Growing and analyzing static biofilms
TL;DR: In this article, the early stages of biofilm formation are examined using static biofilm assays, which are suitable for either small or relatively large-scale studies and can be used individually or in combination for the study of biofilms.
Abstract: Many bacteria can exist as surface-attached aggregations known as biofilms. Presented in this unit are several approaches for the study of these communities. The focus here is on static biofilm systems, which are particularly useful for examination of the early stages of biofilm formation, including initial adherence to the surface and microcolony formation. Furthermore, most of the techniques presented are easily adapted to the study of biofilms under a variety of conditions and are suitable for either small- or relatively large-scale studies. Unlike assays involving continuous-flow systems, the static biofilm assays described here require very little specialized equipment and are relatively simple to execute. In addition, these static biofilm systems allow analysis of biofilm formation with a variety of readouts, including microscopy of live cells, macroscopic visualization of stained bacteria, and viability counts. Used individually or in combination, these assays provide useful means for the study of biofilms.
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TL;DR: In this paper , a review article summarizes the main features of biofilms, with particular focus on parameters affecting biofilm composition and mechanical properties and a thorough overview of the in vitro biofilm models recently developed is presented, focusing on both traditional and advanced approaches.
Abstract: Bacterial infections are a growing concern to the health care systems. Bacteria in the human body are often found embedded in a dense 3D structure, the biofilm, which makes their eradication even more challenging. Indeed, bacteria in biofilm are protected from external hazards and are more prone to develop antibiotic resistance. Moreover, biofilms are highly heterogeneous, with properties dependent on the bacteria species, the anatomic localization, and the nutrient/flow conditions. Therefore, antibiotic screening and testing would strongly benefit from reliable in vitro models of bacterial biofilms. This review article summarizes the main features of biofilms, with particular focus on parameters affecting biofilm composition and mechanical properties. Moreover, a thorough overview of the in vitro biofilm models recently developed is presented, focusing on both traditional and advanced approaches. Static, dynamic, and microcosm models are described, and their main features, advantages, and disadvantages are compared and discussed.
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TL;DR: Isothermal calorimetry can be useful in this context by allowing measurements of the metabolic activity of biofilm grown and maintained on solid medium and working with mycobacterial biofilms is notoriously difficult.
Abstract: Measuring metabolic activity and response of biofilm to different conditions or compounds is of general interest but is also expected to help in developing new antibiofilm compounds and potentially new treatments. Current culture-based and microscopic methods although of much use have several drawbacks. Isothermal calorimetry can be useful in this context by allowing measurements of the metabolic activity of biofilm grown and maintained on solid medium. Biofilms prepared on membranes were placed in calorimetry vials containing solid medium. Sealed vials were introduced in an isothermal calorimeter, and the rate of metabolic heat production was monitored over time. We chose mycobacteria as an example for this paper as working with mycobacterial biofilms is notoriously difficult.
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29 Aug 2014
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01 Jan 2015
TL;DR: The laboratory has identified a novel gene product, morphogenesis protein C (MorC), deletion of which leads to multiple pleotropic effects pertaining to membrane structure and function, and suggests a role of MorC as an accessory or a scaffold protein involved in secretion.
