published_or_final_version / Endodontics / Master / Master of Dental Surgery

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

This study was conducted to investigate the biofilm formation by using advanced microscopic and high-throughput sequencing techniques. The major tasks were (1) to quantitatively evaluate the initial bacterial attachment processes by Atomic Force Microscopy (AFM); (2) to characterize the chemical variation during biofilm formation by Raman microscopy; (3) to analyze the microbial structure and functions in the wastewater and drinking water biofilms by metagenomic analysis. To determine the lateral detachment force for bacteria, a quantitative method using contact mode of AFM was developed. The established method had good repeatability and sensitivity to various bacteria and substrata, and was applied to evaluate the roles of bacterial surface polymers in Phase I and II attachment, i.e. lipopolysaccharides, type 1 fimbria and capsular colanic acid. The results indicated lipopolysaccharides largely enhanced Phases I and II attachment. Fimbriae increased Phase I attachment but not significantly influence the adhesion strength in Phase II. Moreover, colanic acid had negative effect on attachment in both of Phases I and II. Surface-enhanced Raman scattering was applied to evaluate the chemical components in the biofilm matrix at different growth phases, including initial attached bacteria, colonies and mature biofilm. Three model bacteria, including Escherichia coli, Pseudomonas putida, and Bacillus subtilis, were used to cultivate biofilms. The results showed that the content of carbohydrates, proteins, and nucleic acids in biofilm matrix increased significantly along with the biofilm growth of three bacteria judging from the intensities and appearance probabilities of related marker peaks in the spectra. The content of lipids, however, only increased in the Gram-negative biofilms. Moreover, metagenomic data, coupled with PCR-based 454 pyrosequencing reads, were generated for activated sludge and biofilm from a full-scale hybrid reactor to study the microbial taxonomic and functional differences/connections between activated sludge and biofilm. The results showed that the dominant bacteria co-existed in two samples. Global functions in activated sludge and biofilm metagenomes showed quite similar pattern, revealing the limited differences of overall functions existed in two samples. For nitrogen removal, the diversity and abundance of nitrifiers and denitrifiers in biofilm did not surpass that in activated sludge. Whilst, higher abundances of nitrification and denitrification genes were indeed found in biofilm, suggesting the increased nitrogen removal by applying biofilm might be attributed to removal efficiency rather than biomass accumulation of nitrogen removal bacteria. To investigate the bacterial structure and functions of drinking water biofilm, PCR-based 454 pyrosequencing of 16S rRNA gene and Illumina metagenomic data were generated and analyzed. Significant differences of bacterial diversity and taxonomic structure were found between biofilms formed on stainless steel and plastics. Moreover, ecological succession could be obviously observed during biofilm formation. The metabolic network analysis for drinking water biofilm constructed for the first time. Moreover, the occurrence and abundance of specific genes involving in the bacterial pathway of glutathione metabolism and production/degradation of extracellular polymeric substances were also evaluated. / published_or_final_version / Civil Engineering / Doctoral / Doctor of Philosophy

Pseudomonas chlororaphis (aureofaciens) 30-84 is a biocontrol bacterium effective against take-all disease of wheat. Phenazine (PZ) production by strain 30-84 is the primary mechanism responsible for pathogen inhibition and the rhizosphere persistence of 30-84. The PhzR/PhzI system of strain 30-84 directly regulates PZ production and mutations in this QS system are defective in biofilm formation. Genetic complementation or direct addition of AHL signal restored biofilm formation to a phzI mutant. Mutations in PZ biosynthesis were equally defective in biofilm formation. Addition of PZ or genetic complementation of the PZ biosynthetic mutation restored biofilm formation. QS and PZ production also were involved in the establishment of populations on wheat seeds and plant roots. Presence of 10% wild type strain 30-84 in mixtures with QS or PZ mutants restored root colonization. These data demonstrate that QS and specifically PZ production are essential for biofilm formation by strain 30-84. This is a new role for PZs in the rhizosphere community.Strain 30-84 produces primarily phenazine-1-carboxylic acid (PCA) and 2-hydroxy-PCA (2-OH-PCA). We generated derivatives of strain 30-84 that produced the same total amount of PZs as the wild type but produced only PCA, or more efficiently converted PCA to 2-OH-PCA. These derivatives with altered PZ ratios differed from the wild type in initial attachment, biofilm architecture, and dispersal. Increased 2-OH-PCA production increased initial attachment, although both alterations resulted in thicker biofilms and reduced dispersal rates. Loss of 2-OH-PCA production resulted in a significant reduction in pathogen inhibition. My findings indicate that alterations in the endogenous ratios of PZs have wide-ranging effects on the biology of strain 30-84. I initiated studies to understand the mechanisms by which PZs affect surface attachment and biofilm development. Addition of PZs to metabolically inactivated cells improved adhesion compared to the inactive cells alone, suggesting that PZs may improve initial binding to surfaces. Results from whole genome transcription profiles of wild type strain 30-84 to a PZ mutant indicate that genes potentially involved in biofilm formation were up-regulated in the presence of PZs. These results provide initial evidence that PZs may modulate cell adhesion and biofilm formation via multiple mechanisms.

