Assessment of a chemostat-coupled modified Robbins device to study biofilms

The combination of a modified Robbins device (MRD) attached to the effluent line of a continuous cultivation vessel was assessed by the adhesion of planktonic bacteria maintained at a controlled growth rate. This combination of a chemostat and an MRD provides a large number of sample surfaces for monitoring both the formation and control of biofilms over extended periods of time. This apparatus was used to monitor the colonization of two soil isolates,Pseudomonas fluorescens (EX101) andPseudomonas putida (EX102) onto silastic rubber surfaces. At a similar _rel, both bacteria attached to the silastic, howeverP. fluorescens formed confluent, dense biofilms in less than 24 h, whereasP. putida adhered as single cells or microcolonies after the same period. The metabolic activity, measured by INT-formazan formation, was similar for both organisms with a peak at 6 h of colonization and a subsequent decrease after 24 h. Long term colonization studies ofP. fluorescens produced a population of greater than 9.5 log cfu cm^–2 at 28 days demonstrating the advantages of the chemostat-MRD association. This technique proved to be successful for studying bacterial adhesion and biofilm formation in tubular devices by bacterial populations at controlled and low growth rates.     

Inhibition of <Emphasis Type="Italic">Candida</Emphasis> <Emphasis Type="Italic">albicans</Emphasis> Biofilm Formation by Antimycotics Released from Modified Polydimethyl Siloxane

Unlike various disinfectants, antifungals have not been commonly incorporated so far in medical devices, such as catheters or prostheses, to prevent biofilm formation by Candida spp. In the present study, five antimycotics were added to polydimethyl siloxane (PDMS) disks via admixture (nystatin) or impregnation (trimethylsilyl-nystatin (TMS-nystatin), miconazole, tea tree oil (TTO), zinc pyrithione). Nystatin-medicated PDMS disks exhibited a concentration-dependent inhibitory effect on biofilm formation in a microtiter plate (MTP) but not in a Modified Robbins Device (MRD). This observation, together with HPLC data and agar diffusion tests, indicates that a small fraction of free nystatin is released, which kills Candida albicans cells in the limited volume of a MTP well. In contrast, biofilm inhibition amounted to more than one log unit in the MRD on disks impregnated with miconazole, TTO, and zinc pyrithione. It is hypothesized that the reduction in biofilm formation by these compounds in a flow system occurs through a contact-dependent effect.     

Binary culture biofilm formation by Stenotrophomonas maltophilia and Fusarium oxysporum

Binary culture biofilm formation by Stenotrophomonas maltophilia and Fusarium oxysporum was investigated using the recirculating modified Robbins device batch culture system. Sequential attachment studies were carried out in the Robbins device on PVC and glass surfaces, with each species as either the first or the second colonizer. Different surfaces had no significant effect on total numbers of S. maltophilia and F. oxysporum in the binary population biofilm. The attachment of the second colonizer was not influenced significantly by the previous attachment of the first colonizer. These results were confirmed using scanning electron micrographs. Journal of Industrial Microbiology & Biotechnology (2001) 26, 178–183. Received 06 July 1999/ Accepted in revised form 02 November 2000     

The Development of a Bioluminescence Assay to Compare the Efficacy of Biocides Incorporated into Plasticised PVC

A bioluminescence assay was developed using the expression of the luxAB genes in Pseudomonas veronii to allow the efficacy of biocides incorporated into plasticised polyvinylchloride (pPVC) to be determined in situ. A maximum number of cells was found to adhere to the surface after 18 h as measured by bioluminescence, radiolabelling and viable cell counts. A positive correlation was found between the level of bioluminescence and numbers of viable cells attached to the pPVC. When the biocide 10, 10-oxybisphenoxyarsine (OBPA) was incorporated into the pPVC, both bioluminescence and viable cell number were reduced by ca 60% at a concentration of 750 ppm and by >99% at 2250 ppm. When the biocide 2,3,5,6-tetrachloro-4-(methylsulphonyl)pyridine (TCMP) was incorporated into the pPVC, no reduction in viability or bioluminescence was seen after 18 h. However, over a period of 72 h at a concentration of 2250 ppm TCMP, both viable cell number and bioluminescence decreased steadily after 36 h until after 72 h, both bioluminescence and viable cell counts were less than 1% of the initial values. The viability of attached cells can therefore be measured in situ in a sensitive real-time assay by measuring bioluminescence allowing the efficacy of biocides incorporated into plastics to be compared.     

