Calgary Biofilm Device Research Articles

Enhanced enamel benefits from a novel fluoride toothpaste

Objectives: Through the understanding of tooth enamel science and insights into the beneficial role calcium can play in the caries process, a novel fluoride toothpaste has been developed containing micro-calcium. This paper describes a series of in vitro studies to assess delivery of the micro-calcium to a plaque biofilm, delivery of radiolabelled micro-calcium to subsurface enamel lesions and the rehardening of acid softened enamel by this novel toothpaste. Two clinical studies evaluated the delivery of calcium to the mouth. Methods: Uptake of micro-calcium to a plaque biofilm was assessed using a Calgary Biofilm Device and measuring the calcium levels delivered to the biofilm from the micro-calcium containing toothpaste, a calcium carbonate toothpaste, a silica toothpaste or water controls. Sound and subsurface enamel lesions were treated with 45 Ca labelled micro-calcium toothpaste in an in vitro pH cycling study and the uptake of labelled calcium determined. Acid softened enamel specimens were treated with either the micro-calcium containing toothpaste, a calcium carbonate toothpaste or a non-fluoride silica toothpaste in an in vitro remineralisation protocol and the changes in surface microhardness measured. Calcium delivery in vivo was determined in two double-blind, randomised cross-over studies. Subjects brushed their teeth for one minute with either the micro-calcium containing toothpaste or a silica toothpaste. Immediately after brushing and at fixed time intervals up to one hour, unstimulated saliva samples were taken and the total calcium concentration determined. Results: Significantly (p<0.05) more calcium was delivered to a plaque biofilm from the micro-calcium containing toothpaste than the controls. The radiolabelled micro-calcium study demonstrated the uptake of calcium to subsurface enamel lesions. In the remineralisation study, acid softened enamel became significantly harder (p<0.05) following treatment with the micro-calcium containing toothpaste than compared to the control toothpastes. It was shown in the two clinical studies that more calcium was delivered to the mouth following the use of the micro-calcium containing toothpaste than compared to the silica toothpaste. The product differences were in excess of 50% and were of statistical significance (p<0.001). Conclusions: The studies show that the new toothpaste containing micro-calcium delivered elevated levels of calcium to the mouth, promoted enhanced remineralisation of demineralised enamel lesions and thus can help repair early signs of tooth decay.

Influence of biofilm growth age, media, antibiotic concentration and exposure time on Staphylococcus aureus and Pseudomonas aeruginosa biofilm removal in vitro

BackgroundBiofilm is known to be tolerant towards antibiotics and difficult to eradicate. Numerous studies have reported minimum biofilm eradication concentration (MBEC) values of antibiotics for many known biofilm pathogens. However, the experimental parameters applied in these studies differ considerably, and often the rationale behind the experimental design are not well described. This makes it difficult to compare the findings. To demonstrate the importance of experimental parameters, we investigated the influence of biofilm growth age, antibiotic concentration and treatment duration, and growth media on biofilm eradication. Additionally, OSTEOmycin™, a clinically used antibiotic containing allograft bone product, was tested for antibiofilm efficacy.ResultsThe commonly used Calgary biofilm device was used to grow 24 h and 72 h biofilms of Staphylococcus aureus and Pseudomonas aeruginosa, which were treated with time-dependent vancomycin (up to 3000 mg L− 1) and concentration-dependent tobramycin (up to 80 mg L− 1), respectively. Two common bacteriological growth media, tryptic soy broth (TSB) and cation-adjusted Mueller Hinton broth (CaMHB), were tested. We found for both species that biofilms were more difficult to kill in TSB than in CaMHB. Furthermore, young biofilms (24 h) were easier to eradicate than old biofilms (72 h). In agreement with vancomycin being time-dependent, extension of the vancomycin exposure increased killing of S. aureus biofilms. Tobramycin treatment of 24 h P. aeruginosa biofilms was found concentration-dependent and time-independent, however, increasing killing was indicated for 72 h P. aeruginosa biofilms. Treatment with tobramycin containing OSTEOmycin T™ removed 72 h and 168 h P. aeruginosa biofilms after 1 day treatment, while few 72 h S. aureus biofilms survived after 2 days treatment with vancomycin containing OSTEOmycin V™.ConclusionsThis study demonstrated biofilm removal efficacy was influenced by media, biofilm age and antibiotic concentration and treatment duration. It is therefore necessary to taking these parameters into consideration when designing experiments. The results of OSTEOmycin™ products indicated that simple in vitro biofilm test could be used for initial screening of antibiofilm products. For clinical application, a more clinically relevant biofilm model for the specific biofilm infection in question should be developed to guide the amount of antibiotics used for local antibiofilm treatment.

