Biofilms

Development of bacterial biofilms on artificial corals in comparison to surface-associated microbes of hard corals.

by Michael Sweet

Numerous studies have demonstrated the differences in bacterial communities associated with corals versus those in... more Numerous studies have demonstrated the differences in bacterial communities associated with corals versus those in their surrounding environment. However, these environmental samples often represent vastly different microbial micro-environments with few studies having looked at the settlement and growth of bacteria on surfaces similar to corals. As a result, it is difficult to determine which bacteria are associated specifically with coral tissue surfaces. In this study, early stages of passive settlement from the water column to artificial coral surfaces (formation of a biofilm) were assessed. Changes in bacterial diversity (16S rRNA gene), were studied on artificially created resin nubbins that were modelled from the skeleton of the reef building coral Acropora muricata. These models were dip-coated in sterile agar, mounted in situ on the reef and followed over time to monitor bacterial community succession. The bacterial community forming the biofilms remained significantly different (R = 0.864 p<0.05) from that of the water column and from the surface mucus layer (SML) of the coral at all times from 30 min to 96 h. The water column was dominated by members of the α-proteobacteria, the developed community on the biofilms dominated by γ-proteobacteria, whereas that within the SML was composed of a more diverse array of groups. Bacterial communities present within the SML do not appear to arise from passive settlement from the water column, but instead appear to have become established through a selection process. This selection process was shown to be dependent on some aspects of the physico-chemical structure of the settlement surface, since agar-coated slides showed distinct communities to coral-shaped surfaces. However, no significant differences were found between different surface coatings, including plain agar and agar enhanced with coral mucus exudates. Therefore future work should consider physico-chemical surface properties as factors governing change in microbial diversity.

Development of bacterial biofilms on artificial corals in comparison to surface-associated microbes of hard corals.

by Michael Sweet

Numerous studies have demonstrated the differences in bacterial communities associated with corals versus those in... more Numerous studies have demonstrated the differences in bacterial communities associated with corals versus those in their surrounding environment. However, these environmental samples often represent vastly different microbial micro-environments with few studies having looked at the settlement and growth of bacteria on surfaces similar to corals. As a result, it is difficult to determine which bacteria are associated specifically with coral tissue surfaces. In this study, early stages of passive settlement from the water column to artificial coral surfaces (formation of a biofilm) were assessed. Changes in bacterial diversity (16S rRNA gene), were studied on artificially created resin nubbins that were modelled from the skeleton of the reef building coral Acropora muricata. These models were dip-coated in sterile agar, mounted in situ on the reef and followed over time to monitor bacterial community succession. The bacterial community forming the biofilms remained significantly different (R = 0.864 p<0.05) from that of the water column and from the surface mucus layer (SML) of the coral at all times from 30 min to 96 h. The water column was dominated by members of the α-proteobacteria, the developed community on the biofilms dominated by γ-proteobacteria, whereas that within the SML was composed of a more diverse array of groups. Bacterial communities present within the SML do not appear to arise from passive settlement from the water column, but instead appear to have become established through a selection process. This selection process was shown to be dependent on some aspects of the physico-chemical structure of the settlement surface, since agar-coated slides showed distinct communities to coral-shaped surfaces. However, no significant differences were found between different surface coatings, including plain agar and agar enhanced with coral mucus exudates. Therefore future work should consider physico-chemical surface properties as factors governing change in microbial diversity.

Susceptibility of Enterococcus faecalis biofilm to antibiotics and calcium hydroxide

by Chai Wen Lin

The purpose of this study was to investigate the antimicrobial efficacy of six groups of antibiotics and calcium... more The purpose of this study was to investigate the antimicrobial efficacy of six groups of antibiotics and calcium hydroxide against Enterococcus faecalis biofilm in a membrane filter model. Two-day-old E. faecalis (ATCC 29212) biofilm was exposed to ampicillin, co-trimoxazole, erythr omycin, oxytetracycline, vancomycin, vancomycin followed by gentamicin, Ca(OH)2, and phosphate-buffered saline (control). After 1 h of exposure, the antimicrobial activity was neutralized by washing each disc five times in PBS, and then the colony-forming units of the remaining viable bacteria on each disc were counted. The results revealed that only erythromycin, oxytetracycline and Ca(OH)2 showed 100% biofilm kill. An ANOVA with a Bonferroni post hoc test (P < 0.05) detected significant differences among the test agents, except in the ampicillin group versus the co-trimoxazole group. It is concluded that erythromycin, oxytetracycline and Ca(OH)2 are 100% effective in eliminating E. faecalis biofilm, whereas ampicillin, co-trimoxazole, vancomycin, and vancomycin followed by gentamicin are ineffective.

