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: In this article, a transposon mutagenesis method and a high-throughput screening assay were developed to identify mutants of Geobacter sulfurreducens PCA interrupted in the initial stages of surface colonization (attachment and monolayer formation) and the vertical growth and maturation of multilayered biofilms.
Abstract: Geobacter biofilms synthesize an electroactive exopolysaccharide matrix with conductive pili and c-cytochromes that spatially organizes cells optimally for growth and electron transport to iron oxide substrates, soluble metal contaminants, and current-harvesting electrodes. Despite its relevance to bioremediation and bioenergy applications, little is known about the developmental stages leading to the formation of mature (>20 μm thick) electroactive biofilms. Thus, we developed a transposon mutagenesis method and a high-throughput screening assay and identified mutants of Geobacter sulfurreducens PCA interrupted in the initial stages of surface colonization (attachment and monolayer formation) and the vertical growth and maturation of multilayered biofilms. The molecular dissection of biofilm formation demonstrated that cells undergo a regulated developmental program to first colonize the surface to saturation and then synthesize an electroactive matrix to support optimal cell growth within structured communities. Transitioning from a monolayer to a multilayered, mature biofilm required the expression of conductive pili, consistent with the essential role of these extracellular protein appendages as electronic conduits across all layers of the biofilms. The genetic screening also identified cell envelope processes, regulatory pathways, and electron transport components not previously linked to biofilm formation. These genes provide much-needed understanding of the cellular reprogramming needed to build electroactive biofilms. Importantly, they serve as predictive markers of the physiology and reductive capacity of Geobacter biofilms during the bioremediation of toxic metals and radionuclides and current harvesting in bioelectrochemical systems.
5 citations
TL;DR: In this paper , the levels of cytokine secretory inhibitors and the microbiota biofilms of semen from healthy and infertile subjects were determined based on the difference in cytokine content between the control and experimental samples of isolated microorganisms.
Abstract: In this study, we determined the levels of cytokine secretory inhibitors and the microbiota biofilms of semen from healthy and infertile subjects. A total of 118 clinical bacterial isolates were isolated and tested. Cytokine secretory inhibitors were determined based on the difference in cytokine content between the control and experimental samples of cell-free supernatants of isolated microorganisms. Biofilm formation was studied by determining the adhesion of microorganisms to the surface of a 96-well sterile plate and expressed as the optical density at 630 nm (OD630). Cell-free supernatants of Staphylococcus contained higher levels of secretory inhibitor of cytokines in conditionally healthy than in infertile patients. In contrast, in infertile men, the ability to reduce cytokine levels was more characteristic of Enterococcus and Corynebacterium. Seminal Staphylococcus, Corynebacterium, and Enterococcus isolated from infertile subjects showed a greater ability to form biofilms than the same bacteria isolated from healthy men. Further research is needed on this topic, since it is necessary to determine the relationships between decreased secretory inhibitors of cytokines, production of biofilms by bacteria in semen, and infertility. It is likely that the ability of microorganisms to change the concentration of cytokines and increase the level of biofilm formation in semen may be associated with minimal impairments of fertilizing ability, which are not detected using other methods.
5 citations
Dissertation•
28 Sep 2017
TL;DR: The realisation of the Antibiofilmogramme® on isolats cliniques mucoviscidosiques (nouvel outil evaluant la sensibilite des bacteries sessiles aux antibiotiques) a permis de mettre en evidence des phenomenes dinhibition and dinduction de the formation of biofilm as mentioned in this paper.
Abstract: Les patients mucoviscidosiques sont predisposes a une colonisation chronique de l’arbre bronchique par P. aeruginosa. Ce pathogene opportuniste se caracterise par sa capacite a adherer a une surface et a y former un biofilm protecteur, hautement tolerant aux agents antimicrobiens. En routine, les antibiogrammes sont effectues sur des cultures bacteriennes planctoniques. L’efficacite des antibiotherapies ainsi selectionnees est donc peu probante pour l’eradication des biofilms bacteriens. La realisation d’Antibiofilmogrammes® sur des isolats cliniques mucoviscidosiques (nouvel outil evaluant la sensibilite des bacteries sessiles aux antibiotiques) a permis de mettre en evidence des phenomenes d’inhibition et d’induction de la formation du biofilm. Plus precisement, les aminosides sont capables de retarder l’adherence bacterienne. A l’inverse, la famille des β-lactamines presente la capacite de stimuler l’adhesion precoce des micro-organismes. Ces differents effets de l’antibiotherapie generale sur le comportement microbien se verifient par l’intermediaire de techniques conventionnelles in vitro (Cristal Violet, traitement enzymatique a la DNase I) et cellulaires (modele de co-culture statique cellules eucaryotes/bacteries). La pertinence clinique de l’Antibiofilmogramme® se confirme donc par sa capacite a detecter l’initiation precoce de l’adhesion bacterienne, a selectionner les molecules l’inhibant et a ecarter celles pouvant l’induire. Associee aux antibiogrammes traditionnels, son application peu permettre d’affiner les strategies therapeutiques pour le traitement des infections pulmonaires chroniques developpees au cours de la mucoviscidose.