Mucosal Thrombin Alters Gut Microbiota Biofilms Structure And Promote Dispersion Of Bacteria With Aggressive Behavior

Alterations of gut microbiota have been implicated in a broad variety of intestinal diseases. The mechanisms whereby this may occur remains elusive. Gut mucosal microbiota is naturally organized as a polymicrobial biofilm, separated from the intestinal epithelium by a sterile mucus layer. We recently discovered that intestinal epithelium releases active thrombin, which plays a key role in segregation of intestinal biofilms from host tissue. Furthermore, we detected an upregulation of active thrombin in inflamed human patients and in models of colitis.ObjectivesOur study objective was to determine whether exposure to high thrombin, such as this occurring during inflammation, will alter commensal microbiota biofilms and promote the dispersion of bacteria predisposed to damage the intestinal epithelium.MethodsMicrobiota extracted from healthy human colon biopsies were seeded into the Calgary Biofilm Device and polystyrene coupons to develop, a multispecies anaerobic biofilm. Biofilms were exposed to various concentrations of thrombin (10 to 1000 Unit/ml). Dispersed bacteria released from thrombin‐treated biofilms were collected and their composition was assessed by 16S sequencing. These bacteria were apically exposed to human epithelial monolayers on transwells (Caco2 and HT29MTX). Adhesion (90 minutes), invasion (gentamicin assay, 3 hours) and translocation (4 hours) to basolateral side was quantified by plating on agar. Transwells were processed for fluorescent in situ hybridization for bacteria staining and phalloidin antibody for host cell cytoskeleton. Motility phenotype of biofilmdispersed bacteria was assessed on soft agarose gels (swarming and swimming). Mice (B6) were treated intracolonically with thrombin (5U per day for 10 days) or boiled thrombin (similar dose). Rats (Wistar) were treated with TNBS to induce colitis, and were treated for 3 days intracolonically with dabigatran (thrombin inhibitor, 1 μg/kg) or vehicle.ResultsBiofilm‐dispersed bacteria from thrombin‐treated biofilms attached more importantly to the epithelial monolayers compared to untreated biofilm. 3D reconstruction images confirmed such thrombin‐induced phenotype. Thrombin alters swarming and swimming motility in soft agarose gel. 16S analysis further precise the specific composition of biofilm‐dispersed bacteria after thrombin exposure. In mice, intrarectal administration of thrombin caused alterations of gut microbiota biofilms structure (16S sequencing) and organization ( in situ imaging of gut microbiota). During colitis in rats, local inhibition of thrombin activity prevented gut microbiota biofilms alterations associated with colitis (in situ imaging of gut microbiota and 16S analysis).ConclusionsThese data suggest that high concentration of thrombin released at gut mucosal surface during inflammation alters gut biofilm organization and modifies the phenotype of biofilm‐dispersed bacteria, which were able to invade and cross the epithelial barrier, thus increasing their likelihood to trigger inflammatory flares.

Modeling Normal and Dysbiotic Subgingival Microbiomes: Effect of Nutrients

Screening for microbiome modulators requires availability of a high-throughput in vitro model that replicates subgingival dysbiosis and normobiosis, with a tool to measure microbial dysbiosis. Here, we tested various formulations to grow health- and periodontitis-associated subgingival microbiomes in parallel, and we describe a new subgingival dysbiosis index. Subgingival plaque samples pooled from 5 healthy subjects and, separately, 5 subjects with periodontitis were used to inoculate a Calgary Biofilm Device containing saliva-conditioned, hydroxyapatite-coated pegs. Microbiomes were grown for 7 d on either nutrient-rich media—including a modification of SHI medium, brain-heart infusion (BHI) supplemented with hemin and vitamin K, and a blend of SHI and BHI, each at 3 sucrose concentrations (0%, 0.05% and 0.1%)—or nutrient-limited media (saliva with 5%, 10%, or 20% inactivated human serum). The microbiomes were assessed for biomass, viability, and 16S rRNA profiles. In addition to richness and diversity, a dysbiosis index was calculated as the ratio of the sum of relative abundances of disease-associated species to that of health-associated species. The supplemented BHI and blend of SHI and BHI resulted in the highest biomass, whereas saliva-serum maximized viability. Distinct groups of bacteria were enriched in the different media. Regardless of medium type, the periodontitis-derived microbiomes showed higher species richness and alpha diversity and clustered with their inoculum separate from the health-derived microbiomes. Microbiomes grown in saliva-serum showed the highest species richness and the highest similarity to the clinical inocula in both health and disease. However, inclusion of serum reduced alpha diversity and increased dysbiosis in healthy microbiomes in a dose-dependent manner, mainly due to overenrichment of Porphyromonas species. The modification of SHI stood second in terms of species richness and diversity but resulted in low biomass and viability and significantly worsened dysbiosis in the periodontitis-derived microbiomes. Overall, saliva with 5% human serum was optimal for replicating subgingival microbiomes from health and disease.