Methicillin Resistance Alters the Biofilm Phenotype and Attenuates Virulence in Staphylococcus aureus Device-Associated Infections

by Justine Rudkin

Clinical isolates of Staphylococcus aureus can express biofilm phenotypes promoted by the major cell wall autolysin... more Clinical isolates of Staphylococcus aureus can express biofilm phenotypes promoted by the major cell wall autolysin and the fibronectin-binding proteins or the icaADBC-encoded polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG). Biofilm production in methicillin-susceptible S. aureus (MSSA) strains is typically dependent on PIA/PNAG whereas methicillin-resistant isolates express an Atl/FnBP-mediated biofilm phenotype suggesting a relationship between susceptibility to β-lactam antibiotics and biofilm. By introducing the methicillin resistance gene mecA into the PNAG-producing laboratory strain 8325-4 we generated a heterogeneously resistant (HeR) strain, from which a homogeneous, high-level resistant (HoR) derivative was isolated following exposure to oxacillin. The HoR phenotype was associated with a R602H substitution in the DHHA1 domain of GdpP, a recently identified c-di-AMP phosphodiesterase with roles in resistance/tolerance to β-lactam antibiotics and cell envelope stress. Transcription of icaADBC and PNAG production were impaired in the 8325-4 HoR derivative, which instead produced a proteinaceous biofilm that was significantly inhibited by antibodies against the mecA-encoded penicillin binding protein 2a (PBP2a). Conversely excision of the SCCmec element in the MRSA strain BH1CC resulted in oxacillin susceptibility and reduced biofilm production, both of which were complemented by mecA alone. Transcriptional activity of the accessory gene regulator locus was also repressed in the 8325-4 HoR strain, which in turn was accompanied by reduced protease production and significantly reduced virulence in a mouse model of device infection. Thus, homogeneous methicillin resistance has the potential to affect agr- and icaADBC-mediated phenotypes, including altered biofilm expression and virulence, which together are consistent with the adaptation of healthcare-associated MRSA strains to the antibiotic-rich hospital environment in which they are frequently responsible for device-related infections in immuno-compromised patients.

The Vibrio cholerae Pst2 phosphate transport system is upregulated in biofilms and contributes to biofilm-induced hyperinfectivity

by Ben Mudrak

Co-authored with Rita Tamayo

Vibrio cholerae is the causative agent of the deadly diarrheal disease cholera. As part of its life cycle, V. cholerae... more Vibrio cholerae is the causative agent of the deadly diarrheal disease cholera. As part of its life cycle, V. cholerae persists in marine environments, where it forms surface-attached communities commonly described as biofilms. Evidence indicates that these biofilms constitute the infectious form of the pathogen during outbreaks. Previous work has shown that biofilm-derived V. cholerae cells, even when fully dispersed from the biofilm matrix, are vastly more infectious than planktonic (free-living) cells. Here, we sought to identify factors that contribute to biofilm-induced hyperinfectivity in V. cholerae, and we present evidence for one aspect of the molecular basis of this phenotype. We identified proteins upregulated during growth in biofilms and determined their contribution to the hyperinfectivity phenotype. We found that PstS2, the periplasmic component of the Pst2 phosphate uptake system, was enriched in biofilms. Another gene in the pst2 locus was transcriptionally upregulated in biofilms. Using the infant mouse model, we found that mutation of two pst2 components results in impaired colonization. Importantly, deletion of the Pst2 inner membrane complex caused a greater colonization defect after growth in a biofilm as compared to shaking culture. Based on these data, we propose that V. cholerae cells in biofilms upregulate the Pst2 system and therefore gain an advantage upon entry into the host. Further characterization of factors contributing to biofilm-induced hyperinfectivity in V. cholerae will improve our understanding of the transmission of the bacteria from natural aquatic habitats to the human host.

