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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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Citations
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Journal ArticleDOI
TL;DR: Findings will help veterinarians and farmers to understand the epidemiology of S. aureus so that they can monitor the transmission and spread of this pathogen and control it more effectively.
Abstract: The aim of this study was to characterize the phenotypes and genotypes of Staphylococcus aureus isolated from raw bovine milk in Hokkaido, Japan. S. aureus isolates were identified in 135 of 436 milk samples from cows with and without signs of mastitis from three farms in Hokkaido. These clinical isolates were characterized for antimicrobial susceptibility patterns, molecular typing using phage-open-reading frame typing (POT), coagulase gene type, virulence genes, and biofilm-associated genes and were evaluated for biofilm-forming ability. Most isolates were susceptible to the antimicrobial agents tested. The highest rate of resistance was to ampicillin. Molecular typing of all S. aureus isolates indicated a predominance of coagulase type VI and 0-17-34 POT type, and virulence genes were highly prevalent in the isolates from all farms. Moreover, a high percentage of the 0-17-34 POT type isolates showed extensive formation of biofilm. These findings will help veterinarians and farmers to understand the epidemiology of S. aureus so that they can monitor the transmission and spread of this pathogen and control it more effectively.

7 citations


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

  • ...POT is useful in epidemiologic studies because of its rapid and highly discriminatory power (Nada et al. 2009)....

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Journal ArticleDOI
TL;DR: In this paper, the authors investigated the relationship between the molecular characteristics and the ability to form biofilms in the presence of blood plasma (plasma-biofilms) of MRSA strains isolated from bloodstream infections.
Abstract: In Japan, Staphylococcal cassette chromosome mec (SCCmec) type IV methicillin-resistant Staphylococcus aureus (MRSA) is an increasingly prominent cause of bacteremia, but the virulence of most of these strains is unclear. We aimed to investigate the relationship between the molecular characteristics and the ability to form biofilms in the presence of blood plasma (plasma-biofilms) of MRSA strains isolated from bloodstream infections. In this study, the molecular characteristics and biofilms of MRSA strains isolated from blood cultures between 2015 and 2017 were analyzed by PCR-based assays, crystal violet staining, and confocal reflection microscopy methods. Among the 90 MRSA isolates, the detection rate of SCCmec type II clones decreased from 60.7 to 20.6%. The SCCmec type IV clone replaced the SCCmec type II clone as the dominant clone, with a detection rate increasing from 32.1 to 73.5%. The plasma-biofilm formation ability of the SCCmec type IV clone was higher than the SCCmec type II clone and even higher in strains harboring the cna or arcA genes. Plasma-biofilms, mainly composed of proteins, were formed quickly and strongly. Our study demonstrated the increased plasma-biofilm formation ability of SCCmec type IV strains.

7 citations

Journal ArticleDOI
01 Jan 2016
TL;DR: Investigation of the biofilm production, antimicrobial susceptibility, and biocidal activity of commonly used antiseptics and disinfectants in a hospital setting against Pseudomonas aeruginosa and Acinetobacter baumannii isolates found them to be significantly associated with multi-drug resistant A. baumANNii and P. aerug inosa strains.
Abstract: Objective: Nosocomial infections caused by resistant Gram-negative bacilli have become a major problem for hospitals in recent years. Antiseptics and disinfectants play an important role in the prevention of nosocomial infections and in the management of infections. Some Gram-negative bacilli also show resistance to antiseptics and disinfectants. Therefore, the selection of proper antiseptics and disinfectants is crucial to prevent nosocomial infections produced by these resistant organisms. In this study, we investigated the biofilm production, antimicrobial susceptibility, and biocidal activity of commonly used antiseptics and disinfectants in our hospital setting against Pseudomonas aeruginosa and Acinetobacter baumannii isolates. Methods: Between January and December-2014, a total of 50 Pseudomonas aeruginosa and 50 Acinetobacter baumannii strains, which were multidrug-resistant (MDR) strains, were included in this study. Biofilm production was identified spectrophotometrically by the microplate assay. Activity of sodium hypochlorite, chlorhexidine, orthophthalaldehyde (OPA), peracetic acid (PA), and peracetic acid/hydrogen peroxide was studied with suspension tests. Results: Commonly used disinfectant-antiseptics were found to be effective against multi-drug resistant A. baumannii and P. aeruginosa strains as follows, chlorhexidine 98%, sodium hypochlorite 90%, OPA 96%, PA and peracetic acid/hydrogen peroxide 94%. The rates of efficacy against the antibioticsusceptible A. baumannii and P. aeruginosa were found to be 100% for chlorhexidine, OPA and PA, 98% for sodium hypochlorite, and 94% for peracetic acid/ hydrogen peroxide. Considering the relationship between the biofilm production and biocidal activity, 22% of biofilm-producing strains of A. baumannii were found to be resistant to any all disinfectants-antiseptics tested, while this rate was 2% in the P. aeruginosa strains. Disinfectant resistance rates were 2% and 6% for biofilm-negative A. baumannii and, P. aeruginosa strains, respectively. Biofilm production and disinfectant resistance were found to be significantly associated with A. baumannii, compared to P. aeruginosa (p<0.05). Conclusion: Tested antiseptics-disinfectants showed 90% efficacy to Gram-negative non-fermentative bacteria isolated in the intensive care unit in our hospital. It would be reasonable to perform further efficacy tests for commonly used antiseptics and disinfectants on a regular basis.