Abstract: The cell envelope of Aggregatibacter actinomycetemcomitans, a Gram-negative pathogenic bacterium implicated in human oral and systemic disease, plays a critical role in maintenance of cellular homeostasis, resistance to external stress, and host–pathogen interactions Our laboratory has identified a novel gene product, morphogenesis protein C (MorC), deletion of which leads to multiple pleotropic effects pertaining to membrane structure and function The MorC sequence was determined to be conserved in Gammaproteobacteria Based on this bioinformatic analysis, the functional conservation of this protein was investigated utilizing an A actinomycetemcomitans morC mutant as a model system to express homologs from four phylogenetically diverse representatives of the Gammaproteobacteria: Haemophilus influenzae, Escherichia coli, Pseudomonas aeruginosa, and Moraxella catarrhalis MorC from all organisms restored at least one of the A actinomycetemcomitans mutant phenotypes, implying that the protein is functionally conserved across Gammaproteobacteria Further, deletion mutagenesis indicated that the last 10 amino acids of the carboxyl terminus were necessary to maintain the integrity of the membrane The observed pleiotropic effects suggested alterations in the membrane protein composition of the morC mutant Stable isotope dimethyl labeling in conjunction with mass spectrometry was employed to quantitatively determine the differences in the abundance of membrane proteins of the isogenic mutant and wild-type strains A total of 665 envelope associated proteins were identified and functionally annotated using bioinformatic tools All proteins, except MorC, were detected in the mutant strain However, 12 proteins were found in lesser (10) or greater (2) abundance in the membrane preparation of the mutant strain These proteins were ascribed functions associated with protein quality control, oxidative stress response, and protein secretion systems One protein found to be reduced was a component of the fimbrial secretion system of A actinomycetemcomitans The significance of this finding was unclear due to the afimbriated nature of the laboratory strain used in the study Therefore, the defect in fimbriation was identified and complemented in trans The transformed strain displayed all of the hallmarks of a naturally fimbriated strain including: a distinct star-like colony morphology; robust biofilm formation; and presence of fimbriae as detected by electron microscopy The isogenic morC mutant strain transformed with an identical plasmid did not display any fimbriated phenotypes The role of MorC in fimbriae production of a naturally fimbriated strain was investigated by inactivation of morC in a clinical isolate The mutant strain displayed phenotypes typically associated with inactivation of morC However, fimbriae were still observed on the surface, although in lesser amounts on some individual bacteria, and this strain formed a biofilm with volume similar to the parent Interestingly, significant changes in microcolony architecture of the biofilm were observed by confocal microscopy MorC plays a critical role in maintaining secretion of major virulence determinants of A actinomycetemcomitans Specific changes in the protein composition of the cell envelope indicate a direct or compensatory role of these proteins in maintaining membrane physiology The functional conservation of MorC also implies an important role for this protein in other Gram-negative bacteria This work suggests a role of MorC as an accessory or a scaffold protein involved in secretion ii CITATIONS Material from Chapter 2 of this dissertation has been submitted for publication to Molecular Oral Microbiology in June 2015 in the following form: Smith, KP, Voogt, RD, Ruiz, T, and Mintz, KP The conserved carboxyl domain of MorC, an inner membrane protein of Aggregatibacter actinomycetemcomitans, is essential for membrane function Material from Chapter 3 of this dissertation has been published in the following form: Smith, K P, Fields, J G, Voogt, R D, Deng, B, Lam, Y, and Mintz, KP (2014) The cell envelope proteome of Aggregatibacter actinomycetemcomitans Molecular Oral Microbiology DOI: 101111/omi12074 Material from Chapter 4 of this dissertation has been accepted for publication in the following form: Smith, K P, Fields, J G, Voogt, R D, Deng, B, Lam, Y, and Mintz, KP (2015) Alteration in abundance of specific membrane proteins of Aggregatibacter actinomycetemcomitans is attributed to deletion of the inner membrane protein MorC Proteomics In press Material from Chapter 5 of this dissertation has been submitted for publication to Journal of Bacteriology in the following form: Smith, KP, Ruiz, T, and Mintz, KP The Inner Membrane Protein, MorC, is involved in fimbriae production and biofilm formation in Aggregatibacter actinomycetemcomitans
1 citations
TL;DR: The results obtained indicated that the extract and fractions of T. gigas had significant detachment mode of actions for antifouling activities, which may provide new insight into the defence mechanism of the horseshoe crabs against biofilm formation.
Abstract: Horseshoe crabs have survived for a long period due to their myriad defence mechanism. Thorough studies were conducted on their adaptive defence mechanisms but few highlighted their primary innate ...
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TL;DR: The optical densities of stained bacterial films adherent to plastic tissue culture plates serve as a quantitative model for the study of the adherence of coagulase-negative staphylococci to medical devices, a process which may be important in the pathogenesis of foreign body infections.