Relationship between antimicrobial activity and amphipathic structure of ginsenosides

Ginsenoside has a wide range of pharmacological activities, but little research has been conducted on its microbial pathogen inhibition and the relationship between its antimicrobial activity and saponin structure. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) of a series of ginsenosides were used to investigate the antimicrobial activity of the ginsenosides. And the value of hydrophile–lipophile balance (HLB) was introduced as physicochemical factors to clarify the difference in bacteriostatic activity of saponins. The effects of active ginsenoside Rh2 on the Fusobacterium nucleatum and Bacillus cereus biofilm system were observed by calgary biofilm device (CBD) and fluorescence confocal microscopy (CLSM). The morphological characteristics were examined by using transmission electron microscope (TEM) to explore the cell damage. The antimicrobial effects were in the following order from high to low: saponins containing one glycosyl group > aglycones > two ligands > more than two ligands. Ginsenoside Rh2 and RK3 (hydrophile lipophilic balance value were both 1.15) showed high activity against the tested strains. Correlation analysis shows that HLB is associated with antimicrobial activity (p < 0.01). The antimicrobial activity increased with the HLB value of ginsenosides determined by changes in the length, the number, and the composition of sugar side chains. Specific polar ginsenosides cause bacterial death by destroying the membrane system. Based on their good broad-spectrum antibacterial activity, less polar ginsenosides have potential as bacteriostatic agents.

Inhibition of Mycobacterium abscessus, M. chelonae, and M. fortuitum biofilms by Methylobacterium sp.

Methylobacterium sp. is isolated from water distribution systems and has been linked in the biofilms of the systems with a lower presence of Mycobacterium avium. In this study we aimed to determine the in vitro activity of Methylobacterium sp. in the development of rapidly growing mycobacteria (RGM) biofilms. Methylobacterium sp. CECT 7805 was added as a suspension of living bacteria (LB), an autoclaved suspension (AS), and an extract obtained after sonication (ES) at different times (24, 48, and 72 h), to preformed biofilms of Mycobacterium abscessus DSM 44196, Mycobacterium chelonae ATCC 19235, and Mycobacterium fortuitum ATCC 6841, using a 96 h control of each species. The biofilms were analyzed by confocal laser scanning microscopy and by the Calgary biofilm device using the plates MBECTM Biofilm Inoculator. A statistically significant reduction in the thickness and covered surface was observed in all mycobacterial biofilms with all forms of Methylobacterium sp. A statistically significant increase in the autofluorescence was observed in M. abscessus biofilms but not in other biofilms. The increased percentage of dead mycobacteria was statistically significant in all cases. The reduced log CFU (colony-forming units)/peg recount was statistically significant in M. chelonae biofilms after treatment with AS and ES, but in M. fortuitum biofilms the recount decreased only with AS. M. abscessus biofilms were always significantly reduced with AS at 72 h and with ES. Methylobacterium sp. could inhibit RGM biofilm formation. Living cells of Methylobacterium sp. were not necessary to inhibit the growth of a preformed biofilm. M. chelonae biofilms were the most greatly reduced.

Synergism of cationic antimicrobial peptide WLBU2 with antibacterial agents against biofilms of multi-drug resistant Acinetobacter baumannii and Klebsiella pneumoniae.

PurposeThe activity of the cationic antimicrobial peptide WLBU2 was evaluated against planktonic cells and biofilms of multi-drug resistant (MDR) Acinetobacter baumannii and Klebsiella pneumoniae, alone and in combination with classical antimicrobial agents.MethodsControl American Type Culture Collection (ATCC) strains and MDR clinical isolates of A. baumannii and K. pneumoniae were utilized. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of WLBU2 alone and in combination with antimicrobials were determined by classical methods. The Calgary biofilm device was used to determine the minimum biofilm eradication concentration (MBEC). The MTT assay was used to determine the cytotoxicity of agents on eukaryotic cells. The electrophoretic mobility shift assay was used to evaluate the ability of WLBU2 to bind bacterial DNA.ResultsThe WLBU2 MIC and MBC values were identical indicating bactericidal activity. The MIC/MBC values ranged from 1.5625 to 12.5 µM. At these concentrations, Vero cells and human skin fibroblasts were viable. The MBEC of WLBU2 ranged from 25 to 200 µM. A significant loss of eukaryotic cell viability was observed at the MBEC range. The combination of sub-inhibitory concentrations of WLBU2 with amoxicillin-clavulanate or ciprofloxacin for K. pneumoniae, and with tobramycin or imipenem for A. baumannii, demonstrated synergism, leading to a significant decrease in MIC and MBEC values for some isolates and ATCC strains. However, all combinations were associated with considerable loss in eukaryotic cells’ viability. WLBU2 did not demonstrate the ability to bind bacterial plasmid DNA.ConclusionWLBU2 in combination with antimicrobials holds promise in eradication of MDR pathogens.

