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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: Experimental evolution found that diversity, partly related to altered cyclic-di-GMP levels, arose and persisted due to the emergence of ecological interdependencies related to attachment patterns, which points to the need to account for diversity in infections.
Abstract: The ecological and evolutionary forces that promote and maintain diversity in biofilms are not well understood. To quantify these forces, three Pseudomonas aeruginosa populations were experimentally evolved from strain PA14 in a daily cycle of attachment, assembly, and dispersal for 600 generations. Each biofilm population evolved diverse colony morphologies and mutator genotypes defective in DNA mismatch repair. This diversity enhanced population fitness and biofilm output, owing partly to rare, early colonizing mutants that enhanced attachment of others. Evolved mutants exhibited various levels of the intracellular signal cyclic-di-GMP, which associated with their timing of adherence. Manipulating cyclic-di-GMP levels within individual mutants revealed a network of interactions in the population that depended on various attachment strategies related to this signal. Diversification in biofilms may therefore arise and be reinforced by initial colonists that enable community assembly. IMPORTANCE How biofilm diversity assembles, evolves, and contributes to community function is largely unknown. This presents a major challenge for understanding evolution during chronic infections and during the growth of all surface-associated microbes. We used experimental evolution to probe these dynamics and found that diversity, partly related to altered cyclic-di-GMP levels, arose and persisted due to the emergence of ecological interdependencies related to attachment patterns. Clonal isolates failed to capture population attributes, which points to the need to account for diversity in infections. More broadly, this study offers an experimental framework for linking phenotypic variation to distinct ecological strategies in biofilms and for studying eco-evolutionary interactions.

68 citations


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

  • ...5) by modulating EPS production through the pel operon and by affecting flagellar function (39, 65)....

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Journal ArticleDOI
TL;DR: The application of polymyxin B and GS in combination is a promising option for a topical medication and in the prevention of acute and chronic infections caused by multidrug-resistant or biofilm-forming P. aeruginosa.
Abstract: Multidrug-resistant Pseudomonas aeruginosa is a major cause of severe hospital-acquired infections. Currently, polymyxin B (PMB) is a last-resort antibiotic for the treatment of infections caused by Gram-negative bacteria, despite its undesirable side effects. The delivery of drug combinations has been shown to reduce the required therapeutic doses of antibacterial agents and thereby their toxicity if a synergistic effect is present. In this study, we investigated the synergy between two cyclic antimicrobial peptides, PMB and gramicidin S (GS), against different P. aeruginosa isolates, using a quantitative checkerboard assay with resazurin as a growth indicator. Among the 28 strains that we studied, 20 strains showed a distinct synergistic effect, represented by a fractional inhibitory concentration index (FICI) of ≤0.5. Remarkably, several clinical P. aeruginosa isolates that grew as small-colony variants revealed a nonsynergistic effect, as indicated by FICIs between >0.5 and ≤0.70. In addition to inhibiting the growth of planktonic bacteria, the peptide combinations significantly decreased static biofilm growth compared with treatment with the individual peptides. There was also a faster and more prolonged effect when the combination of PMB and GS was used compared with single-peptide treatments on the metabolic activity of pregrown biofilms. The results of the present study define a synergistic interaction between two cyclic membrane-active peptides toward 17 multidrug-resistant P. aeruginosa and biofilms of P. aeruginosa strain PAO1. Thus, the application of PMB and GS in combination is a promising option for a topical medication and in the prevention of acute and chronic infections caused by multidrug-resistant or biofilm-forming P. aeruginosa.

68 citations


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

  • ...The medium was aspirated to remove planktonic cells, the wells were washed with water, and the formation of sessile biofilms was evaluated by crystal violet (CV) staining, as described previously (38)....