Abstract: The adherence of coagulase-negative staphylococci to smooth surfaces was assayed by measuring the optical densities of stained bacterial films adherent to the floors of plastic tissue culture plates. The optical densities correlated with the weight of the adherent bacterial film (r = 0.906; P less than 0.01). The measurements also agreed with visual assessments of bacterial adherence to culture tubes, microtiter plates, and tissue culture plates. Selected clinical strains were passed through a mouse model for foreign body infections and a rat model for catheter-induced endocarditis. The adherence measurements of animal passed strains remained the same as those of the laboratory-maintained parent strain. Spectrophotometric classification of coagulase-negative staphylococci into nonadherent and adherent categories according to these measurements had a sensitivity, specificity, and accuracy of 90.6, 80.8, and 88.4%, respectively. We examined a previously described collection of 127 strains of coagulase-negative staphylococci isolated from an outbreak of intravascular catheter-associated sepsis; strains associated with sepsis were more adherent than blood culture contaminants and cutaneous strains (P less than 0.001). We also examined a collection of 84 strains isolated from pediatric patients with cerebrospinal fluid (CSF) shunts; once again, pathogenic strains were more adherent than were CSF contaminants (P less than 0.01). Finally, we measured the adherence of seven endocarditis strains. As opposed to strains associated with intravascular catheters and CSF shunts, endocarditis strains were less adherent than were saprophytic strains of coagulase-negative staphylococci. The optical densities of bacterial films adherent to plastic tissue culture plates serve as a quantitative model for the study of the adherence of coagulase-negative staphylococci to medical devices, a process which may be important in the pathogenesis of foreign body infections. Images
1,980 citations
"Growing and analyzing static biofil..." refers methods in this paper
...While popularized in the mid-to-late 1990s (Mack et al., 1994; O’Toole et al., 1999), the assay in its typically used form is derived from a protocol published by Christensen et al. (1985)....
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TL;DR: Results show that oxygen limitation and low metabolic activity in the interior of the biofilm, not poor antibiotic penetration, are correlated with antibiotic tolerance of this P. aeruginosa biofilm system.
Abstract: The roles of slow antibiotic penetration, oxygen limitation, and low metabolic activity in the tolerance of Pseudomonas aeruginosa in biofilms to killing by antibiotics were investigated in vitro. Tobramycin and ciprofloxacin penetrated biofilms but failed to effectively kill the bacteria. Bacteria in colony biofilms survived prolonged exposure to either 10 μg of tobramycin ml−1or 1.0 μg of ciprofloxacin ml−1. After 100 h of antibiotic treatment, during which the colony biofilms were transferred to fresh antibiotic-containing plates every 24 h, the log reduction in viable cell numbers was only 0.49 ± 0.18 for tobramycin and 1.42 ± 0.03 for ciprofloxacin. Antibiotic permeation through colony biofilms, indicated by a diffusion cell bioassay, demonstrated that there was no acceleration in bacterial killing once the antibiotics penetrated the biofilms. These results suggested that limited antibiotic diffusion is not the primary protective mechanism for these biofilms. Transmission electron microscopic observations of antibiotic-affected cells showed lysed, vacuolated, and elongated cells exclusively near the air interface in antibiotic-treated biofilms, suggesting a role for oxygen limitation in protecting biofilm bacteria from antibiotics. To test this hypothesis, a microelectrode analysis was performed. The results demonstrated that oxygen penetrated 50 to 90 μm into the biofilm from the air interface. This oxic zone correlated to the region of the biofilm where an inducible green fluorescent protein was expressed, indicating that this was the active zone of bacterial metabolic activity. These results show that oxygen limitation and low metabolic activity in the interior of the biofilm, not poor antibiotic penetration, are correlated with antibiotic tolerance of this P. aeruginosa biofilm system.
918 citations
TL;DR: The results suggest that some other resistance mechanism is involved for both agents and contributed to wild-type biofilm resistance to ampicillin but not to ciprofloxacin.