Formation of Pseudomonas aeruginosa inhibition zone during tobramycin disk diffusion is due to transition from planktonic to biofilm mode of growth

Pseudomonas aeruginosa PAO1 (tobramycin MIC = 0.064 µg/mL) was used to perform agar diffusion tests employing tobramycin-containing tablets. Bacterial growth and formation of inhibition zones were studied by stereomicroscopy and by blotting with microscope slides and staining with methylene blue, Alcian blue and a fluorescent lectin for the P. aeruginosa PSL, which was studied by confocal laser scanning microscopy. Diffusion of tobramycin from the deposit was modelled using a 3D geometric version of Fick's second law of diffusion. The time-dependent gradual increase in the minimum biofilm eradication concentration (MBEC) was studied using a Calgary Biofilm Device. The early inhibition zone was visible after 5 h of incubation. The corresponding calculated tobramycin concentration at the border was 1.9 µg/mL, which increased to 3.2 µg/mL and 6.3 µg/mL after 7 h and 24 h, respectively. The inhibition zone increased to the stable final zone after 7 h of incubation. Bacterial growth and small aggregate formation (young biofilms) took place inside the inhibition zone until the small aggregates contained less than ca. 64 cells and production of polysaccharide matrix including PSL had begun; thereafter, the small bacterial aggregates were killed by tobramycin. Bacteria at the border of the stable inhibition zone and beyond continued to grow to a mature biofilm and produced large amount of polysaccharide-containing matrix. Formation of the inhibition zone during agar diffusion antimicrobial susceptibility testing is due to a switch from a planktonic to biofilm mode of growth and gives clinically important information about the increased antimicrobial tolerance of biofilms.

Potential for repurposing the personal care product preservatives bronopol and bronidox as broad-spectrum antibiofilm agents for topical application.

Bacterial biofilms represent a major impediment to healing in chronic wounds and are largely refractory to the antibacterial agents currently used in wound management. From a repurposing screen of compounds considered safe for topical application in humans, we report the identification of the personal care product preservatives bronopol and bronidox as broad-spectrum antibiofilm agents and potential candidates for reducing biofilm burden in chronic wounds. Antibiofilm activity was assessed by viable counting against single-species biofilms of Staphylococcus aureus and Pseudomonas aeruginosa in the Calgary Biofilm Device, and against mixed-species biofilms of the two organisms growing on nitrocellulose discs. Bronopol and bronidox exhibited broad-spectrum antibiofilm activity that encompassed the two major wound pathogens, S. aureus and P. aeruginosa. When impregnated into gauze dressings at their existing maximum authorized concentrations for safe use and placed onto an established mixed-species biofilm, bronopol and bronidox completely eradicated P. aeruginosa and achieved an ∼5 log10 reduction in the S. aureus population. The antibiofilm action of bronopol and bronidox was attributed to their ability to kill slow- or non-growing bacteria found in biofilms, and both compounds exhibited synergistic antibiofilm effects in combination with established wound-treatment agents. Bronopol and bronidox kill bacteria regardless of growth state, a property that endows them with broad-spectrum antibiofilm activity. As this effect is observed at concentrations authorized for use on human skin, these compounds represent promising candidates for the treatment of chronic wounds.

Tomatidine and analog FC04\u2013100 possess bactericidal activities against Listeria, Bacillus and Staphylococcus spp

BackgroundTomatidine (TO) is a plant steroidal alkaloid that possesses an antibacterial activity against the small colony variants (SCVs) of Staphylococcus aureus. We report here the spectrum of activity of TO against other species of the Bacillales and the improved antibacterial activity of a chemically-modified TO derivative (FC04–100) against Listeria monocytogenes and antibiotic multi-resistant S. aureus (MRSA), two notoriously difficult-to-kill microorganisms.MethodsBacillus and Listeria SCVs were isolated using a gentamicin selection pressure. Minimal inhibitory concentrations (MICs) of TO and FC04–100 were determined by a broth microdilution technique. The bactericidal activity of TO and FC04–100 used alone or in combination with an aminoglycoside against planktonic bacteria was determined in broth or against bacteria embedded in pre-formed biofilms by using the Calgary Biofilm Device. Killing of intracellular SCVs was determined in a model with polarized pulmonary cells.ResultsTO showed a bactericidal activity against SCVs of Staphylococcus aureus, Bacillus cereus, B. subtilis and Listeria monocytogenes with MICs of 0.03–0.12 μg/mL. The combination of an aminoglycoside and TO generated an antibacterial synergy against their normal phenotype. In contrast to TO, which has no relevant activity by itself against Bacillales of the normal phenotype (MIC > 64 μg/mL), the TO analog FC04–100 showed a MIC of 8–32 μg/mL. Furthermore, FC04–100 showed a strong bactericidal activity against L. monocytogenes SCVs in kill kinetics experiments, while TO did not. The addition of FC04–100 (4 μg/mL) to a cefalexin:kanamycin (3:2) combination improved the activity of the combination by 32 fold against cefalexin and kanamycin-resistant MRSA strains. In combination with gentamicin, FC04–100 also exhibited a strong bactericidal activity against biofilm-embedded S. aureus. Also, FC04–100 and TO showed comparable intracellular killing of S. aureus SCVs.ConclusionsChemical modifications of TO allowed improvement of its antibacterial activity against prototypical S. aureus and of its bactericidal activity against L. monocytogenes. Antibacterial activities against such prominent pathogens could be useful to prevent Listeria contamination in the food chain or as treatment for MRSA infections.