In vitro efficacy of bismuth thiols against biofilms formed by bacteria isolated from human chronic wounds

by James Folsom

Folsom, J.P., B. Baker, and P. S. Stewart. 2011. In vitro efficacy of bismuth thiols against biofilms formed by bacteria isolated from human chronic wounds. Journal of Applied Microbiology 111:989-996.

Aims:  The purpose of this study was to evaluate the antimicrobial efficacy of thirteen bismuth thiol preparations for... more Aims:  The purpose of this study was to evaluate the antimicrobial efficacy of thirteen bismuth thiol preparations for bactericidal activity against established biofilms formed by two bacteria isolated from human chronic wounds.

Methods:  Single species biofilms of a Pseudomonas aeruginosa or a methicillin-resistant Staphylococcus aureus were grown in either colony biofilm or drip-flow reactors systems. Biofilms were challenged with bismuth thiols, antibiotics or silver sulfadiazine, and log reductions were determined by plating for colony formation.

Conclusions:  Antibiotics were ineffective or inconsistent against biofilms of both bacterial species tested. None of the antibiotics tested were able to achieve >2 log reductions in both biofilm models. The 13 different bismuth thiols tested in this investigation achieved widely varying degrees of killing, even against the same micro-organism in the same biofilm model. For each micro-organism, the best bismuth thiol easily outperformed the best conventional antibiotic. Against P. aeruginosa biofilms, bismuth-2,3-dimercaptopropanol (BisBAL) at 40–80 μg ml−1 achieved >7·7 mean log reduction for the two biofilm models. Against MRSA biofilms, bismuth-1,3-propanedithiol/bismuth-2-mercaptopyridine N-oxide (BisBDT/PYR) achieved a 4·9 log reduction.

Significance and Impact of the Study:  Bismuth thiols are effective antimicrobial agents against biofilms formed by wound bacteria and merit further development as topical antiseptics for the suppression of biofilms in chronic wounds.

Investigations of Nitrogen Removal Pathways in a Biological Packed Bed Reactor Using Elementary Mass Balances

by Antonio Albuquerque

A. Albuquerque, J. Makinia, K. Pagilla
WEF Nutrient Removal Conference 2009, Washington, USA, 28 Jun-1 Jul 2009; 117-135; 2009

Nitrogen (N) cycle involves a complex set of potential biochemical pathways with reactions catalyzed by different... more Nitrogen (N) cycle involves a complex set of potential biochemical pathways with reactions catalyzed by different microorganisms. Elementary mass balances for COD, DO, NH4-N and alkalinity were conducted and stoichiometric relationships were investigated to explain possible pathways of the nitrogen removal mechanisms in a lab-scale submerged down flow biological packed bed (BPB) reactor. Four sets of experiments were performed by modifying the organic loading and C/N ratio in comparison with steady-state conditions. Approximately 90% of COD and NH4-N removal occurred in two upper sections occupying 1/5 of the reactor height. The elementary
mass balances could not explain all the experimental results with respect to nitrogen removal and oxygen consumption by known mechanisms. The mass balance calculations, excluding the possibility of nitrification, were in general in accordance with the observations indicating no or minimal NO3-N production. The theoretical stoichiometric requirements for nitrification reaction
were satisfied in 6 experiments and in another 8 experiments nitrification may have occurred, but stoichiometry was not satisfied. Using C/N ratio as the variable, only at C/N ratio = 10, the predictions confirmed the possibility of nitrification in the same 3 (out of 4) assays as observed in practice. The results of this study reveal that the nitrogen transformations occurring in the studied reactor are complex and cannot be explained by simple mechanisms of microbial assimilation and
nitrification.