Abstract: The penetration of two antibiotics, ampicillin and ciprofloxacin, through biofilms developed in an in vitro model system was investigated. The susceptibilities of biofilms and corresponding freely suspended bacteria to killing by the antibiotics were also measured. Biofilms of Klebsiella pneumoniae were developed on microporous membranes resting on agar nutrient medium. The susceptibilities of planktonic cultures and biofilms to 10 times the MIC were determined. Antibiotic penetration through biofilms was measured by assaying the concentration of antibiotic that diffused through the biofilm to an overlying filter disk. Parallel experiments were performed with a mutant K. pneumoniae strain in which beta-lactamase activity was eliminated. For wild-type K. pneumoniae grown in suspension culture, ampicillin and ciprofloxacin MICs were 500 and 0.18 microgram/ml, respectively. The log reductions in the number of CFU of planktonic wild-type bacteria after 4 h of treatment at 10 times the MIC were 4.43 +/- 0.33 and 4.14 +/- 0.33 for ampicillin and ciprofloxacin, respectively. Biofilms of the same strain were much less susceptible, yielding log reductions in the number of CFU of -0.06 +/- 0.06 and 1.02 +/- 0.04 for ampicillin and ciprofloxacin, respectively, for the same treatment. The number of CFU in the biofilms after 24 h of antibiotic exposure was not statistically different from the number after 4 h of treatment. Ampicillin did not penetrate wild-type K. pneumoniae biofilms, whereas ciprofloxacin and a nonreactive tracer (chloride ion) penetrated the biofilms quickly. The concentration of ciprofloxacin reached the MIC throughout the biofilm within 20 min. Ampicillin penetrated biofilms formed by a beta-lactamase-deficient mutant. However, the biofilms formed by this mutant were resistant to ampicillin treatment, exhibiting a 0.18 +/- 0.07 log reduction in the number of CFU after 4 h of exposure and a 1.64 +/- 0.33 log reduction in the number of CFU after 24 h of exposure. Poor penetration contributed to wild-type biofilm resistance to ampicillin but not to ciprofloxacin. The increased resistance of the wild-type strain to ciprofloxacin and the mutant strain to ampicillin and ciprofloxacin could not be accounted for by antibiotic inactivation or slow diffusion since these antibiotics fully penetrated the biofilms. These results suggest that some other resistance mechanism is involved for both agents.
885 citations
"Growing and analyzing static biofil..." refers methods in this paper
...Colony biofilms Colony biofilms (see Basic Protocol 3) have typically been used for the purpose of determining antibiotic resistance (Anderl et al., 2000; Walters et al., 2003)....
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TL;DR: This article operationally defines a biofilm as bacteria that are attached to a surface in sufficient numbers to be detected macroscopically.
Abstract: Interest in the study of microbial biofilms has increased greatly in recent
years due in large part to the profound impact biofilms have in clinical,
industrial, and natural settings. Traditionally, the study of biofilms has
been approached from an ecological or engineering perspective, using a
combination of classical microbiology and advanced microscopy. We and
others have begun to use genetic approaches to understand the development
of these complex communities. To begin we must answer the question:
What is a biofilm? This definition, by necessity, may be quite broad because
it is clear that many organisms can attach to a variety of surfaces under
diverse environmental conditions. Therefore, in the context of this article
we will operationally define a biofilm as bacteria that are attached to a
surface in sufficient numbers to be detected macroscopically.
820 citations
TL;DR: The results demonstrate that the mutants were impaired in the accumulative phase of biofilm production by S. epidermidis by mediating intercellular adhesion.
Abstract: The primary attachment to polymer surfaces followed by accumulation in multilayered cell clusters leads to production of Staphylococcus epidermidis biofilms, which are thought to contribute to virulence in biomaterial-related infections. We isolated Tn917 transposon mutants of biofilm-producing S. epidermidis 13-1, which were completely biofilm negative. In pulsed-field gel electrophoresis no obvious deletions of the mutants were noted. The Tn917 insertions of mutants M10 and M11 were located on different EcoRI fragments but on identical 60-kb SmaI and 17-kb BamHI chromosomal fragments. Linkage of transposon insertions of mutants M10 and M11 with the altered phenotype was demonstrated by phage transduction, whereas the several other mutants apparently represented spontaneous variants. In a primary attachment assay with polystyrene spheres, no significant difference between any of the mutants and the wild type could be detected. Cell clustering as an indication of intercellular adhesion, which is a prerequisite for accumulation in multilayered cell clusters, was not detected with any mutant. These results demonstrate that the mutants were impaired in the accumulative phase of biofilm production. Mutants M10 and M11 did not produce detectable amounts of a specific polysaccharide antigen (D. Mack, N. Siemssen, and R. Laufs, Infect. Immun. 60:2048-2057, 1992), whereas substantially reduced amounts of antigen were produced by the spontaneous variants. Hexosamine was determined as the major specific component of the antigen enriched by gel filtration of biofilm-producing S. epidermidis 1457 because almost no hexosamine was detected in material prepared from the isogenic biofilm-negative transductant 1457-M11, which differentiates the antigen from other S. epidermidis polysaccharide components. Our results provide direct genetic evidence for a function of the antigen in the accumulative phase of biofilm production by S. epidermidis by mediating intercellular adhesion.
343 citations
"Growing and analyzing static biofil..." refers methods in this paper
...While popularized in the mid-to-late 1990s (Mack et al., 1994; O’Toole et al., 1999), the assay in its typically used form is derived from a protocol published by Christensen et al. (1985)....
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