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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13 May 2020-Journal of Environmental Science and Health Part A-toxic\/hazardous Substances & Environmental Engineering
TL;DR: Results showed promising results and these bacterial strains can be used as potential tools for bioremediation of PAH in contaminated sites and scaling up of the biofilm bioreactor can prove beneficial in large scale treatment ofPAH contaminated water.
Abstract: Degradation studies of phenanthrene and naphthalene as a mixture was carried out using a developed bacterial consortium. The isolates used in consortium were identified as Chryseobacterium sp., Sph...
16 citations
TL;DR: Encapsulation of MAHMA NONOate and molsidomine within a hydrogel composed of cellulose nanocrystals has shown a synergistic effect in dispersing well-established biofilms, suggesting that the combined treatments with nitric oxide donor and hydrogels may allow for new sustainable cleaning strategies.
Abstract: Protected by extracellular polymers, microbes within biofilms are significantly more resistant to disinfectants. Current research has been instrumental in identifying nitric oxide donors and hydrogels as potential disinfectant additives. Nitric oxide (NO) donors are considered a very promising molecule as biofilm dispersal agents and hydrogels have recently attracted a lot of interest due to their biocompatible properties and ability to form stable thin films. When the NO donor MAHMA NONOate was dissolved in phosphate saline buffer, it was able to reduce the biomass of well-established biofilms up to 15% for at least 24 h of contact time. Encapsulation of MAHMA NONOate and molsidomine within a hydrogel composed of cellulose nanocrystals (CNC) has shown a synergistic effect in dispersing well-established biofilms: after 2 h of exposure, moderate but significant dispersion was measured. After 6 h of exposure, the number of cells transitioning from the biofilm to the planktonic state was up to 0.6 log higher when compared with non-treated biofilms. To further explore the transport processes of NO donors within hydrogels, we measured the nitric oxide flux from gels, at 25°C for a composite of 0.1 µM MAHMA NONOate–CNC. Nitric oxide diffuses up to 500 µm from the hydrogel surface, with flux decreasing according to Fick’s law. 60% of NO was released from the hydrogel composite during the first 23 min. These data suggest that the combined treatments with nitric oxide donor and hydrogels may allow for new sustainable cleaning strategies.
16 citations
TL;DR: A series of N-octaneamino-4-aminoquinoline derivatives were synthesized to optimize this chemotype's anti-virulence activity against P. aeruginosa via inhibition of pyocyanin production and structure-activity data was rationalized using molecular docking studies.
Abstract: Pseudomonas aeruginosa is a leading cause of nosocomial infections that are becoming increasingly difficult to treat due to the occurrence of antibiotic resistant strains. Since P. aeruginosa virulence is controlled through quorum sensing, small molecule treatments inhibiting quorum sensing signaling pathways provide a promising therapeutic option. Consequently, we synthesized a series of N-octaneamino-4-aminoquinoline derivatives to optimize this chemotype’s antivirulence activity against P. aeruginosa via inhibition of pyocyanin production. The most potent derivative, which possesses a benzofuran substituent, provided effective inhibition of pyocyanin production (IC50 = 12 μM), biofilm formation (BFIC50 = 50 μM), and motility. Experimentally, the compound’s activity is achieved through competitive inhibition of PqsR, and structure–activity data were rationalized using molecular docking studies.
16 citations
TL;DR: The results showed the potential use of cellulose fibers from vegetal for surgical sutures due to excellent mechanical properties, rapid degradation, and no bacterial adhesion.
Abstract: Suture biomaterials are critical in wound repair by providing support to the healing of different tissues including vascular surgery, hemostasis, and plastic surgery. Important properties of a suture material include physical properties, handling characteristics, and biological response for successful performance. However, bacteria can bind to sutures and become a source of infection. For this reason, there is a need for new biomaterials for suture with antifouling properties. Here we report two types of cellulose fibers from coconut (Cocos nucifera) and sisal (Agave sisalana), which were purified with a chemical method, characterized, and tested in vitro and in vivo. According to SEM images, the cellulose fiber from coconut has a porous surface, and sisal has a uniform structure without internal spaces. It was found that the cellulose fiber from sisal has mechanical properties closer to silk fiber biomaterial using Ultimate Tensile Strength. When evaluating the cellulose fibers biodegradability, the cellulose from coconut showed a rapid weight loss compared to sisal. The antifouling test was negative, which demonstrated that neither possesses intrinsic microbicidal activity. Yet, a weak biofilm was formed on sisal cellulose fibers suggesting it possesses antifouling properties compared to cellulose from coconut. In vivo experiments using healthy mice demonstrated that the scarring and mechanical connection was like silk for both cellulose fibers. Overall, our results showed the potential use of cellulose fibers from vegetal for surgical sutures due to excellent mechanical properties, rapid degradation, and no bacterial adhesion.
16 citations
Cites background or methods from "Growing and analyzing static biofil..."
...The general protocol was established using microtiter plates; however, in this experiment spectrophotometer cuvettes were used, since fibers size (1–2 cm) were too large to use common plate [47]....
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...We measured the optical density (OD) of each sample at a wavelength of 600 nm using a spectrophotometer according to the bibliography [47,48]....
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...At this stage, the staining of biofilm was stable [47,48]....
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TL;DR: In this paper, Bacillus subtilis, isolated and characterized in this work, exhibited growth inhibition against B. cinerea of more than 40% in in vitro cultures, by studying several of their biochemical properties, such as production of lipopeptides, potassium solubilization, proteolytic and amylolytic capacity, production of siderophores, biofilm assays, and so on.
Abstract: Plant diseases are one of the main factors responsible for food loss in the world, and 20-40% of such loss is caused by pathogenic infections. Botrytis cinerea is the most widely studied necrotrophic phytopathogenic fungus. It is responsible for incalculable economic losses due to the large number of host plants affected. Today, B. cinerea is controlled mainly by synthetic fungicides whose frequent application increases risk of resistance, thus making them unsustainable in terms of the environment and human health. In the search for new alternatives for the biocontrol of this pathogen, the use of endophytic microorganisms and their metabolites has gained momentum in recent years. In this work, we isolated endophytic bacteria from Zea mays cultivated in Colombia. Several strains of Bacillus subtilis, isolated and characterized in this work, exhibited growth inhibition against B. cinerea of more than 40% in in vitro cultures. These strains were characterized by studying several of their biochemical properties, such as production of lipopeptides, potassium solubilization, proteolytic and amylolytic capacity, production of siderophores, biofilm assays, and so on. We also analyzed: (i) its capacity to promote maize growth (Zea mays) in vivo, and (ii) its capacity to biocontrol B. cinerea during in vivo infection in plants (Phaseolus vulgaris).
16 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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