Pharmacodynamics of amoxicillin against field isolates ofStreptococcus parauberisfrom olive flounder (Paralichthys olivaceus)

Olive flounder is the most important species for the Northeast Asian fish farming industry. However, this species is substantially affected by multiple infectious agents, including Streptococcus parauberis. Evaluation of antibiotics before their application is critical to treat infections and prevent drug resistance. Therefore, in this study, the pharmacodynamics of amoxicillin (AMX) and other antimicrobials against the planktonic- and biofilm-forming bacteria were assessed. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and Time–kill curve assay were analysed using micro-dilution method. The minimum biofilm eradicating concentration (MBEC) was determined using the Calgary Biofilm device. The effects of temperature, pH, hardness and salinity were detected for both planktonic- and biofilm-forming bacteria. The MIC of AMX ranged from 0.015 to 2 μg/ml, whereas that of cephalexin (CEP), enrofloxacin (ENR) and oxytetracycline (OTC) ranged from 0.125 to 256, 0.125 to >512 and 0.25 to >512 μg/ml respectively. No bacteria were resistant against AMX, while the percentage of resistance to CEP, OTC and ENR were 68.7%, 52.6% and 11.1% respectively. The IC50 of AMX, CEP, ENR and OTC was 0.03, 0.091, 0.015 and 0.213 μg/ml respectively. The MBEC of amoxicillin against S. parauberis ranged from 0.5 to 16 μg/ml. Higher rates of bacterial growth were obtained at 30°C, pH = 8 and salinity of 7.5–10 ppt. The hardness of the media suppressed the bacterial growth. In conclusion, AMX was found to be effective against both the planktonic and the biofilm forms of the prominent fish pathogen, S. parauberis.

Biofilm formation and antimicrobial susceptibility of staphylococci and enterococci from osteomyelitis associated with percutaneous orthopaedic implants.

Staphylococci and enterococci account for most deep infections associated with bone-anchored percutaneous implants for amputation treatment. Implant-associated infections are difficult to treat; therefore, it is important to investigate if these infections have a biofilm origin and to determine the biofilm antimicrobial susceptibility to improve treatment strategies. The aims were: (i) to test a novel combination of the Calgary biofilm device and a custom-made susceptibility MIC plate (Sensititre® ), (ii) to determine the biofilm formation and antimicrobial resistance in clinical isolates causing implant-associated osteomyelitis, and (iii) to describe the associated clinical outcome. Enterococci and staphylococci were characterized by microtitre plate assay, Congo Red Agar plate test, and PCR. Biofilm susceptibility to 10 antimicrobials and its relationship to treatment outcomes were determined. The majority of the strains produced biofilm in vitro showing inter- and intraspecies differences. Biofilms showed a significantly increased antimicrobial resistance compared with their planktonic counterparts. Slime-producing strains tolerated significantly higher antimicrobial concentrations compared with non-producers. All seven staphylococcal strains carried ica genes, but two did not produce slime. The degree of biofilm formation and up-regulated antibiotic resistance may translate into a variable risk of treatment failure. This new method set-up allows for the reproducible determination of minimum biofilm eradication concentration of antimicrobial agents, which may guide future antimicrobial treatment decisions in orthopaedic implant-associated infection. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2630-2640, 2017.

Tert-butyl benzoquinone: mechanism of biofilm eradication and potential for use as a topical antibiofilm agent.

ObjectivesTert-butyl benzoquinone (TBBQ) is the oxidation product of tert-butyl hydroquinone (TBHQ), an antimicrobial food additive with >40 years of safe use. TBBQ displays potent activity against Staphylococcus aureus biofilms in vitro. Here, we report on studies to further explore the action of TBBQ on staphylococcal biofilms, and provide a preliminary preclinical assessment of its potential for use as a topical treatment for staphylococcal infections involving a biofilm component.MethodsThe antibacterial properties of TBBQ were assessed against staphylococci growing in planktonic culture and as biofilms in the Calgary Biofilm Device. Established assays were employed to measure the effects of TBBQ on biofilm structure and bacterial membranes, and to assess resistance potential. A living-skin equivalent was used to evaluate the effects of TBBQ on human skin.ResultsTBBQ eradicated biofilms of S. aureus and other staphylococcal species at concentrations ≤64 mg/L. In contrast to other redox-active agents exhibiting activity against biofilms, TBBQ did not cause substantial destructuring of the biofilm matrix; instead, the antibiofilm activity of the compound was attributed to its ability to kill slow- and non-growing cells via membrane perturbation. TBBQ acted synergistically with gentamicin, did not damage a living-skin equivalent following topical application and exhibited low resistance potential.ConclusionsThe ability of TBBQ to eradicate biofilms appears to result from its ability to kill bacteria regardless of growth state. Preliminary evaluation suggests that TBBQ represents a promising candidate for development as a topical antibiofilm agent.

