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 paper , the antimicrobial, bio-film-preventing and biofilm-eradicating activity of 2(5H)-furanone derivative F131, containing an l-borneol fragment against S. aureus and C. albicans mixed biofilms was investigated.
Abstract: Candida albicans and Staphylococcus aureus are human pathogens that are able to form mixed biofilms on the surface of mucous membranes, implants and catheters. In biofilms, these pathogens have increased resistance to antimicrobials, leading to extreme difficulties in the treatment of mixed infections. The growing frequency of mixed infections caused by S. aureus and C. albicans requires either the development of new antimicrobials or the proposal of alternative approaches to increase the efficiency of conventional ones. Here, we show the antimicrobial, biofilm-preventing and biofilm-eradicating activity of 2(5H)-furanone derivative F131, containing an l-borneol fragment against S. aureus–C. albicans mixed biofilms. Furanone F131 is also capable of inhibiting the formation of monospecies and mixed biofilms by S. aureus and C. albicans. The minimal biofilm-prevention concentration (MBPC) of this compound was 8–16 μg/mL for S. aureus and C. albicans mono- and two-species biofilms. While the compound demonstrates slightly lower activity compared to conventional antimicrobials (gentamicin, amikacin, fluconazole, terbinafine and benzalkonium chloride), F131 also increases the antimicrobial activity of fluconazole–gentamicin and benzalkonium chloride against mixed biofilms of S. aureus–C. albicans, thus reducing MBPC of fluconazole–gentamicin by 4–16 times and benzalkonium chloride twofold. F131 does not affect the transcription of the MDR1, CDR1 and CDR2 genes, thus suggesting a low risk of micromycete resistance to this compound. Altogether, combined use of antibiotics with a F131 could be a promising option to reduce the concentration of fluconazole used in antiseptic compositions and reduce the toxic effect of benzalkonium chloride and gentamicin. This makes them an attractive starting point for the development of alternative antimicrobials for the treatment of skin infections caused by S. aureus–C. albicans mixed biofilms.
2 citations
Dissertation•
28 Feb 2018
TL;DR: In this article, the authors propose a strategy to examine and comprendre tous les different composants of the biofilm in the ecosystem of the cavite buccale.
Abstract: L’ecosysteme buccal est un environnement complexe dans lequel cohabitent plus de 700 especes de bacteries differentes. Un desequilibre au sein de ce biofilm est a l’origine des principales maladies de la cavite buccale : les maladies carieuses et parodontales.Pendant plusieurs annees, l’etude de l’ecosysteme buccal s’est faite par une approche reductionniste : les microbiologistes etudiaient les especes bacteriennes individuellement. Cette strategie a permis d’examiner et de comprendre tous les differents composants de cet ecosysteme, sans pour autant pouvoir appliquer les conclusions de ces etudes au biofilm buccal dans son ensemble. En effet, les bacteries ne se comportent pas de la meme facon lorsqu’elles sont a l’etat planctonique, ou lorsqu’elles sont organisees en biofilm.La flore microbienne buccale est reconnue comme etant l’une des plus complexes dans le corps humain. La multitude d’especes en presence complique l’etude de ce biofilm. En effet, sa reproduction in vitro est rendue difficile par la complexite des relations entre chacune des especes. De plus, son recueil, et ses decomptes qualitatif et quantitatif restent tres delicats.Dans la litterature sont decrits plusieurs modeles de biofilm.Les modeles pluri-especes dynamiques in vitro ont l’avantage de se rapprocher des conditions retrouvees in vivo, permettant un certain flux de milieu, le controle de parametres tels que le pH et la temperature, ainsi que l’elimination des dechets produits.Cependant, ces modeles restent tres onereux et difficiles de mise en place, ce qui complique l’etude du biofilm buccal. Egalement, l’identification des bacteries mises en presence reste un sujet d’etude delicat : en effet, les methodes traditionnelles de culture montrent des limites, et ne permettent pas une analyse quantitative des resultats, essentielle a la comprehension des phenomenes mis en jeu dans cet ecosysteme.Le but de notre travail ici est la mise en place de modele de biofilm pluri-especes dynamique, fiable et reproductible, facile a mettre en place et moins onereux que ceux decrits dans la litterature. Ce modele de biofilm doit permettre l’etude de l’influence de variations de conditions environnementales sur ce dernier, ainsi que celle de candidats probiotiques ayant deja prouve leur efficacite sur des supports in vitro statiques. Enfin, toujours dans une optique de simplification, les differentes methodes d’identification des biofilms formes sont comparees (methodes de culture traditionnelle, PCR conventionnelle, spectrometrie de masse MALDI-TOF, et qPCR), afin d’etablir un protocole d’identification reproductible permettant une analyse qualitative et quantitative des resultats.
2 citations
Cites background from "Growing and analyzing static biofil..."
...La culture sur gélose (agar plate) est l'un des modèles de culture microbienne les plus simples au laboratoire [90]....
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TL;DR: The results validate the usefulness of the three plants as traditional medicine in some instances, and show the anti-biofilm properties of crude methanolic extracts from three medicinal plants used in Namibia.
