In-Vitro Model of Scardovia wiggsiae Biofilm Formation and Effect of Nicotine

Abstract Recently, Scardovia wiggsiae has been reported to be strongly associated with caries formation. This study aimed to establish an in vitro model of S. wiggsiae biofilm and to investigate the effect of nicotine on S. wiggsiae colony-forming units (CFUs) growth. S. wiggsiae biofilm was grown overnight using brain-heart infusion (BHI) broth supplemented with 5 g of yeast extract/L (BHI-YE). The overnight culture was used as an inoculum to grow S. wiggsiae biofilm on standardized enamel and dentin samples. Samples were incubated with different nicotine concentrations (0, 0.5, 1, 2, 4, 8, 16 and 32 mg/mL) for 3 days. The dissociated biofilms were diluted, spiral plated on blood agar plates, and incubated for 24 h. CFUs/mL were quantified using an automated colony counter. A two-way ANOVA was used to compare the effect of different nicotine concentrations on S. wiggsiae CFUs. This study demonstrated that S. wiggsiae biofilm could be initiated and formed in vitro. Increased CFUs was observed through 0.5-4 mg/mL and 0.5-8 mg/mL of nicotine using enamel and dentin substrates, respectively. 16 and 32 mg/mL of nicotine were determined as the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC), respectively. S. wiggsiae formed greater biofilm on enamel than dentin specimens in response to the nicotine stimulus. This study demonstrated the negative effect of smoking on increasing S. wiggsiae biofilm. Establishing S. wiggsiae biofilm in vitro may allow researchers in the future to have a better understanding of caries pathogenesis and bacterial interaction.

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Epidural Catheter Reconnection

Anesthesiology ◽

10.1097/00000542-199610000-00025 ◽

1996 ◽

Vol 85 (4) ◽

pp. 883-888 ◽

Cited By ~ 39

Author(s):

Paul B. Langevin ◽

Nikolaus Gravenstein ◽

Sharon O. Langevin ◽

Paul A. Gulig

Keyword(s):

Epidural Catheter ◽

In Vitro Model ◽

Brain Heart Infusion ◽

Vertical Position ◽

Fluid Displacement ◽

Colony Forming Units ◽

Vitro Model ◽

Epidural Catheters ◽

Preservative Free

Background An in vitro model of epidural catheter contamination was used to determine if disconnected catheters can be safely reconnected. Methods Epidural catheters were filled with brain-heart infusion (BHI) broth or preservative-free saline containing 5 micrograms/ml fentanyl. Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus (1.10(5) colony-forming units) was injected into the initial 1.1 +/- 0.24 inch (2.75 +/- 0.60 cm) of the catheters. To study the effect of bacteria settling in a vertically oriented catheter on the advancement of bacteria along the catheter, bacteria were incubated with catheters in the vertical and the horizontal positions. To determine if bacteria are swept further into a catheter when fluid in it is displaced, catheters were inclined 30 degrees and the fluid in them was allowed to drain from the distal end to various extents. Bacteria were incubated with the catheter held horizontally. After incubation, the catheters were serially sectioned, and the resulting segments were eluted with buffered saline-containing gelatin (BSG), which was collected on BHI agar plates for colony counts. This determined if a segment of the catheter remained internally sterile distal to the point of disconnection. Effectiveness of decontaminating the exterior of the catheter was also tested as follows: Catheters (n = 10) were first immersed in BSG containing 1.10(5) S, aureus, immediately immersed in betadine for 2 min, exposed to air for 3 min, cut with a sterile instrument, and reconnected to a sterile screw cap catheter connector. Reconnected catheters were perfused with 10 ml BSG for 1 hr. Collected perfusate (100 microliters) was removed for direct colony count; the remaining perfusate was mixed with an equal volume of BHI and incubated overnight. A 100 microliters aliquot of BHI-BSG mixture was sampled the next day. No bacteria were cultured from either the perfusate or BHI-BSG mixture. Results Eight hours after contamination, as long as the fluid in the catheter was static, no bacteria were detected more than 13 inches (32.5 cm) from the contaminated end of catheters filled with BHI and no more than 8 inches (20 cm) from the end of those filled with fentanyl solution. This finding was not affected by incubation of the catheter in the vertical position. Fluid displacement less than 8 inches (20 cm) had no effect on dissemination, but when fluid was displaced 13 inches (32.5 cm), bacteria were found at the end of the catheter, 35 inches (87.5 cm) away. No bacteria were recovered from the perfusate of reconnected catheters after the catheters were cleaned with betadine and cut with a sterile instrument. Conclusions There may be an area distal to the disconnected end of an epidural catheter where its interior remains sterile for at least 8 hr. Such an area exists only when the fluid in the catheter remains static. Furthermore, the exterior of the catheter can be adequately cleaned to prevent bacteria from entering the catheter when reconnected at that point.