Genes involved in Listeria monocytogenes biofilm formation at a simulated food processing plant temperature of 15 °C

Listeria monocytogenes is a pathogenic foodborne bacterium whose persistence in food processing environments is in part attributed to its biofilm formation. Most biofilm studies have been carried out at 30–37°C rather than at temperatures found in the food processing plants (i.e., 10–20°C). The objective of the present study was to mine for novel genes that contribute to L. monocytogenes biofilm formation at 15°C using the random insertional mutagenesis approach. A library of 11,024 L. monocytogenes 568 (serotype 1/2a) Himar1 insertional mutants was created. Mutants with reduced or enhanced biofilm formation at 15°C were detected in microtiter plate assays with crystal violet and safranin staining. Fourteen mutants expressed enhanced biofilm phenotypes, and harbored transposon insertions in genes encoding cell wall biosynthesis, motility, metabolism, stress response, and cell surface associated proteins. Deficient mutants (n=5) contained interruptions in genes related to peptidoglycan, teichoic acid, or lipoproteins. Enhanced mutants produced significantly (p<0.05) higher cell densities in biofilm formed on stainless steel (SS) coupons at 15°C (48h) than deficient mutants, which were also more sensitive to benzalkonium chloride. All biofilm deficient mutants and four enhanced mutants in the microtiter plate assay (flaA, cheR, lmo2563 and lmo2488) formed no biofilm in a peg lid assay (Calgary biofilm device) while insertions in lmo1224 and lmo0543 led to excess biofilm in all assays. Two enhanced biofilm formers were more resistant to enzymatic removal with DNase, proteinase K or pectinase than the parent strain. Scanning electron microscopy of individual biofilms made by five mutants and the parent on SS surfaces showed formation of heterogeneous biofilm with dense zones by immotile mutants, while deficient mutants exhibited sparse growth. In conclusion, interruptions of 9 genes not previously linked to biofilm formation in L. monocytogenes (lmo2572, lmo2488 (uvrA), lmo1224, lmo0434 (inlB), lmo0263 (inlH), lmo0543, lmo0057 (EsaA), lmo2563, lmo0453), caused enhanced biofilm formation in the bacterium at 15°C. The remaining mutants harbored interruptions in 10 genetic loci previously associated with biofilm formation at higher temperatures, indicating some temperature driven differences in the formation of biofilm by L. monocytogenes.

Prospective study on the diagnostics and susceptibility testing of infections associated with osseointegrated percutaneous implants

Event Abstract Back to Event Prospective study on the diagnostics and susceptibility testing of infections associated with osseointegrated percutaneous implants Magdalena Zaborowska1, 2, Rickard Brånemark2, 3, 4, Joakim Strömberg3, Peter Thomsen1, 2 and Margarita Trobos1, 2 1 Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Department of Biomaterials, Sweden 2 University of Gothenburg, BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Sweden 3 Center for Advanced Reconstruction of Extremities, Sahlgrenska University Hospital, Department of Orthopaedics, Sweden 4 Department of Orthopaedics, University of California, International Center for Osseointegration Research Education and Surgery (iCORES), United States Introduction: Biomaterial-associated infections (BAI) are among the main reasons for implant failure, often leading to long-term antimicrobial treatment and implant removal. BAI are mainly caused by biofilm producing bacteria with increased antimicrobial resistance. Proper and rapid diagnosis is important for successful treatment. Sonication is an alternative method for the diagnosis of prosthetic infections with higher sensitivity than standard tissue cultures[1]. The aims of the study were to: i) identify and quantify bacteria causing osteomyelitis associated with osseointegrated percutaneous amputation prostheses by sonication of explanted implants; ii) characterise the isolated strains by their biofilm production abilities and antimicrobial susceptibility. In parallel, the diagnostic laboratory at the hospital performed standard culturing techniques of bone tissue and blood marrow samples. Materials and Methods: A bone-anchored part (fixture) of a prosthesis was extracted due to suspicion of infection according to clinical evaluation. The retrieved fixture was placed in sterile saline, sonicated at 47 KHz for 5 min and vortexed for 1 min. The implant was removed, the remaining solution was centrifuged, pellet was re-suspended in saline and quantitative cultures were made on general purpose agar (blood agar for aerobic and brucella agar for anaerobic cultures) and selective agar plates (staphylococci agar, enterococci agar, streptococci agar, chromogenic orientation agar and pseudomonas agar). Crystal violet (CV) staining and Congo Red (CR) agar plate test was performed to evaluate the biofilm production. Identification of the staphylococcal strains was performed using API Staph. The Calgary Biofilm Device and a custom-made antimicrobial susceptibility plate were used to determine minimum biofilm eradication concentrations (MBEC) and minimum inhibitory concentrations (MIC) to 10 antimicrobial agents. Results and Discussion: We report the first results of an ongoing prospective study where the method has been tested real-time. In the first patient, Enterococcus faecalis (5x104 CFU/implant) and Escherichia coli (102 CFU/implant) were detected after sonication. These strains were both biofilm-producing strains according to CV and CR tests. Biofilms showed increased tolerance to the antimicrobials compared with the standard planktonic MIC values (Table 1). Escherichia coli was the only species detected by standard tissue cultures; in contrast, by the use of sonication, Enterococcus faecalis was further identified indicating the possibility of a polymicrobial infection with potential implications for the choice of the optimal treatment strategy for the patient. Conclusions: An increased detection of the potential causative agents by sonication of retrieved implants may improve the current diagnostics of BAI. In addition, determination of MBEC for clinically relevant antimicrobial agents has the potential to optimize the treatment choices for patients. The BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, the Västra Götaland Region, the Swedish Research Council (K2015-52X-09495-28-4), an LUA/ALF Research Grant (ALFGBG-138721), the IngaBritt and Arne Lundberg Foundation, the Wilhelm and Martina Lundgren Foundation, the Handlanden Hjalmar Svensson Foundation, Doctor Felix Neubergh Foundation and the Area of Advance Materials of Chalmers and GU Biomaterials within the Strategic Research Area initiative launched by the Swedish Government

