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: Antibiofilm potential of AgNPs showed that they were highly effective at sub MIC concentrations in causing 50% biofilm inhibition against food borne pathogen S. Typhi implying that antibiofilm effect is independent of antibacterial effect, which was evidenced by fluorescent imaging and SEM imaging.
28 citations
TL;DR: This article showed that E. coli displays persistence against a widely used disinfectant, benzalkonium chloride (BAC), which is associated with reduced cell surface charge and mutations in the lpxM locus, encoding an enzyme for lipid A biosynthesis.
Abstract: Biocides used as disinfectants are important to prevent the transmission of pathogens, especially during the current antibiotic resistance crisis. This crisis is exacerbated by phenotypically tolerant persister subpopulations that can survive transient antibiotic treatment and facilitate resistance evolution. Here, we show that E. coli displays persistence against a widely used disinfectant, benzalkonium chloride (BAC). Periodic, persister-mediated failure of disinfection rapidly selects for BAC tolerance, which is associated with reduced cell surface charge and mutations in the lpxM locus, encoding an enzyme for lipid A biosynthesis. Moreover, the fitness cost incurred by BAC tolerance turns into a fitness benefit in the presence of antibiotics, suggesting a selective advantage of BAC-tolerant mutants in antibiotic environments. Our findings highlight the links between persistence to disinfectants and resistance evolution to antimicrobials.
28 citations
Journal Article•
TL;DR: It is found that the broad-spectrum antibiotic tropodithietic acid (TDA) causes the same regulatory effects in quorum sensing (QS) as the common signaling molecule N-acyl-homoserine lactone (AHL) at concentrations 100-fold lower than the minimal inhibitory concentration against bacteria.
Abstract: Antibiotics are typically regarded as microbial weapons, but whereas their function at concentrations lethal for bacteria is often well characterized, the role of antibiotics at much lower concentrations as possibly found under natural conditions remains poorly understood. By using whole-transcriptome analyses and phenotypic screenings of the marine bacterium Phaeobacter inhibens we found that the broad-spectrum antibiotic tropodithietic acid (TDA) causes the same regulatory effects in quorum sensing (QS) as the common signaling molecule N-acyl-homoserine lactone (AHL) at concentrations 100-fold lower than the minimal inhibitory concentration against bacteria. Our results show that TDA has a significant impact on the expression of ~10% of the total genes of P. inhibens, in the same manner as the AHL. Furthermore, TDA needs the AHL associated LuxR-type transcriptional regulator, just as the AHL molecule. Low concentrations of antibiotics can obviously have a strong influence on the global gene expression of the bacterium that produces it and drastically change the metabolism and behaviour of the bacterium. For P. inhibens this includes motility, biofilm formation and antibiotic production, all important for settlement on new host-associated surfaces. Our results demonstrate that bacteria can produce antibiotics not only to antagonise other bacteria, but also to mediate QS like endogenous AHL molecules.
28 citations
TL;DR: The pel operon is identified in more than 125 proteobacteria, demonstrating that its distribution was previously underestimated and insight is provided into the unique molecular architecture and export mechanism of the Pel apparatus, a widespread exopolysaccharide secretion system found in environmental and pathogenic bacteria.
Abstract: Secreted polysaccharides are important functional and structural components of bacterial biofilms. The opportunistic pathogen Pseudomonas aeruginosa produces the cationic exopolysaccharide Pel, which protects bacteria from aminoglycoside antibiotics and contributes to biofilm architecture through ionic interactions with extracellular DNA. A bioinformatics analysis of genome databases suggests that gene clusters for Pel biosynthesis are present in >125 bacterial species, yet little is known about how this biofilm exopolysaccharide is synthesized and exported from the cell. In this work, we characterize PelC, an outer membrane lipoprotein essential for Pel production. Crystal structures of PelC from Geobacter metallireducens and Paraburkholderia phytofirmans coupled with structure-guided disulfide cross-linking in P. aeruginosa suggest that PelC assembles into a 12- subunit ring-shaped oligomer. In this arrangement, an aromatic belt in proximity to its lipidation site positions the highly electronegative surface of PelC toward the periplasm. PelC is structurally similar to the Escherichia coli amyloid exporter CsgG; however, unlike CsgG, PelC does not possess membrane-spanning segments required for polymer export across the outer membrane. We show that the multidomain protein PelB with a predicted C-terminal β-barrel porin localizes to the outer membrane, and propose that PelC functions as an electronegative funnel to guide the positively charged Pel polysaccharide toward an exit channel formed by PelB. Together, our findings provide insight into the unique molecular architecture and export mechanism of the Pel apparatus, a widespread exopolysaccharide secretion system found in environmental and pathogenic bacteria.
28 citations
TL;DR: Aggregated Methylobacterium oryzae CBMB20 cells exhibited enhanced tolerance to UV irradiation, heat, desiccation, different temperature regimes, oxidative stress, starvation and supported higher population in media.
Abstract: Salinity is one of the major factors contributing to the loss of crop productivity and thereby impacting livelihood of people in more than 100 countries of the world and the area of land affected by salinity is increasing day by day. This will worsen due to various factors such as drought that might result in high soil salinity. Use of plant growth promoting rhizobacteria is one of the promising eco-friendly strategies for salinity stress management as part of sustainable agricultural practices. However, it requires selecting rhizobacteria with good survivability and adaptation to salt stress. In this study we report aggregation of Methylobacterium oryzae CBMB20 cells grown in media containing high C/N ratio (30:1) than in media containing low C/N ratio (7:1). Aggregated Methylobacterium oryzae CBMB20 cells exhibited enhanced tolerance to UV irradiation, heat, desiccation, different temperature regimes, oxidative stress, starvation and supported higher population in media. Poly-β-hydroxybutyrate accumulation, exopolysaccharide production, proline accumulation and biofilm formation were good at 100 mM salt concentration with good microbial cell hydrophobicity at both 50 and 100 mM than other concentrations. Both the aggregated and non-aggregated cells grown under 0–200 mM salt concentrations produced IAA even at 200 mM salt concentration with a peak at 100 mM concentration with aggregated cells producing significantly higher quantities. ACC deaminase activity was observed in all NaCl concentrations studied with gradual and drastic reduction in aggregated and non-aggregated cells over increased salt concentrations.
28 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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