Holdfast formation in motile swarmer cells optimizes surface attachment during Caulobacter crescentus development

by Assaf Levi

Genetic dissection of" Caulobacter crescentus" surface colonization

by Assaf Levi

Spontaneous Gac Mutants of Pseudomonas Biological Control Strains: Cheaters or Mutualists?

by William Driscoll

Bacteria rely on a range of extracellular metabolites to suppress competitors, gain access to resources, and exploit... more Bacteria rely on a range of extracellular metabolites to suppress competitors, gain access to resources, and exploit plant or animal hosts. The GacS/GacA two-component regulatory system positively controls the expression of many of these beneficial external products in pseudomonad bacteria. Natural populations often contain variants with defective Gac systems that do not produce most external products. These mutants benefit from a decreased metabolic load but do not appear to displace the wild type in nature. How could natural selection maintain the wild type in the presence of a mutant with enhanced growth? One hypothesis is that Gac mutants are “cheaters” that do not contribute to the public good, favored within groups but selected against between groups, as groups containing more mutants lose access to ecologically important external products. An alternative hypothesis is that Gac mutants have a mutualistic interaction with the wild type, so that each variant benefits by the presence of the other. In the biocontrol bacterium Pseudomonas chlororaphis strain 30-84, Gac mutants do not produce phenazines, which suppress competitor growth and are critical for biofilm formation. Here, we test the predictions of these alternative hypotheses by quantifying interactions between the wild type and the phenazine- and biofilm-deficient Gac mutant within growing biofilms. We find evidence that the wild type and Gac mutants interact mutualistically in the biofilm context, whereas a phenazine-defective structural mutant does not. Our results suggest that the persistence of alternative Gac phenotypes may be due to the stabilizing role of local mutualistic interactions.

Nanomaterials Can Influence Living Biological Systems with Nanometer Sensitivity

by Giuseppe Vecchio

S. Sabella, V. Brunetti, G. Maiorano, L. Rizzello, B. Sorce, G. Vecchio, A. Galeone, R. Cingolani, P.P. Pompa

Nanotech 2011, Vol 3, Chapter 3, Bio Nano Materials, Pages 167-170
ISBN: 978-1-4398-7138-6

Conference Technical Proceedings, TechConnectWorldConference and Expo 2011, June 13-16, Boston (MA)

We show the different response of human neuroblastoma cells line to gold surfaces with different levels of... more We show the different response of human neuroblastoma cells line to gold surfaces with different levels of nanoroughness, finding out that neurons are capable to sense and actively respond to these nanotopography features, with a surprising sensitivity to variations of few nanometers. Focal adhesion complexes, that allows cellular sensing, cannot properly assemble onto nanostructured surfaces, leading to a marked decrease in cell adhesion. Moreover, apoptosis/necrosis assays established that nanoscale features induce cell death by necrosis, with a trend directly related to roughness values. Finally, by seeding SH-SY5Y cells onto micropatterned flat and nanorough gold surfaces, we demonstrated the possibility to realize substrates with cytophilic or cytophobic behavior, simply by fine tuning their surface topography at nanometer scale, inducing a clear self-alignment of neurons. These nanostructured substrates were also investigated to explore their use as novel materials which can prevent bacterial colonization.

Cholate-Stimulated Biofilm Formation by Lactococcus lactis Cells

by arsalan zaidi

On the paradigm of altruistic suicide in the unicellular world

by William Driscoll

Theory for the evolution of diffusible external goods

by William Driscoll

Identifying new variables during infection: proximity to the host epithelium and epigenetic programs alter the expression of virulence factors in Vibrio cholerae

by Ben Mudrak

An opinion piece highlighting recent research by the Schoolnik lab, co-authored with Rita Tamayo

Formation of Biofilm at Different Nutrient Levels by Various Genotypes of Listeria monocytogenes

by James Folsom

Strains of Listeria monocytogenes differ in their ability to form biofilms. The objectives of this study were to... more Strains of Listeria monocytogenes differ in their ability to form biofilms. The objectives of this study were to determine whether genetically related strains have similar biofilm-forming capacities and what effect nutrient concentration has on the ability of different strains to produce biofilms. Biofilms of 30 strains of L. monocytogenes, obtained from a variety of sources were grown on stainless steel in tryptic soy broth (TSB) or in a 1:10 dilution of TSB (DTSB) for 24 h at 32°C. The amount of biofilm formed was determined with image analysis after cells were stained with bisBenzimide H 33258 (Hoechst 33258). The strains were genetically subtyped by repetitive element sequence–based PCR (rep-PCR) with the primer set rep-PRODt and rep-PROG5. Data were analyzed with an analysis of variance and Duncan's multiple range test. Eleven strains produced the same amount of biofilm in both media. Fourteen strains produced more biofilm in TSB than in DTSB. Five strains produced more biofilm in DTSB than in TSB. Serotype 4b strains produced more biofilm in TSB than did serotype 1/2a strains, whereas serotype 1/2a strains produced more biofilm in DTSB than did serotype 4b strains. Growth in DTSB resulted in decreased biofilm accumulation for serotype 4b strains. There was no correlation between genetic subtype and the amount of biofilm accumulation. These results indicate that strains of serotype 1/2a and serotype 4b differ in the regulation of their biofilm phenotype. The poor biofilm accumulation of serotype 4b isolates when grown in DTSB could be a factor in the predominance of serogroup 1/2 strains in food processing plants, where nutrients may be limited.