In vitro culture of previously uncultured oral bacterial phylotypes.

Around a third of oral bacteria cannot be grown using conventional bacteriological culture media. Community profiling targeting 16S rRNA and shotgun metagenomics methods have proved valuable in revealing the complexity of the oral bacterial community. Studies investigating the role of oral bacteria in health and disease require phenotypic characterizations that are possible only with live cultures. The aim of this study was to develop novel culture media and use an in vitro biofilm model to culture previously uncultured oral bacteria. Subgingival plaque samples collected from subjects with periodontitis were cultured on complex mucin-containing agar plates supplemented with proteose peptone (PPA), beef extract (BEA), or Gelysate (GA) as well as on fastidious anaerobe agar plus 5% horse blood (FAA). In vitro biofilms inoculated with the subgingival plaque samples and proteose peptone broth (PPB) as the growth medium were established using the Calgary biofilm device. Specific PCR primers were designed and validated for the previously uncultivated oral taxa Bacteroidetes bacteria HOT 365 and HOT 281, Lachnospiraceae bacteria HOT 100 and HOT 500, and Clostridiales bacterium HOT 093. All agar media were able to support the growth of 10 reference strains of oral bacteria. One previously uncultivated phylotype, Actinomyces sp. HOT 525, was cultivated on FAA. Of 93 previously uncultivated phylotypes found in the inocula, 26 were detected in in vitro-cultivated biofilms. Lachnospiraceae bacterium HOT 500 was successfully cultured from biofilm material harvested from PPA plates in coculture with Parvimonas micra or Veillonella dispar/parvula after colony hybridization-directed enrichment. The establishment of in vitro biofilms from oral inocula enables the cultivation of previously uncultured oral bacteria and provides source material for isolation in coculture.

Interspecies interactions result in enhanced biofilm formation by co-cultures of bacteria isolated from a food processing environment

Bacterial attachment and biofilm formation can lead to poor hygienic conditions in food processing environments. Furthermore, interactions between different bacteria may induce or promote biofilm formation. In this study, we isolated and identified a total of 687 bacterial strains from seven different locations in a meat processing environment and evaluated their biofilm formation capability. A diverse group of bacteria was isolated and most were classified as poor biofilm producers in a Calgary biofilm device assay. Isolates from two sampling sites, the wall and the meat chopper, were further examined for multispecies biofilm formation. Eight strains from each sampling site were chosen and all possible combinations of four member co-cultures were tested for enhanced biofilm formation at 15 °C and 24 °C. In approximately 20% of the multispecies consortia grown at 15 °C, the biofilm formation was enhanced when comparing to monospecies biofilms. Two specific isolates (one from each location) were found to be present in synergistic combinations with higher frequencies than the remaining isolates tested. This data provides insights into the ability of co-localized isolates to influence co-culture biofilm production with high relevance for food safety and food production facilities.

Effects of azithromycin, metronidazole, amoxicillin, and metronidazole plus amoxicillin on an in vitro polymicrobial subgingival biofilm model.