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Highly variable effect of sonication to dislodge biofilm-embedded Staphylococcus epidermidis directly quantified by epifluorescence microscopy: an in vitro model study

Journal of Orthopaedic Surgery and Research ◽

10.1186/s13018-020-02052-3 ◽

2020 ◽

Vol 15 (1) ◽

Author(s):

Erik T. Sandbakken ◽

Eivind Witsø ◽

Bjørnar Sporsheim ◽

Kjartan W. Egeberg ◽

Olav A. Foss ◽

...

Keyword(s):

Staphylococcus Epidermidis ◽

Biofilm Formation ◽

In Vitro Model ◽

Laser Scanning Microscopy ◽

Epifluorescence Microscopy ◽

Variable Effect ◽

Prosthetic Joint Infections ◽

Before And After ◽

Vitro Model

Abstract Background In cases of prosthetic joint infections, culture of sonication fluid can supplement culture of harvested tissue samples for correct microbial diagnosis. However, discrepant results regarding the increased sensitivity of sonication have been reported in several studies. To what degree bacteria embedded in biofilm are dislodged during the sonication process has to our knowledge not been fully elucidated. In the present in vitro study, we have evaluated the effect of sonication as a method to dislodge biofilm by quantitative microscopy. Methods We used a standard biofilm method to cover small steel plates with biofilm forming Staphylococcus epidermidis ATCC 35984 and carried out the sonication procedure according to clinical practice. By comparing area covered with biofilm before and after sonication with epifluorescence microscopy, the effect of sonication on biofilm removal was quantified. Two series of experiments were made, one with 24-h biofilm formation and another with 72-h biofilm formation. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were used to confirm whether bacteria were present after sonication. In addition, quantitative bacteriology of sonication fluid was performed. Results Epifluorescence microscopy enabled visualization of biofilm before and after sonication. CLSM and SEM confirmed coccoid cells on the surface after sonication. Biofilm was dislodged in a highly variable manner. Conclusion There is an unexpected high variation seen in the ability of sonication to dislodge biofilm-embedded S. epidermidis in this in vitro model.

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Impact of sarA on Antibiotic Susceptibility of Staphylococcus aureus in a Catheter-Associated In Vitro Model of Biofilm Formation

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01432-08 ◽

2009 ◽

Vol 53 (6) ◽

pp. 2475-2482 ◽

Cited By ~ 33

Author(s):

Elizabeth C. Weiss ◽

Horace J. Spencer ◽

Sonja J. Daily ◽

Brian D. Weiss ◽

Mark S. Smeltzer

Keyword(s):

Staphylococcus Aureus ◽

Clinical Isolate ◽

Antimicrobial Susceptibility ◽

Antibiotic Susceptibility ◽

Biofilm Formation ◽

In Vitro Model ◽

Susceptibility Testing ◽

Specific Context ◽

Vitro Model

ABSTRACT Mutation of the staphylococcal accessory regulator (sarA) in Staphylococcus aureus limits but does not abolish the capacity of the organism to form a biofilm. As a first step toward determining whether this limitation is therapeutically relevant, we carried out in vitro studies comparing the relative susceptibility of an S. aureus clinical isolate (UAMS-1) and its isogenic sarA mutant (UAMS-929) in the specific context of a catheter-associated biofilm. The antibiotics tested were daptomycin, linezolid, and vancomycin, all of which were evaluated by using concentrations based on the MIC defined as the breakpoint for a susceptible strain of S. aureus (≤1.0, ≤2.0, and ≤4.0 μg/ml for daptomycin, vancomycin, and linezolid, respectively). Mutation of sarA had no significant impact on the MIC of UAMS-1 for any of the targeted antibiotics, as defined by Etest antimicrobial susceptibility testing. However, mutation of sarA did result in a significant increase in antimicrobial susceptibility to all targeted antibiotics when they were tested in the specific context of a biofilm. Additionally, whether susceptibility was assessed by using UAMS-1 or its sarA mutant, daptomycin was found to be more effective against established S. aureus biofilms than either linezolid or vancomycin.