5 citations
Additional excerpts
...Développés initialement pour l’étude de l’adhésion bactérienne, les systèmes de culture statique peuvent, de manière plus approfondie, être appliqués à l’analyse du développement de micro-organismes sessiles (Merritt et al., 2011)....
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TL;DR: It is suggested that antibody raised against dextransucrase exhibit inhibitory effects on the growth of S. mutans and biofilm formation with no reactivity with various mammalian tissues, thus it could be an effective anticariogenic agent.
Abstract: Streptococcus mutans is a common principal causative agent of dental caries. In this communication, we describe that the antibodies raised against purified dextransucrase effectively inhibited the growth of S. mutans. The purified enzyme showed 58-fold enrichment, 17.5% yield and a specific activity of 3.96 units/mg protein. Purified IgG fraction of the antibody showed significant affinity with the antigenic protein. Immunotritation of the enzyme with dextransucrase antibody showed a gradual increase in inhibition of dextransucrase activity. The growth of S. mutans was also inhibited by 85% in the presence of 28 μg of IgG fraction of the antibody. Antibodies also impaired glucosyltransferase activity (72.8%) and biofilm formation by 92.6% in S. mutans. Western blot analysis revealed no cross reactivity with the various tissues of mice, rat, rabbit and humans. Dot blot analysis showed little reactivity with Lactobacillus acidophilus and Staphylococcus aureus and there was no reactivity with other bacterial strains like Enterococcus faecalis, Escherichia coli and Salmonella typhimurium. These findings suggest that antibody raised against dextransucrase exhibit inhibitory effects on the growth of S. mutans and biofilm formation with no reactivity with various mammalian tissues, thus it could be an effective anticariogenic agent.
5 citations
Cites methods from "Growing and analyzing static biofil..."
...To examine the inhibitory effect of serum antibody on biofilm formation by S. mutans, biofilm microplate assay was performed by standard protocol (Merritt et al. 2005)....
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...mutans, biofilm microplate assay was performed by standard protocol (Merritt et al. 2005)....
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TL;DR: In this paper, the authors developed a lipoglycopeptide therapy customized for pulmonary delivery that not only demonstrates potent activity against planktonic MRSA, but also against protected colonies of MRSA in biofilms and within cells.
Abstract: Chronic pulmonary methicillin-resistant Staphylococcus aureus (MRSA) disease in cystic fibrosis (CF) has a high probability of recurrence following treatment with standard-of-care antibiotics and represents an area of unmet need associated with reduced life expectancy. We developed a lipoglycopeptide therapy customized for pulmonary delivery that not only demonstrates potent activity against planktonic MRSA, but also against protected colonies of MRSA in biofilms and within cells, the latter of which have been linked to clinical antibiotic failure. A library of next-generation potent lipoglycopeptides was synthesized with an emphasis on attaining superior pharmacokinetics (PK) and pharmacodynamics to similar compounds of their class. Our strategy focused on hydrophobic modification of vancomycin, where ester and amide functionality were included with carbonyl configuration and alkyl length as key variables. Candidates representative of each carbonyl attachment chemistry demonstrated potent activity in vitro, with several compounds being 30 to 60 times more potent than vancomycin. Selected compounds were advanced into in vivo nose-only inhalation PK evaluations in rats, where RV94, a potent lipoglycopeptide that utilizes an inverted amide linker to attach a 10-carbon chain to vancomycin, demonstrated the most favorable lung residence time after inhalation. Further in vitro evaluation of RV94 showed superior activity to vancomycin against an expanded panel of Gram-positive organisms, cellular accumulation and efficacy against intracellular MRSA, and MRSA biofilm killing. Moreover, in vivo efficacy of inhaled nebulized RV94 in a 48 h acute model of pulmonary MRSA (USA300) infection in neutropenic rats demonstrated statistically significant antibacterial activity that was superior to inhaled vancomycin.
4 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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