7 citations


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

  • ...aeruginosa PAO-JP3 strain as negative control.[10-12] Clinical strains incubated overnight in the MacConkey agar (BioMerieux, France) and a colony of control strains were inoculated into tubes containing a 5-ml fresh Luria Bertani (LB, Sigma, USA) liquid medium at 37°C for 24 hours to calculate the biofilm production....

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  • ...A mean + two standard deviations were considered as the cut-off value.([11]) Sodium hypochloride (1%), OPA (Orto-Phthalaldehyde) (0....

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Journal ArticleDOI
TL;DR: This study sought to determine if LGS can enhance the antimicrobial activity and biofilm disruption capability of topical antibiotic therapy.
Abstract: Background and objectives Bacterial biofilm formation within chronic wound beds, which provides an effective barrier against antibiotics, is a known cause of recalcitrant infections and a significant healthcare burden, often requiring repeated surgical debridements. Laser-generated shockwaves (LGS) is a novel, minimally invasive, and nonthermal modality for biofilm mechanical debridement which utilizes compressive stress waves, generated by photonic absorption in thin titanium films to mechanically disrupt the biofilm. Prior studies have demonstrated LGS monotherapy to be selectively efficacious for biofilm disruption and safe for host tissues. In this study, we sought to determine if LGS can enhance the antimicrobial activity and biofilm disruption capability of topical antibiotic therapy. Study design/materials and methods Staphylococcus epidermidis biofilms grown in vitro on glass were treated with topical gentamicin (31, 62, and 124 μg/ml) with and without LGS (n = 3-11/treatment group). Mechanical shockwaves were generated with a 1,064 nm Nd:YAG laser (laser fluence 110.14 mJ/mm2 , pulse duration 5 ns, spot size 3 mm). Following a 24-hour incubation period, bacterial viability was assessed by determining the number of colony-forming units (CFU) via the Miles and Misra method. Residual biofilm bioburden was analyzed using the crystal violet biofilm assay. Results With gentamicin monotherapy, CFU density (CFU/mm2 ) at 31, 62, and 124 μg/ml were (282 ± 84) × 104 , (185 ± 34) × 104 , and (113 ± 9) × 104 , respectively. With LGS and gentamicin therapy, CFU density decreased to (170 ± 44) × 104 , (89 ± 24) × 104 , and (43 ± 3) × 104 , respectively (P = 0.1704, 0.0302, and 0.0004 when compared with gentamicin alone). Biofilm burden as measured by the assay in the gentamicin 31, 62, and 124 μg/ml groups was reduced by 80%, 95%, and 98% when LGS was added (P = 0.0102, >0.0001, and 0.0001 for all groups when compared with gentamicin alone). Furthermore, samples treated with LGS saw an increase in susceptibility to gentamicin, in terms of reduced biofilm bioburden and CFU densities. Conclusion LGS enhances the efficacy of topical antibiotics in an in vitro model. This has significant implications for clinical applications in the management of chronic soft tissue infections and recalcitrant chronic rhinosinusitis. Lasers Surg. Med. 49:539-547, 2017. © 2017 Wiley Periodicals, Inc.

7 citations

Book ChapterDOI
04 Jun 2015
TL;DR: The resistance phenomenon also applies to fungal biofilms and has been documented with Candida albicans as discussed by the authors, and multiple explanations have been advanced for the high resistance of bio-films to antimicrobials.
Abstract: Biofilms constitute a protected mode of growth that allows survival in a hostile environment leading to their inherent resistance to antimicrobial agents and host immune responses, which are at the root of many persistent and chronic bacterial infections. In both the healthcare and the food industry, the number of bacterial infections and noninfectious complications are rising due to the bacterial biofilms formation and the subsequent failure of many medical devices. Microscopy, especially light microscopy , and transmission and scanning electron microscopy played a pivotal role in the realization of the biofilm concept. The process of biofilm formation starts with colonization by microorganisms. The arrangement of bacterial biofilm can be extremely variable and complex. The resistance phenomenon also applies to fungal biofilms and has been documented with Candida albicans. Multiple explanations have been advanced for the high resistance of biofilms to antimicrobials.

7 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)....

    [...]