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Microbial Adhesion on Different Bracket Types in vitro

The Angle Orthodontist ◽

10.2319/092908-507.1 ◽

2009 ◽

Vol 79 (5) ◽

pp. 915-921 ◽

Cited By ~ 16

Author(s):

Janvan Gastel ◽

Marc Quirynen ◽

Wim Teughels ◽

Martine Pauwels ◽

Wim Coucke ◽

...

Keyword(s):

Biofilm Formation ◽

Brain Heart Infusion ◽

In Vitro Study ◽

Microbial Adhesion ◽

Type D ◽

High Adhesion ◽

Colony Forming Units ◽

Orthodontic Patients ◽

Pigmented Bacteria

Abstract Objective: To test the hypothesis that there are differences in total bacterial counts and capacity for biofilm formation between seven different bracket types. Material and Methods: By means of an in vitro experiment, seven commercially available bracket systems (Damon [A], Clarity [B], Mystique [C], Speed [D], Victory MBT [E], Micro-loc [F], and Generus [G]) were compared. A total of 25 premolar brackets of each bracket system were incubated in brain heart infusion medium containing the saliva and bacteria of two orthodontic patients. After 72 hours, the amounts of aerobe and anaerobe bacteria were determined by counting the colony-forming units (CFU). The CFU ratio (aerobe/anaerobe) also was calculated, and the black pigmented bacteria were analyzed. Results: Significant differences between the different bracket types in terms of biofilm formation were found. Bracket types can be arbitrarily divided into low, intermediate, and high plaque-retaining brackets. The group with low adhesion consists of bracket types E, F, and G; the group with high adhesion of bracket types A, B, and C; and type D exhibits intermediate adhesion. The group with high microbial adhesion (A, B, and C) did present significantly lower CFU ratios (aerobe/anaerobe) than were exhibited by the other bracket systems (P < .05). Conclusion: The hypothesis is accepted. Orthodontic brackets serve as different loci for biofilm formation; in this in vitro study, significant differences were noted between the different types of brackets.

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Biofilm Formation and Effect of Caspofungin on Biofilm Structure of Candida Species Bloodstream Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00316-09 ◽

2009 ◽

Vol 53 (10) ◽

pp. 4377-4384 ◽

Cited By ~ 58

Author(s):

J. A. G. Ferreira ◽

J. H. Carr ◽

C. E. F. Starling ◽

M. A. de Resende ◽

R. M. Donlan

Keyword(s):

Biofilm Formation ◽

In Vitro Model ◽

Drug Exposure ◽

Sem Analysis ◽

Paradoxical Growth ◽

Vitro Model ◽

Species Specific ◽

Biofilm Structure ◽

Lipid Formulations

ABSTRACT Candida biofilms are microbial communities, embedded in a polymeric matrix, growing attached to a surface, and are highly recalcitrant to antimicrobial therapy. These biofilms exhibit enhanced resistance against most antifungal agents except echinocandins and lipid formulations of amphotericin B. In this study, biofilm formation by different Candida species, particularly Candida albicans, C. tropicalis, and C. parapsilosis, was evaluated, and the effect of caspofungin (CAS) was assessed using a clinically relevant in vitro model system. CAS displayed in vitro activity against C. albicans and C. tropicalis cells within biofilms. Biofilm formation was evaluated after 48 h of antifungal drug exposure, and the effects of CAS on preformed Candida species biofilms were visualized using scanning electron microscopy (SEM). Several species-specific differences in the cellular morphologies associated with biofilms were observed. Our results confirmed the presence of paradoxical growth (PG) in C. albicans and C. tropicalis biofilms in the presence of high CAS concentrations. These findings were also confirmed by SEM analysis and were associated with the metabolic activity obtained by biofilm susceptibility testing. Importantly, these results suggest that the presence of atypical, enlarged, conical cells could be associated with PG and with tolerant cells in Candida species biofilm populations. The clinical implications of these findings are still unknown.