Chronic periodontitis is one of the most prevalent human diseases and is caused by dysbiosis of the subgingival microbiota. Treatment involves primarily mechanical disruption of subgingival biofilms and, in certain cases, adjunctive use of systemic antibiotic therapy. In vitro biofilm models have been developed to study antimicrobial agents targeting subgingival species. However, these models accommodate a limited number of taxa, lack reproducibility, and have low throughput. We aimed to develop an in vitro multispecies biofilm model that mimics subgingival plaque, to test antimicrobial agents. Biofilms were cultivated using the Calgary Biofilm Device and were exposed to amoxicillin (AMX), metronidazole (MTZ), azithromycin (AZM), and AMX-MTZ at four different concentrations for 12, 24, or 36 h. Chlorhexidine (CHX) (0.12%) was used as the positive control. The compositions of the biofilms were analyzed by checkerboard DNA-DNA hybridization, and the percent reduction in biofilm metabolic activity was determined using 2,3,5-triphenyltetrazolium chloride and spectrophotometry. Thirty-five of the 40 species used in the inoculum were consistently recovered from the resulting in vitro biofilms. After 36 h of exposure at the 1:27 dilution, AMX-MTZ reduced metabolic activity 11% less than CHX (q = 0.0207) but 54% more than AMX (q = 0.0031), 72% more than MTZ (q = 0.0031), and 67% more than AZM (q = 0.0008). Preliminary evidence of a synergistic interaction between AMX and MTZ was also observed. In summary, we developed reproducible biofilms with 35 subgingival bacterial species, and our results suggested that the combination of AMX and MTZ had greater antimicrobial effects on these in vitro multispecies biofilms than expected on the basis of the independent effects of the drugs.

Development and pyrosequencing analysis of an in-vitro oral biofilm model.

BackgroundDental caries and periodontal disease are the commonest bacterial diseases of man and can result in tooth loss. The principal method of prevention is the mechanical removal of dental plaque augmented by active agents incorporated into toothpastes and mouthrinses. In-vitro assays that include complex oral bacterial biofilms are required to accurately predict the efficacy of novel active agents in vivo. The aim of this study was to develop an oral biofilm model using the Calgary biofilm device (CBD) seeded with a natural saliva inoculum and analysed by next generation sequencing. The specific objectives were to determine the reproducibility and stability of the model by comparing the composition of the biofilms over time derived from (i) the same volunteers at different time points, and (ii) different panels of volunteers.ResultsPyrosequencing yielded 280,093 sequences with a mean length of 432 bases after filtering. A mean of 320 and 250 OTUs were detected in pooled saliva and biofilm samples, respectively. Principal coordinates analysis (PCoA) plots based on community membership and structure showed that replicate biofilm samples were highly similar and clustered together. In addition, there were no significant differences between biofilms derived from the same panel at different times using analysis of molecular variance (AMOVA). There were significant differences between biofilms from different panels (AMOVA, P < 0.002). PCoA revealed that there was a shift in biofilm composition between seven and 14 days (AMOVA, P < 0.001). Veillonella parvula, Veillonella atypica/dispar/parvula and Peptostreptococcus stomatis were the predominant OTUs detected in seven-day biofilms, whilst Prevotella oralis, V. parvula and Streptococcus constellatus were predominant in 14-day biofilms.ConclusionsDiverse oral biofilms were successfully grown and maintained using the CBD. Biofilms derived from the same panel of volunteers were highly reproducible. This model could be used to screen both antimicrobial-containing oral care products and also novel approaches aiming to modify plaque composition, such as pre- or probiotics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0364-1) contains supplementary material, which is available to authorized users.

A novel approach for harnessing biofilm communities in moving bed biofilm reactors for industrial wastewater treatment

Moving bed biofilm reactors (MBBRs) are an effective biotechnology for treating industrial wastewater. Biomass retention on moving bed biofilm reactor (MBBR) carriers (biofilm support materials), allows for the ease-of-operation and high treatment capacity of MBBR systems. Optimization of MBBR systems has largely focused on aspects of carrier design, while little attention has been paid to enhancing strategies for harnessing microbial biomass. Previously, our research group demonstrated that mixed-species biofilms can be harvested from an industrial wastewater inoculum [oil sands process water (OSPW)] using the Calgary Biofilm Device (CBD). Moreover, the resultant biofilm communities had the capacity to degrade organic toxins (naphthenic acids—NAs) that are found in OSPW. Therefore, we hypothesized that harnessing microbial communities from industrial wastewater, as biofilms, on MBBR carriers may be an effective method to bioremediate industrial wastewater. Here, we detail our methodology adapting the workflow employed for using the CBD, to generate inoculant carriers to seed an MBBR. In this study, OSPW-derived biofilm communities were successfully grown, and their efficacy evaluated, on commercially available MBBR carriers affixed within a modified CBD system. The resultant biofilms demonstrated the capacity to transfer biomass to recipient carriers within a scaled MBBR. Moreover, MBBR systems inoculated in this manner were fully active 2 days post-inoculation, and readily degraded a select population of NAs. Together, these findings suggest that harnessing microbial communities on carriers affixed within a modified CBD system may represent a facile and rapid method for obtaining functional inoculants for use in wastewater MBBR treatment systems.