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Reference EntryDOI

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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Dissertation
01 Jul 2018
TL;DR: The asymmetric design improves debris and biofilm removal especially when used with a constant taper, and was verified as an ideal system for assessing root canal treatment in vitro.
Abstract: The remaining debris and biofilm in the anatomical complexities of root canal systems can affect treatment outcomes. Files with asymmetric cross-section design may improve debris and biofilm removal from these difficult spaces during canal preparation. Tooth opacity and different densities of the remaining materials prevent the direct systematic assessment of the preparation process. The present study assessed remaining debris and biofilm using a novel transparent root canal model with novel approaches. Natural and simulated root canal samples with isthmus space were evaluated. Canal preparation by ProTaper Next and Revo-S asymmetric systems was evaluated in comparison to the standard ProTaper Universal symmetric system. The root canals were investigated by microcomputed tomography (micro-CTL confocal laser scanning microscopy (CLSML and optical coherence tomography (OCT) imaging tools. Data analysis was undertaken with SPSS (V. 24). Files with asymmetric cross-section and constant taper removed more debris and biofilm from the complex root canal system. The model allowed direct assessment of remaining materials and confirmed the novel imaging approach with the OCT. In conclusion, the asymmetric design improves debris and biofilm removal especially when used with a constant taper. The model was verified as an ideal system for assessing root canal treatment in vitro.

2 citations


Cites background from "Growing and analyzing static biofil..."

  • ...Also they can be used to study the biofilm at different developmental stage periods (Merritt et al., 2005)....

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  • ...In addition, the biofilm maturation stage can be easily achieved in comparison to the static method (Merritt et al., 2005, Lei et al., 2016)....

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Dissertation
01 Jan 2016

2 citations


Cites methods from "Growing and analyzing static biofil..."

  • ...The biofilm biomass was quantified by measuring OD590 (Ding et al. 2014a; Merritt et al. 2005)....

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Journal ArticleDOI
25 Mar 2019
TL;DR: According to the biological activity results, it can be concluded that these natural agents may be potentially used for veterinary sciences, food industry or pharmaceutical applications that aim to manage S. maltophilia biofilm.
Abstract: Aerobic, non-fermentative and Gram-negative Stenotrophomonas maltophilia is a multidrug-resistant bacilli that is known to be pathogen for human and animals. S. maltophilia has been isolated from different animal species and also found in a variety of environments including soil, water, and plants. S. maltophilia , which has the ability to form biofilms on surfaces that cause environmental problems, is resistant to many antibiotic classes such as cephalosporins, carbapenems, and aminoglycosides. Here in this study, its aimed to determine the inhibition activities of natural phenolic compounds eugenol and pulegone against S. maltophilia MU69. Antibacterial activities of eugenol and pulegone were initially determined by disc diffusion method. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were also determined by tube dilution assay. Antibiofilm activities of the compounds were investigated by crystal violet staining and also monitorized using Scanning Electron Microscopy (SEM). Zone of inhibition measurements were found to be 39 mm and 28 mm for eugenol and pulegone, respectively. MIC and MBC values were found to be 3.12 mg/mL for eugenol while these were 2.5 mg/mL and 5 mg/mL for pulegone, respectively. The highest antibiofilm activity was found to be 56.30±0.45% for MIC of eugenol. To our knowledge, this is the first report of the antibacterial and antibiofilm activity of eugenol and pulegone against S. maltophilia . According to the biological activity results, it can be concluded that these natural agents may be potentially used for veterinary sciences, food industry or pharmaceutical applications that aim to manage S. maltophilia biofilm.

2 citations

Posted ContentDOI
15 Dec 2020-bioRxiv
TL;DR: Using Pseudovibrio as a model, encoded metabolites, which are termed pseudovibriamides, promote motility and decrease biofilms and are not surfactants, but instead are linear peptides with a reversal in chain polarity.
Abstract: Collective behavior is a common feature of life. Although swarming motility and biofilms are opposed collective behaviors, both contribute to bacterial survival and host colonization. We have identified a link between motility/biofilms and a nonribosomal peptide synthetase-polyketide synthase gene cluster family (ppp) conserved in Pseudovibrio and Pseudomonas Proteobacteria known to interact with diverse eukaryotes. After developing reverse genetics for Pseudovibrio, we discovered two pseudovibriamide families, heptapeptides with a reversal in chain polarity via an ureido linkage 1-6 and related nonadepsipeptides 7-12. Imaging mass spectrometry showed that 1 was excreted whereas 7 was colony-associated. Deletion of pppA abolished production of 1-12 leading to reduced motility and increased biofilm production. pppD mutants that produced only 1-6 showed motility comparable to the wild-type and reduced biofilm formation, indicating that the excreted heptapeptides play a role in promoting motility. In contrast to lipopeptides widely known to affect swarming and biofilms, pseudovibriamides are not surfactants. Our results expand current knowledge on metabolites mediating bacterial collective behavior. Moreover, the establishment of reverse genetics will enable future exploration of the ecological and biotechnological potential of Pseudovibrio bacteria which have been proposed to contribute to marine sponge health.

2 citations

08 Dec 2017
TL;DR: This thesis aims to explore the role of natural compound collections in the discovery of anti-biofilm drug discovery and their role in the treatment of biofilm infections.
Abstract: .................................................................................................................. vii Svensk sammanfattning ........................................................................................ ix List of original publications .................................................................................. xi Contribution of the author ................................................................................... xii Abbreviations ........................................................................................................ xiii 1 Introduction ......................................................................................................... 1 2 Literature review ................................................................................................. 3 2.1 Bacterial biofilms .......................................................................................... 3 2.1.1 Biofilm lifecycle .................................................................................. 3 2.1.1.1 Attachment ............................................................................ 5 2.1.1.2 Maturation ............................................................................. 5 2.1.1.3 Dispersal ................................................................................ 6 2.1.1.4 Quorum sensing (QS) .......................................................... 6 2.1.2 Specific features of biofilms.............................................................. 8 2.1.3 Clinical impact of biofilms .............................................................. 10 2.1.4 Treatment of biofilm infections...................................................... 13 2.1.4.1 Preventative approaches ................................................... 13 2.1.4.2 Treatment using conventional antibiotics ....................... 14 2.1.4.3 Treatment using enzyme-based products ....................... 15 2.2 Antimicrobial drug discovery .................................................................. 16 2.2.1 General aspects ................................................................................ 17 2.2.1.1 Lead discovery process ...................................................... 18 2.2.1.2 Compound libraries ........................................................... 21 2.2.1.3 Statistical analysis ............................................................... 22 2.2.2 Drug discovery from natural products ......................................... 23 2.2.2.1 Natural compound collections explored in this thesis .. 24 2.2.3 Anti-biofilm drug discovery .......................................................... 26 2.2.3.1 Targets and mechanisms of anti-biofilm agents ............ 26

2 citations

References
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Journal ArticleDOI
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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Journal ArticleDOI
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

Journal ArticleDOI
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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Book ChapterDOI
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

Journal ArticleDOI
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)....

    [...]