Chlorine Resistance of Listeria monocytogenes Biofilms and Relationship to Subtype, Cell Density, and Planktonic Cell Chlorine Resistance

by James Folsom

Strains of Listeria monocytogenes vary in their ability to produce biofilms. This research determined if cell density,... more Strains of Listeria monocytogenes vary in their ability to produce biofilms. This research determined if cell density, planktonic chlorine resistance, or subtype are associated with the resistance of L. monocytogenes biofilms to chlorine. Thirteen strains of L. monocytogenes were selected for this research based on biofilm accumulation on stainless steel and rep-PCR subtyping. These strains were challenged with chlorine to determine the resistance of individual strains of L. monocytogenes. Planktonic cells were exposed to 20 to 80 ppm sodium hypochlorite in 20 ppm increments for 5 min in triplicate per replication, and the experiment was replicated three times. The number of tubes with surviving L. monocytogenes was recorded for each isolate at each level of chlorine. Biofilms of each strain were grown on stainless steel coupons. The biofilms were exposed 60 ppm of sodium hypochlorite. When in planktonic culture, four strains were able to survive exposure to 40 ppm of chlorine, whereas four strains were able to survive 80 ppm of chlorine in at least one of three tubes. The remaining five strains survived exposure to 60 ppm of chlorine. Biofilms of 11 strains survived exposure to 60 ppm of chlorine. No association of biofilm chlorine resistance and planktonic chlorine resistance was observed; however, biofilm chorine resistance was similar for strains of the same subtype. Biofilm cell density was not associated with chlorine resistance. In addition, biofilms that survived chlorine treatment exhibited different biofilm morphologies. These data suggest that chlorine resistance mechanisms of planktonic cells and biofilms differ, with planktonic chlorine resistance being more affected by inducible traits, and biofilm chlorine resistance being more affected by traits not determined in this study.

Proteomic Analysis of a Hypochlorous Acid-Tolerant Listeria monocytogenes Cultural Variant Exhibiting Enhanced Biofilm Production

by James Folsom

Following exposure of Listeria monocytogenes Scott A (SA) to hypochlorous acid, rough colony variants were identified... more Following exposure of Listeria monocytogenes Scott A (SA) to hypochlorous acid, rough colony variants were identified that were tolerant of hypochlorous acid and produced increased amounts of biofilm. A derivative of one of these variants was smooth, produced even more biofilm, and exhibited greater biofilm chlorine resistance. The objective of this research was to compare the protein expression of a cultural variant to SA and to identify proteins that might be associated with biofilm production and chlorine tolerance. Suspension chlorine tolerance for several cultural variants (SAR, SAR5, and SBS) was determined by exposure to 60 to 120 ppm of hypochlorous acid for 5 min. Hypochlorous acid tolerance of biofilms was determined after growing biofilms on stainless steel and then exposing them to 200 ppm of hypochlorous acid for 5 min. All cultural variants were able to survive 120 ppm of hypochlorous acid in suspension. There was little difference in the hypochlorous acid tolerance of the cultural variant planktonic cells. The cultural variants produced greater amounts of biofilm than the common form of L. monocytogenes and were more tolerant of hypochlorous acid. The SBS variant was selected for proteomic comparison because it was the variant that produced the most biofilm and was the most tolerant of hypochlorous acid when grown as a biofilm. Protein expression of planktonic and biofilm cells of SBS was compared to SA by two-dimensional difference gel electrophoresis. The 50s ribosomal protein, L10, was down-regulated in biofilm SBS. Other proteins down-regulated in planktonic SBS were the peroxide resistance protein (Dpr) and a sugar-binding protein (LMO0181). This sugar-binding protein was also up-regulated in biofilm SBS. One protein spot down-regulated in planktonic SBS contained both 50s ribosomal protein L7/L12 and an unknown protein (LMO1888).