Quorum sensing is a cell communication mechanism used to coordinate group behaviour based on population density. A mathematical model of quorum sensing in bacterial biofilms is developed, consisting of a nonlinear diffusion reaction system describing the effects of a growing biofilm on bacterial quorum sensing behaviour. In numerical experiments, the influence of the hydrodynamic environment and nutrient conditions on biofilm growth and quorum sensing behaviour are studied, and flow-facilitated inter-colony communication and spatiotemporal quorum sensing induction patterns are observed. The model is extended to include an impact of quorum sensing on biofilm growth, through the explicit description of EPS, the protective biomass layer surrounding bacterial biofilm cells. The circumstances under which quorum sensing-regulated EPS production is a beneficial strategy for cells are identified. Biofilm colonies that use this strategy have lower cell populations than non-quorum sensing colonies, but may secure nutrients in a space-limited environment and outcompete neighbouring colonies.

This thesis is an investigation of Pseudomonas aeruginosa bacterial biofilms. The objective of the first study was to evaluate the biofilm-forming capacity of canine otitis isolates of P. aeruginosa and to compare the minimum inhibitory concentrations (MICs) of antimicrobials for planktonic versus biofilm-embedded bacteria. Biofilm forming ability was assessed using a microtitre plate assay. Broth microdilution was used to assess the MICs of neomycin, polymyxin B, enrofloxacin and gentamicin for the planktonic and biofilm-embedded bacteria of eighty-three isolates. Thirty-three (40%) isolates were biofilm producers and MICs for biofilm-embedded bacteria were significantly higher than their planktonic counterparts for all antimicrobials (all P<0.05). The objective of the second study was to evaluate the impact of Tromethamine edetate disodium dihydrate (Triz-EDTA®) in combination with antimicrobials on antimicrobial susceptibility of P. aeruginosa biofilm-embedded bacteria. MICs of the four antimicrobials for the biofilm embedded bacteria and biofilm-embedded bacteria with added Triz-EDTA® were assessed with broth microdilution for thirty-one biofilm-producing isolates. Addition of Triz-EDTA® significantly reduced MICs for neomycin (P < 0.008) and gentamicin (P < 0.04) but not enrofloxacin (P = 0.7), or polymyxin B (P = 0.5). The objective of the third study was to determine the presence of biofilm-associated genes in biofilm forming and non-biofilm forming isolates. Four genes involved with carbohydrate matrix production (pelA), irreversible attachment (sadB) and quorum sensing (lasB, rhlA) were selected. DNA was extracted and polymerase chain reaction (PCR) was performed for all isolates. All isolates possessed lasB and sadB, 74 (90%) possessed pelA and 74 (90%) possessed rhlA. All thirty-two (100%) isolates that were classified as biofilm producers contained all genes. There was an association between the presence of pelA and rhlA and biofilm production (P < 0.017) and between the presence of rhlA and pelA and the quantity of biofilm produced (both P < 0.001). These results highlight that biofilm formation of Pseudomonas aeruginosa otic isolates does occur and can impact antimicrobial therapy. Certain compounds can also influence antimicrobial susceptibility of biofilm-embedded bacteria. Genetics may also play a role in biofilm formation.

No description available.

Biofilm communities were cultivated in rotating annular bioreactors using water from the South Saskatchewan River. The impacts of seasonal variations, nutrients, pollutants and dissolved oxygen on the activity and composition of the biofilms were assessed by using a combination of microcosm assays and molecular biology techniques. / The seasonal pattern in nitrification, denitrification and hexadecane mineralization, and in the occurrence of nirK in the South Saskatchewan River biofilms was: fall greater than winter, which was equivalent to spring. Hexadecane mineralization was higher in fall 1999 than in fall 2001, denitrification was similar in these two years, and no seasonal pattern of nitrification was observed. / The addition of combined nutrients (C, N, and P) resulted in significant increases in the measured bacterial activities and in the predominance of alkB, nirS and nirK in all seasons and years. The addition of individual nutrients did not stimulate hexadecane mineralization, denitrification, and the PCR amplification of nirS and nirK. In fall 1999, CNP and, to a lesser extent P, stimulated nitrification, whereas in fall 2001, no pattern was observed. The results showed that nutrients, especially P, were limiting for bacterial activities, and that the biofilm activities and composition varied with nutrient availability and time of year. / At the concentration assessed (1 ppb), hexadecane partially inhibited denitrification to similar extents in both years, had a negative impact on nitrification and hexadecane mineralization in fall 1999, and a positive impact on these two latter activities in fall 2001. Nickel (0.5 mg liter-1 ) negatively affected denitrification but had no effect on hexadecane mineralization. The alkB and nirS genes were less predominant and absent, respectively, in biofilms grown in the presence of nickel. DGGE analyses indicated that nickel reduced the biofilm bacterial diversity. / The results presented herein provide much needed information on the microbial ecology of river biofilms, and on the impact and interactive effects of pollutant and nutrient inputs on these biofilms. These results and the techniques used in this project can be applied to monitor environmental effects of anthropogenic activities on aquatic biofilms, and can contribute to establish or revise environmental regulations.