Abstract: Traditional medicine is widely used, but its effectiveness is often questioned. Biofilm-producing bacteria and fungi are important in difficult-to-treat persistent and recurrent infections. The present study investigated the anti-biofilm properties of crude methanolic extracts from three medicinal plants used in Namibia, namely Aptosimum albomarginatum (Marloth and Engl.), Albizia anthelmintica (A. Rich Brongn.) and Dicoma schinzii (O. Hoffm.). Biofilm formation, inhibition and eradication were determined using microtiter plate assay. Extracts were tested against Escherichia coli ATCC 700928, Staphylococcus aureus ATCC 12600, S. aureus U3300, Bacillus subtilis ATCC 13933, Streptococcus mutans ATCC 25175, Streptococcus sanguinis ATCC 10556, Pseudomonas aeruginosa and Candida albicans. All isolates were strong biofilm producers. A. albomarginatum root extract moderately inhibited biofilm formation in S. mutans ATCC 25175 (60.0%), E. coli ATCC 700928 (51.6%) and P. aeruginosa (49.1%). A. anthelmintica twigs caused 58.4% biofilm inhibition in C. albicans and eradicated S. aureus U3300 biofilm by 74.8%. D. schinzii leaf extract inhibited P. aeruginosa biofilm by 67.3%, and in addition broke down S. mutans ATCC 25175 biofilm by 44.2%. These results validate the usefulness of the three plants as traditional medicine in some instances.
Key words: Aptosimum albomarginatum, Albizia anthelmintica, Dicoma schinzii, traditional medicine, anti-biofilm activity, flavonoids, saponins.
2 citations
Cites methods from "Growing and analyzing static biofil..."
...The methods of Christensen et al. (1985), Merritt et al. (2011), and Monte et al. (2014) were used for biofilm assays, with some modifications....
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TL;DR: In the two species biofilms, B. subtilis produces two antibiotics, bacillaene and bacilysin, that inhibit C. jejuni growth, which supports the application of the PS-216 strain to pathogen biofilm control.
Abstract: Campylobacter jejuni is a prevalent cause of foodborne infections worldwide, while Bacillus subtilis as a potential probiotic represents an alternative strategy to control this alimentary infection. However, only limited literature exists on the specific mechanisms that shape interactions between B. subtilis and C. jejuni in biofilms. ABSTRACT The foodborne pathogen Campylobacter jejuni is typically found in an agricultural environment; in animals, such as birds, as an intestinal commensal; and also in food products, especially fresh poultry meat. Campylobacter interactions within mixed species biofilms are poorly understood, especially at the microscale. We have recently shown that the beneficial bacterium Bacillus subtilis reduces C. jejuni survival and biofilm formation in coculture by secreting the antibiotic bacillaene. We extend these studies here by providing evidence that besides bacillaene, the antagonistic effect of B. subtilis involves a nonribosomal peptide bacilysin and that the fully functional antagonism depends on the quorum-sensing transcriptional regulator ComA. Using confocal laser scanning microscopy, we also show that secreted antibiotics influence the distribution of C. jejuni and B. subtilis cells in the submerged biofilm and decrease the thickness of the pathogen’s biofilm. Furthermore, we demonstrate that genes encoding structural or regulatory proteins of the efflux apparatus system (cmeF and cmeR), respectively, contribute to the survival of C. jejuni during interaction with B. subtilis PS-216. In conclusion, this study demonstrates a strong potential of B. subtilis PS-216 to reduce C. jejuni biofilm growth, which supports the application of the PS-216 strain to pathogen biofilm control. IMPORTANCE Campylobacter jejuni is a prevalent cause of foodborne infections worldwide, while Bacillus subtilis as a potential probiotic represents an alternative strategy to control this alimentary infection. However, only limited literature exists on the specific mechanisms that shape interactions between B. subtilis and C. jejuni in biofilms. This study shows that in the two species biofilms, B. subtilis produces two antibiotics, bacillaene and bacilysin, that inhibit C. jejuni growth. In addition, we provide the first evidence that specific pathogen efflux pumps contribute to the defense against B. subtilis attack. Specifically, the CmeDEF pump acts during the defense against bacilysin, while CmeR-dependent overexpression of CmeABC nullifies the bacillaene attack. The role of specific B. subtilis antibiotics and these polyspecific pumps, known for providing resistance against medically relevant antibiotics, has not been studied during bacterial competition in biofilms before. Hence, this work broadens our understanding of mechanisms that shape antagonisms and defense during probiotic-pathogen interactions.
2 citations
01 Sep 2018
TL;DR: In this paper, the antimicrobial and antioxidative effect of the essential oil (EO) of immortelle [Helichrysum italicum (Roth) G Don (H italicum)] and its ability to inhibit biofilm of nontuberculous mycobacteria was investigated.
Abstract: Objective: To investigate the antimicrobial and antioxidative effect of the essential oil (EO) of immortelle [Helichrysum italicum (Roth) G Don (H italicum)] and its ability to inhibit biofilm of nontuberculous mycobacteria, M avium and M intracellulare Materials and methods: a broth microdilution method was used to determine the minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the H italicum EO Survival of mycobacteria in Middlebrook 7H9 broth under the influence of H italicum EO was monitored by subcultivation of suspension of 0, 1, 4 and 8 days For determination of the inhibition of biofilm formation in water, the biofilm coloring method with crystal violet was applied The antioxidant effect of MIC concentrations of H italicum EO was determined by DPPH (2,2-diphenyl-1-picrylhydrazil) method Results: MIC and MBC value of immortelle EO was 32 mg/ml Treatment of mycobacteria with H italicum EO at one half times the MIC resulted in approximately 2 log10 reduction of M avium and 25 log10 M intracellulare respectively over 24 h, and complete reduction of both mycobacteria on the fourth day M avium showed a greater tendency to form biofilms The concentration of one-quarter and one-half of the MIC values resulted in statistically significant inhibition of biofilm production of the tested mycobacteria MIC concentrations of H italicum EU after 60 minutes inhibited 382% of DPPH radicals Conclusion: The subinhibitory concentrations of H italicum EO significantly reduce the viability of M avium and M intracellulare and result in substantial inhibition of biofilm production of the tested mycobacteria in water, which would allow the application of low effective concentrations of this EO that do not have a cytotoxic effect as a natural disinfectant in water
2 citations
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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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