moreover, QS-regulated sdsA1 secreted by P. aeruginosa was observed to play a role in conferring community-level resistance of mixed-species biofilm community to SDS. To further extend the relevance of the observations above to in vivo conditions, static mixed-species biofilms were grown in Artificial Sputum Medium (ASM), which replicates the nutritional environment within the CF lung. It was observed that in dual-species biofilms grown in ASM, but not in M9CasGlucose medium, the loss of QS in P. aeruginosa resulted in an increase in coaggregation between P. aeruginosa and with K. pneumoniae KP-1. This effect was not observed when a clinical strain of K. pneumoniae, MGH78578, was substituted for KP-1, suggesting that the QS-regulated coaggregation between P. aeruginosa and K. pneumoniae was strain and medium-dependent. Furthermore, the type 6 secretion (T6SS) gene, tssM1, was observed to be important for monospecies biofilm formation in M9CasGlucose, but not in complex media such as LB and ASM. However, when the K. pneumoniae tssM1 mutant was grown in continuous-culture mixed-species biofilms with P. aeruginosa and P. protegens, the K. pneumoniae ∆tssM1 was not deficient in biofilm formation. In addition to an increase in the proportion of K. pneumoniae after deletion of tssM1, there was also a decrease in the biovolume of P. aeruginosa. It was hypothesized that tit-for-tat duelling between P. aeruginosa and K. pneumoniae was a potential factor in the composition of the mixed-species biofilm. The roles of K. pneumoniae MGH78578 autoinducer-2 (AI-2) transporter proteins, LsrB/LsrD, and the biofilm regulator protein BssR in modulating monospecies biofilm formation and interspecies interactions were also investigated. The lsrB and lsrD genes did not significantly affect monospecies biofilm formation, while inactivation of the bssR gene increased monospecies biofilm formation, suggesting that bssR represses biofilm formation. The bssR gene also controlled motility, surface attachment and the formation of non-surface attached floating aggregates, which could account for the increased monospecies biofilm formation in the ∆bssR mutant. Furthermore, the loss of lsrB, lsrD and bssR resulted in a significant change in composition of the three species biofilm community, which was likely due to the combined effect of individual pairwise interspecies interactions of the K. pneumoniae mutant with both P. aeruginosa and P. protegens. In conclusion, the findings in this thesis expands the understanding of how various genes and signals affect the interspecies interactions within a biofilm consortium, resulting in changes in composition and spatial organization within the mixed-species community. This knowledge is crucial in understanding the complex dynamics within mixed-species communities and could result in new approaches to combat harmful polymicrobial biofilms and unlock the potential of polymicrobial biofilms in various beneficial applications.