Aeromonas spp. isolates, which are fish and opportunistic human pathogens, form biofilms, however the factors involved in and affecting biofilm formation have not been fully elucidated. Biofilm formation is affected by motility, cell surface characteristics, and/or metabolism, thus it is important to identify factors potentially contributing to initial attachment and/or biofilm formation and their correlation with biofilm formation by Aeromonas spp. isolates. With knowledge of the stages of biofilm formation, mechanisms involved in biofilm formation and its physiology, various strategies may be applied to control aeromonad biofilms. Factors potentially involved in initial attachment and/or biofilm formation were investigated for 99 Aeromonas isolates obtained from seawater and cultured fish. Aeromonad biofilm formation was assessed using microtiter plate assays under varying physicochemical conditions. The disk diffusion method was used to determine the antimicrobial susceptibility profiles of isolates, for comparison to clinical and aquaculture isolates reported in other studies. The MICs and MBICs for antimicrobial agents (azithromycin, ceftazidime, ciprofloxacin, gentamicin and tetracycline) of planktonic cells and biofilm cells, respectively, were investigated using the broth microdilution and modified microtiter plate assays. The effect of sub-MIC (0.5 × MIC) and supra-MIC (2 × MIC) exposures on biofilm-forming cells was also determined using microtiter plate assays. The presence of efflux pump-mediated resistance in 45 Aeromonas spp. isolates was determined using the disk diffusion assay incorporating efflux pump inhibitors (EPIs) [carbonyl cyanide 3-chlorophenylhydrazone (CCCP), phenylalanine arginine β-naphthylamide (PAβN) and 1-(1-naphthylmethyl)-piperazine (NMP)]. Modified microtite plate assays were used to determine the effect of EPIs [CCCP, PAβN, and NMP], matrix-degrading DNase I and quorum-sensing inhibitors (QSIs; vanillin, 2(5H)-furanone, S-adenosylhomocysteine and cinnamaldehyde) on initial attachment and mature biofilm. Majority of isolates were motile by swimming and swarming and displayed caseinase, gelatinase, and DNase activities, as well as an A-layer phenotype. Majority of isolates displayed high levels of autoaggregation and were hydrophilic. Isolates showed varying levels of adherence, but majority were strongly adherent in nutrient-rich media at 30 ºC. Motility appeared to be a significant characteristic for biofilm formation. Majority of Aeromonas isolates spp. showed high levels of resistance to β-lactams, trimethoprim and sulphamethoxazole, and were susceptible to augmentin, piperacillin-tazobactam, aztreonam, 2nd and 3rd generation cephalosporins, carbapenems, macrolides, fluoroquinolones and aminoglycosides . High levels of resistance towards ceftazidime (MIC > 32 μg/ml) were observed for isolates, while levels of resistance towards remaining antimicrobial agents tested (tetracycline, azithromycin, ciprofloxacin, and gentamicin) were ≤ 32 μg/ml. There was a ≥16-fold increase in MBICs (4096 μg/ml) compared to the MICs for all the antimicrobial agents. The sub-MIC, MIC, and supra-MIC exposures of all antimicrobial agents had an inhibitory effect on both initial attachment and pre-formed biofilms by Aeromonas spp. isolates. Majority of isolates were more susceptible to tetracycline, norfloxacin, and azithromycin due to CCCP and NMP inhibition of the efflux pumps eliminating these antimicrobial agents. Susceptibility to erythromycin was observed for 51% and 47% of isolates, respectively, due to NMP and PAβN inhibition of the efflux pump/s eliminating erythromycin. In the microtiter plate assays, CCCP, NMP and PABN exposures resulted in significant reduction of biofilm formation by majority of Aeromonas spp. isolates in both initial attachment and mature biofilm assays, with CCCP being more effective. DNase I was more effective in reducing mature biofilm, causing reduction for 60% of isolates, compared to its effect on initial attachment. QSIs were also more effective in reducing mature biofilm compared to inhibiting initial attachment. Although increased biofilm dispersal was observed with all QSIs, vanillin and 2(5H)-furanone were more effective compared to S-adenosylhomocysteine and cinnamaldehyde. Based on data obtained in this study, antimicrobial agents, EPIs and QSIs can be used as potential biofilm-inhibiting compounds in aquaculture to control aeromonad infections and may not only prevent disease outbreaks but they could also increase the effectiveness of existing therapeutic agents. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2012.

No description available.