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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01 Jan 2015
TL;DR: This book presents a meta-analysis of the determinants of infectious disease in eight operation theatres and some of the methods used to treat these diseases have been described.
Abstract: .......................................................................................................................................... ii Acknowledgements ........................................................................................................................ iii List of Figures ................................................................................................................................. v List of Tables ................................................................................................................................ vii
3 citations
Cites background from "Growing and analyzing static biofil..."
...A high absorbance reading corresponded to a high amount of biofilm and, low absorbance readings indicated scarce biofilm formation (Merrit et al., 2011)....
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08 Oct 2021
TL;DR: In this paper, the authors characterized biofilms formed by several Prevotella species and further assessed biofilm inhibition and detachment of preformed biofilm, and found that proteinase was more effective in biofilm detachment than DNase I.
Abstract: Background: Periodontitis, a chronic inflammatory oral infection is the outcome of disturbances in the homeostasis of the oral biofilm microbiota. A number of studies have found the occurrence of Prevotella species in elevated levels in periodontitis compared to healthy subjects. Even though different aspects of Prevotella as part of oral biofilm have been studied, in vitro biofilms formed by these species have not been characterized systematically. The objective of this study was to characterize biofilms formed by several Prevotella species and further to assess biofilm inhibition and detachment of preformed biofilms. Methods: Biofilms were grown in 24-well plates containing brucella broth in anaerobic conditions for 3 days, and were quantified using crystal violet staining. Images of SYTO 9 Green fluorescent stained biofilms were captured using confocal microscopy. Biofilm inhibition and detachment by proteinase and DNase I was tested. The biochemical characterization included quantification of proteins and DNA in the biofilms and biofilm-supernatants. Results: P. loescheii, P. oralis and P. nigrescens showed highest biofilm formation. P. nigrescens formed significantly higher amounts of biofilms than P. loescheii (P=0.005) and P. oralis (P=0.0013). Inhibition of biofilm formation was significant only in the case of P. oralis when treated with proteinase (P=0.037), whereas with DNase I treatment, the inhibition was not significant (P=0.531). Overall, proteinase was more effective in biofilm detachment than DNase I. Protein and DNA content were higher in biofilm than the supernatant with the highest amounts found in P. nigrescens biofilm and supernatants. P. oralis biofilms appeared to secrete large amounts of proteins extracellularly into the biofilm-supernatants. Conclusion: Significant differences among Prevotella species to form biofilms may imply their variable abilities to get integrated into oral biofilm communities. Of the species that were able to grow as biofilms, DNase I and proteinase inhibited the biofilm growth or were able to cause biofilm detachment.
3 citations
Dissertation•
11 Sep 2018
TL;DR: This dissertation describes the development of two mass spectrometric strategies for the detection of metabolites and proteins from a biofilm-forming Pseudomonas aeruginosa chronic wound clinical isolate using laser-based ion sources.
Abstract: Bacterial biofilms are structurally and physiologically heterogenous communities of bacterial cells embedded in a self-produced extracellular polymeric substance. Current studies aim to understand the dynamics and responses of biofilms to various environmental factors especially since they play a pathogenic role in chronic wounds preventing wound healing. One approach to studying biofilms is through mass spectrometry (MS), a versatile analytical tool that provides qualitative and quantitative analyses and has been utilized for various biological applications such as biofilms.
This dissertation describes the development of two mass spectrometric strategies for the detection of metabolites and proteins from a biofilm-forming Pseudomonas aeruginosa chronic wound clinical isolate. Two laser-based ion sources were implemented: atmospheric pressure matrix assisted laser desorption ionization MS and laser diode thermal desorption atmospheric pressure photoionization MS. The latter was demonstrated for the analysis of metabolites within intact P. aeruginosa biofilms. A bottom-up proteomic workflow for planktonic and biofilm cultures was also developed based upon liquid chromatography tandem mass spectrometry. This proteomic protocol was then used to compare the effect of lactate nutrient supplementation on cellular metabolism in both P. aeruginosa planktonic and biofilm cultures.
3 citations
Cites methods from "Growing and analyzing static biofil..."
...The most basic method for static biofilm growth is on nutrient agar plates [18], where the cell culture is either inoculated directly on nutrient agar plates or inoculated onto a polycarbonate membrane which is placed above the nutrient agar....
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TL;DR: T6SS‐containing K. pneumoniae was common in people who had BSIs and the system may play a major role in bacterial competition, and T6SS-positive isolates did not show stronger biofilm-forming activity or a higher survival rate in the presence of normal human serum in comparison to T6 SS-negative isolates.
Abstract: Background Klebsiella pneumoniae (K. pneumoniae) causes bloodstream infection (BSI), which is responsible for a high rate of morbidity and mortality among different populations. In mainland China, data on the correlation and features of the type VI secretion system (T6SS) gene cluster in K. pneumoniae is currently scarce. As a result, we conducted a prospective investigation to determine the involvement of the T6SS in K. pneumoniae pathogenicity and antibiotic resistance. Methods In this prospective analysis, we enrolled 119 individuals who had been diagnosed with K. pneumoniae bloodstream infection between July 2019 and January 2021 and acquired demographic and clinical data from their medical records. The virulence genes rmpA, rmpA2, aerobactin, iroB, hcp, vgrG, and icmF were tested for K1 and K2, antimicrobial susceptibility. Five T6SS-positive and five T6SS-negative isolates were chosen for the competition, serum resistance, and biofilm formation experiments to further gain insights regarding the microbiological properties of T6SS-positive K. pneumoniae isolates. Results Among 119 isolates obtained from patients with BSIs, 20 (16.8%) were T6SS positive K. pneumoniae. T6SS positive strains had four virulence genes and a greater K1 capsular serotypes rate than T6SS negative bacteria. Among hvKP isolates, the T6SS positive rate was substantially greater than the T6SS negative rate (P = 0.001). T6SS-positive K. pneumoniae strains had a lower rate of antimicrobial resistance in comparison to T6SS-negative bacteria. T6SS-positive isolates may be more competitive with Escherichia coli than T6SS-negative isolates. T6SS-positive isolates, on the other hand, did not show stronger biofilm-forming activity or a higher survival rate in the presence of normal human serum in comparison to T6SS-negative isolates. Conclusion T6SS-positive K. pneumoniae was common in people who had BSIs. In T6SS‐containing K. pneumoniae, the system may play a major role in bacterial competition.
3 citations
TL;DR: It is proposed that these herbal combinations could serve as a multifaceted approach to combat the pathogen and also, in turn, reduce antimicrobial resistance development.
3 citations
References
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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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