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Impact of biofilm formation and azoles' susceptibility in Scedosporium/Lomentospora species using an in vitro model that mimics the cystic fibrosis patients' airway environment

Journal of Cystic Fibrosis ◽

10.1016/j.jcf.2020.12.001 ◽

2020 ◽

Author(s):

Thaís P. Mello ◽

Michaela Lackner ◽

Marta H. Branquinha ◽

André L.S. Santos

Keyword(s):

Cystic Fibrosis ◽

Biofilm Formation ◽

In Vitro Model ◽

Vitro Model

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Fusarium and Candida albicans Biofilms on Soft Contact Lenses: Model Development, Influence of Lens Type, and Susceptibility to Lens Care Solutions

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00387-07 ◽

2007 ◽

Vol 52 (1) ◽

pp. 171-182 ◽

Cited By ~ 121

Author(s):

Yoshifumi Imamura ◽

Jyotsna Chandra ◽

Pranab K. Mukherjee ◽

Ali Abdul Lattif ◽

Loretta B. Szczotka-Flynn ◽

...

Keyword(s):

Candida Albicans ◽

Contact Lens ◽

Biofilm Formation ◽

In Vitro Model ◽

Contact Lenses ◽

Fungal Keratitis ◽

Soft Contact ◽

Soft Contact Lenses ◽

Vitro Model

ABSTRACT Fungal keratitis is commonly caused by Fusarium species and less commonly by Candida species. Recent outbreaks of Fusarium keratitis were associated with contact lens wear and with ReNu with MoistureLoc contact lens care solution, and biofilm formation on contact lens/lens cases was proposed to play a role in this outbreak. However, no in vitro model for contact lens-associated fungal biofilm has been developed. In this study, we developed and characterized in vitro models of biofilm formation on various soft contact lenses using three species of Fusarium and Candida albicans. The contact lenses tested were etafilcon A, galyfilcon A, lotrafilcon A, balafilcon A, alphafilcon A, and polymacon. Our results showed that clinical isolates of Fusarium and C. albicans formed biofilms on all types of lenses tested and that the biofilm architecture varied with the lens type. Moreover, differences in hyphal content and architecture were found between the biofilms formed by these fungi. We also found that two recently isolated keratitis-associated fusaria formed robust biofilms, while the reference ATCC 36031 strain (recommended by the International Organization for Standardization guidelines for testing of disinfectants) failed to form biofilm. Furthermore, using the developed in vitro biofilm model, we showed that phylogenetically diverse planktonic fusaria and Candida were susceptible to MoistureLoc and MultiPlus. However, Fusarium biofilms exhibited reduced susceptibility against these solutions in a species- and time-dependent manner. This in vitro model should provide a better understanding of the biology and pathogenesis of lens-related fungal keratitis.

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Using Bacteriophages To Reduce Formation of Catheter-Associated Biofilms by Staphylococcus epidermidis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.4.1268-1275.2006 ◽

2006 ◽

Vol 50 (4) ◽

pp. 1268-1275 ◽

Cited By ~ 157

Author(s):

John J. Curtin ◽

Rodney M. Donlan

Keyword(s):

Staphylococcus Epidermidis ◽

Biofilm Formation ◽

In Vitro Model ◽

Flow Reactor ◽

Divalent Cations ◽

Exposure Period ◽

Model System ◽

Indwelling Catheters ◽

Vitro Model

ABSTRACT Use of indwelling catheters is often compromised as a result of biofilm formation. This study investigated if hydrogel-coated catheters pretreated with a coagulase-negative bacteriophage would reduce Staphylococcus epidermidis biofilm formation. Biofilms were developed on hydrogel-coated silicone catheters installed in a modified drip flow reactor. Catheter segments were pretreated with the lytic S. epidermidis bacteriophage 456 by exposing the catheter lumen to a 10-log-PFU/ml culture of the bacteriophage for 1 h at 37°C prior to biofilm formation. The untreated mean biofilm cell count was 7.01 ± 0.47 log CFU/cm2 of catheter. Bacteriophage treatment with and without supplemental divalent cations resulted in log-CFU/cm2 reductions of 4.47 (P < 0.0001) and 2.34 (P = 0.001), respectively. Divalent cation supplementation without bacteriophage treatment provided a 0.67-log-CFU/cm2 reduction (P = 0.053). Treatment of hydrogel-coated silicone catheters with an S. epidermidis bacteriophage in an in vitro model system significantly reduced viable biofilm formation by S. epidermidis over a 24-h exposure period, suggesting the potential of bacteriophage for mitigating biofilm formation on indwelling catheters and reducing the incidence of catheter-related infections.

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