Physiology of Pseudomonas aeruginosa in biofilms as revealed by transcriptome analysis

by James Folsom

Co-authored with Lee Richards, Betsey Pitts, Frank Roe, Garth D Ehrlich, Albert Parker, Aurélien Mazurie, Philip S Stewart

Background: Transcriptome analysis was applied to characterize the physiological activities of Pseudomonas aeruginosa... more Background: Transcriptome analysis was applied to characterize the physiological activities of Pseudomonas aeruginosa grown for three days in drip-flow biofilm reactors. Conventional applications of transcriptional profiling often compare two paired data sets that differ in a single experimentally controlled variable. In contrast this study obtained the transcriptome of a single biofilm state, ranked transcript signals to make the priorities of the population manifest, and compared rankings for a priori identified physiological marker genes between the biofilm and published data sets.
Results: Biofilms tolerated exposure to antibiotics, harbored steep oxygen concentration gradients, and exhibited stratified and heterogeneous spatial patterns of protein synthetic activity. Transcriptional profiling was performed and the signal intensity of each transcript was ranked to gain insight into the physiological state of the biofilm population. Similar rankings were obtained from data sets published in the GEO database http://www.ncbi.nlm.nih.gov/geo. By comparing the rank of genes selected as markers for particular physiological activities between the biofilm and comparator data sets, it was possible to infer qualitative features of the physiological state of the biofilm bacteria. These biofilms appeared, from their transcriptome, to be glucose nourished, iron replete, oxygen limited, and growing slowly or exhibiting stationary phase character. Genes associated with elaboration of type IV pili were strongly expressed in the biofilm. The biofilm population did not indicate oxidative stress, homoserine lactone mediated quorum sensing, or activation of efflux pumps. Using correlations with transcript ranks, the average specific growth rate of biofilm cells was estimated to be 0.08 h-1.
Conclusions: Collectively these data underscore the oxygen-limited, slow-growing nature of the biofilm population and are consistent with antimicrobial tolerance due to low metabolic activity

Identification of Carbohydrate Metabolism Genes in the Metagenome of a Marine Biofilm Community Shown to Be Dominated by Gammaproteobacteria and Bacteroidetes

by James McDonald

Edwards J.L., Smith D.L., Connolly J., McDonald J.E., Cox M.J., Joint I., Edwards C., McCarthy A.J. Identification of Carbohydrate Metabolism Genes in the Metagenome of a Marine Biofilm Community Shown to Be Dominated by Gammaproteobacteria and Bacteroidetes. Genes. 2010; 1(3):371-384.

Polysaccharides are an important source of organic carbon in the marine environment and degradation of the insoluble... more Polysaccharides are an important source of organic carbon in the marine environment and degradation of the insoluble and globally abundant cellulose is a major component of the marine carbon cycle. Although a number of species of cultured bacteria are known to degrade crystalline cellulose, little is known of the polysaccharide hydrolases expressed by cellulose-degrading microbial communities, particularly in the marine environment. Next generation 454 Pyrosequencing was applied to analyze the microbial community that colonizes and degrades insoluble polysaccharides in situ in the Irish Sea. The bioinformatics tool MG-RAST was used to examine the randomly sampled data for taxonomic markers and functional genes, and showed that the community was dominated by members of the Gammaproteobacteria and Bacteroidetes. Furthermore, the identification of 211 gene sequences matched to a custom-made database comprising the members of nine glycoside hydrolase families revealed an extensive repertoire of functional genes predicted to be involved in cellulose utilization. This demonstrates that the use of an in situ cellulose baiting method yielded a marine microbial metagenome considerably enriched in functional genes involved in polysaccharide degradation. The research reported here is the first designed to specifically address the bacterial communities that colonize and degrade cellulose in the marine environment and to evaluate the glycoside hydrolase (cellulase and chitinase) gene repertoire of that community, in the absence of the biases associated with PCR-based molecular techniques.