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Journal ArticleDOI
TL;DR: The mode of antibacterial effect of Bacillus strains against R. solanacearum is demonstrated, suggesting that the potential antibacterial and anti-biofilm related mechanisms are associated to their ability to secret the corresponding lipopeptides in surrounding niche.
Abstract: Bacillus strains are extensively studied for their beneficial role in plant growth and biological control of tomato bacterial wilt (TBW), however their underlying mechanisms remained unexplored. In this study, four rhizobacterial strains, Bacillus amyloliquefaciens D29, B. amyloliquefaciens Am1, B. subtilis D16 and B. methylotrophicus H8 were investigated for their antibacterial activity against (TBW) pathogen and their ability to stimulate Tomato growth. Results revealed that all four strains were able to form robust biofilm, produce Indole acetic acid (IAA) and siderophores, while only D29, Am1 and H8 have capability to solubilize phosphate. The culture filtrate of each strain significantly suppressed the growth and biofilm of Ralstonia solanacearum, where, the cell wall was severely disrupted, which resulted into cell lysis and subsequent leakage of intracellular cytosolic contents. PCR analysis revealed that all four strains are harboring the antimicrobial associated genes for biosynthesis of Bacyllomicin, Fengycin, Iturin, Surfactin and Bacylisin. Subsequent real-time qPCR analysis revealed that the expression of ituC and srfAA genes in Am1 and D16 was remarkably up-regulated during in vitro interaction with R. solanacearum. This suggest that the potential antibacterial and anti-biofilm related mechanisms are associated to their ability to secret the corresponding lipopeptides in surrounding niche. In greenhouse, a positive correlation (0.777 and 0.686) was noted between the IAA amount produced by Bacillus strains and fresh/dry weight of bacterized tomato plants. This the first report demonstrated the mode of antibacterial effect of Bacillus strains against R. solanacearum, moreover this study will help in understanding the mode of action of Bacillus strains during biological management of TBW and promoting the growth of tomato plants.

68 citations


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

  • ...The ability of selected strains to form biofilm was estimated by using solid-surface-associated biofilm formation with the crystal violet (CV) staining method (Merritt et al. 2005)....

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  • ...2.5 Biofilm assays The ability of selected strains to form biofilm was estimated by using solid-surface-associated biofilm formation with the crystal violet (CV) staining method (Merritt et al. 2005)....

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Journal ArticleDOI
30 Jun 2015-PLOS ONE
TL;DR: Reducing the liposome size and formulating liposomes as positively charged enhanced the penetration and inhibition of S. aureus and P. aeruginosa biofilms.
Abstract: Background Staphylococcus aureus and Pseudomonas aeruginosa are major pathogens in chronic rhinosinusitis (CRS) and their biofilms have been associated with poorer postsurgical outcomes. This study investigated the distribution and anti-biofilm effect of cationic (+) and anionic (-) phospholipid liposomes with different sizes (unilamellar and multilamellar vesicle, ULV and MLV respectively) on S. aureus and P. aeruginosa biofilms. Method Specific biofilm models for S. aureus ATCC 25923 and P. aeruginosa ATCC 15692 were established. Liposomal distribution was determined by observing SYTO9 stained biofilm exposed to DiI labeled liposomes using confocal scanning laser microscopy, followed by quantitative image analysis. The anti-biofilm efficacy study was carried out by using the alamarBlue assay to test the relative viability of biofilm treated with various liposomes for 24 hours and five minutes. Results The smaller ULVs penetrated better than larger MLVs in both S. aureus and P. aeruginosa biofilm. Except that +ULV and –ULV displayed similar distribution in S. aureus biofilm, the cationic liposomes adhered better than their anionic counterparts. Biofilm growth was inhibited at 24-hour and five-minute exposure time, although the decrease of viability for P. aeruginosa biofilm after liposomal treatment did not reach statistical significance. Conclusion The distribution and anti-biofilm effects of cationic and anionic liposomes of different sizes differed in S. aureus and P. aeruginosa biofilms. Reducing the liposome size and formulating liposomes as positively charged enhanced the penetration and inhibition of S. aureus and P. aeruginosa biofilms.

67 citations

Journal ArticleDOI
TL;DR: LAB were able to produce antimicrobial compounds that inhibit S. aureus and form a biofilm, which was successfully inhibited by the CFS of L. bulgaricus FTDC 8611.

67 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)....

    [...]