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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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Journal ArticleDOI
TL;DR: This review will discuss biofilm screening strategies suitable for drug discovery efforts, especially chemical and biological approaches that specifically target biofilm destruction.
Abstract: Biofilms formed by pathogenic bacteria present a serious threat to human health as the efficacy of standard antibiotic therapeutic regimens is compromised by reduced microbial susceptibility within the biofilm environment. The discovery of improved therapies for biofilm elimination requires an understanding of biofilm formation and dispersal, and the development of assays to specifically analyze these dynamic processes. This review will discuss biofilm screening strategies suitable for drug discovery efforts, especially chemical and biological approaches that specifically target biofilm destruction.

22 citations


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

  • ...Several publications [17-19] have provided comprehensive reviews of analytical methods com-...

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Journal ArticleDOI
TL;DR: The antimicrobial and antibiofilm activity of T. sipyleus BOISS.
Abstract: In this study, antimicrobial and antibiofilm activities and the chemical composition of Thymus sipyleus BOISS. subsp. sipyleus BOISS. var. davisianus RONNIGER essential oil was evaluated. The essential oil was obtained by hydro-distillation and analyzed by gas chromatography-mass spectrometry. Fourteen compounds were characterized, having as major components thymol (38.31 %) and carvacrol (37.95 %). Minimum inhibitory concentrations (MICs) of oil and the major components were calculated by serial dilution method, and anti-biofilm effects by microplate biofilm assay against five Gram positive (Staphylococcus aureus MU 38, MU 40, MU 46, MU 47, Stahylococcus epidermidis MU 30) and five Gram negative (Pseudomonas aeruginosa MU 187, MU 188, MU 189, Pseudomonas fluorescens MU 180, MU 181) bacteria. It was found that MICs for essential oil, thymol and carvacrol were between 5 and 50 µl/ml, 0.125–0.5 µg/ml and 0.125–05 µl/ml, respectively. The results showed that doses of MIC produced a greater anti-biofilm influence than 0.5, 0.25 and 0.125 MIC. In the presence of essential oil (MIC), the mean biofilm formation value was equal to 67 ± 5.5 % for P. aeruginosa MU 188, and essential oil (MIC) inhibition exceeds 60 % for P. aeruginosa biofilms. The results also showed that carvacrol (MIC) was able to induce an inhibition 72.9 ± 4.1 % for S.aureus (MU 40) biofilm. In addition, thymol (MIC) showed 68.6 ± 5.3 % reduction in biofilm formation of P. fluorescens MU 181. This study demonstrated the antimicrobial and antibiofilm activity of T. sipyleus BOISS. subsp. sipyleus BOISS. var. davisianus RONNIGER essential oil and points out the exceptional efficiency of thymol and carvacrol, which could represent candidates in the treatment of Pseudomonas and Staphylococcus biofilms.

22 citations

Journal ArticleDOI
10 Feb 2017-PLOS ONE
TL;DR: A flow chamber model optimized for the assessment of dental implant materials is developed and it could be shown that biofilms of the five important oral bacteria can be reproducibly formed on the surface of titanium, a frequent implant material.
Abstract: Since the introduction of modern dental implants in the 1980s, the number of inserted implants has steadily increased. Implant systems have become more sophisticated and have enormously enhanced patients' quality of life. Although there has been tremendous development in implant materials and clinical methods, bacterial infections are still one of the major causes of implant failure. These infections involve the formation of sessile microbial communities, called biofilms. Biofilms possess unique physical and biochemical properties and are hard to treat conventionally. There is a great demand for innovative methods to functionalize surfaces antibacterially, which could be used as the basis of new implant technologies. Present, there are few test systems to evaluate bacterial growth on these surfaces under physiological flow conditions. We developed a flow chamber model optimized for the assessment of dental implant materials. As a result it could be shown that biofilms of the five important oral bacteria Streptococcus gordonii, Streptococcus oralis, Streptococcus salivarius, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans, can be reproducibly formed on the surface of titanium, a frequent implant material. This system can be run automatically in combination with an appropriate microscopic device and is a promising approach for testing the antibacterial effect of innovative dental materials.

22 citations

Journal Article
P. L. Tang, Pui C. F, W. C Wong, A. Noorlis, R. Son 
TL;DR: Since the result showed that Salmonella Typhi formed biofilm on fresh produce surfaces, hygienic practice from farm to fork including handling, processing, distribution and storage of the fresh produce should be of concern.
Abstract: This study aimed to determine the biofilm formation ability by Salmonella Typhi on cucumber, mango and guava surface, as well as to determine the relationship between time contact and biofilm formation. Crystal violet assay was performed to quantify the biofilm formation based on the value of optical density at 570 nm of the destaining crystal violet at the specific interval time. The result showed that the attachment of the bacterial cells on the fresh produce surface increased with the contact time. The readings of OD570 at time 12 h for cucumber, mango and guava surfaces were 0.824, 0.683 and 0.598, respectively, indicating that the biofilm formation by Salmonella Typhi on different fresh produce surface varied with time. Since the result showed that Salmonella Typhi formed biofilm on fresh produce surfaces, hygienic practice from farm to fork including handling, processing, distribution and storage of the fresh produce should be of concern.

21 citations


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

  • ...As a result, crystal violet staining may overestimate the number of adherent bacteria of the biofilm (Merritt et al., 2005)....

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Journal ArticleDOI
TL;DR: In this paper, the anti-bacterial and antibiofilm effects of a GO/AgNPs coated NiTi alloy prepared by Electrophoretic deposition were determined by measuring the biofilm mass and the number of viable cells using a crystal violet assay and colony counting assay.
Abstract: Biofilm formation on medical devices can induce complications. Graphene oxide/silver nanoparticles (GO/AgNPs) coated nickel-titanium (NiTi) alloy has been successfully produced. Therefore, the aim of this study was to determine the anti-bacterial and anti-biofilm effects of a GO/AgNPs coated NiTi alloy prepared by Electrophoretic deposition (EPD). GO/AgNPs were coated on NiTi alloy using various coating times. The surface characteristics of the coated NiTi alloy substrates were investigated and its anti-biofilm and anti-bacterial effect on Streptococcus mutans biofilm were determined by measuring the biofilm mass and the number of viable cells using a crystal violet assay and colony counting assay, respectively. The results showed that although the surface roughness increased in a coating time-dependent manner, there was no positive correlation between the surface roughness and the total biofilm mass. However, increased GO/AgNPs deposition produced by the increased coating time significantly reduced the number of viable bacteria in the biofilm (p < 0.05). Therefore, the GO/AgNPs on NiTi alloy have an antibacterial effect on the S. mutans biofilm. However, the increased surface roughness does not influence total biofilm mass formation (p = 0.993). Modifying the NiTi alloy surface using GO/AgNPs can be a promising coating to reduce the consequences of biofilm formation.

21 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)....

    [...]