Bacterial adhesion measurements on soft contact lenses using a Modified Vortex Device and a Modified Robbins Device

S. marcescens 8100 andP. aeruginosa 15442 were used to study bacterial adhesion to hydrogel contact lenses which had not been worn. Bacterial removal from unworn lens materials was assessed with a calibrated vortex device modified with a digital rpm readout and fitted with a test tube attachment (MVD). The MVD, which relies on a whirlpool-like force to remove the bacteria, showed that bacteria adhered to the same degree to etafilcon A, vifilcon A and polymacon lenses under standardized conditions. Tracking the isoenzyme patterns of these bacterial species over time showed instability ofS. marcescens upon repeated passage. This instability was not evident withP. aeruginosa. Bacterial adhesion ofP. aeruginosa 15442, to human worn and unworn etafilcon A materials was determined with a Modified Robbins Device. The MRD was closed off at both ends stopping medium and bacterial movement after 1 h of fluid flow over the lens surface. The results show that immediately following this 1-h period more bacteria adhere to unworn contact lenses than to worn lenses. However, bacterial counts were equivalent on worn and unworn lenses following 5 h of static incubation.     

Use of the modified Robbins device to study the in vitro biofilm removal efficacy of NitrAdine, a novel disinfecting formula for the maintenance of oral medical devices

Aims:  To evaluate the use of the modified Robbins device (MRD) to test disinfection strategies against biofilms that form on oral medical devices and to test the biofilm removal efficacy of NitrAdine^TM, a disinfectant for the maintenance of oral medical devices.
Methods and Results:  Biofilms were grown on discs using the MRD and biofilms formed in this system were used to evaluate the efficacy of NitrAdine^TM and to determine the optimal disinfection conditions. Our data indicate that the use of the MRD allows for the rapid and reproducible formation of high-density biofilms. Determination of the efficacy of NitrAdine^TM revealed high activity against biofilms tested (e.g. >3 log reduction for Candida albicans and Staphylococcus aureus ) and allowed the determination of the optimal conditions for its use.
Conclusion:  The high reproducibility and flexibility of the MRD make it an excellent candidate for standardized testing of disinfectants aimed at reducing biofilms on oral medical devices. Using this system, we were able to demonstrate that NitrAdine^TM exhibits high activity against biofilms formed by the micro-organisms tested.
Significance and Impact of the Study:  Our data suggest that our procedure is appropriate for standardized testing of disinfectants aimed at reducing biofilms on oral medical devices.     

OmpA is an adhesion factor of Aeromonas veronii, an optimistic pathogen that habituates in carp intestinal tract

Aims: In the present study, we focused on one of the Aeromonas veronii isolates that exhibited marked adhesion onto carp intestine and studied its membrane-associated proteins for their possible involvement in mucosal adhesion. Methods and Results: We isolated a strain of Aer. veronii (CWP11) that exhibited a high degree of temperature-dependent adhesion activity onto carp intestinal tract and studied its adhesion factor. A proteomic analysis of the membrane-associated fraction showed the presence of multiple proteins that were specifically expressed in CWP11 cells cultured at 25°C. Of these, a 30 kDa protein was identified to be OmpA by a mass fingerprint analysis. Cloning and nucleotide sequencing of the ompA region of CWP11 revealed the presence of two tandem ompA homologues (ompAI-ompAII). Escherichia coli that expressed either OmpAI or OmpAII exhibited marked adhesion onto carp intestinal surface. Disruption of ompAI by a homologous recombination technique resulted in marked reduction of the adhesion activity in CWP11. Conclusion: The OmpA homologue plays an important role in the adhesion of the Aer. veronii strain onto the surface of intestinal tract. Significance and Impact of the Study: We successfully identified an OmpA homologue to be an adhesion factor of Aer. veronii, an optimistic pathogen that habituates in carp intestinal tract.     

On-line monitoring of antifouling and fouling-release surfaces using bioluminescence and fluorescence measurements during laminar flow

A laminar flow biofilm-monitoring system was used to determine the efficacies of three antifouling (AF) coatings and five fouling-release (FR) coatings againstVibrio harveyi attachment. On-line measurements of tryptophan fluorescence and bioluminescence from each coating, normalized to an upstream stainless steel coupon, were used to determine the effects of AF and FR surfaces on biofilm formation. The AF coatings consisted of 5, 10, and 35 wt% Sea Nine 211 (C9211) incorporated into a vinyl copolymer. Both the 10 and 35 wt% coatings significantly inhibited biofilm biomass development measured by tryptophan fluorescence compared to the stainless steel control.V. harveyi bioluminescence was significantly greater than tryptophan fluorescence in cells attached to these coatings, suggesting that bioluminescence expression may be a marker for cellular stress or toxicity in biofilms. Five different polydimethylsiloxane (PDMS) FR coatings did not inhibit biofilm formation under low flow conditions. However, four PDMS coatings demonstrated decreased biomass levels compared to stainless steel after exposure to a shear stress of 330 dynes cm^–2. There was no toxic additive in these coatings; bioluminescence and tryptophan fluorescence were proportional.     

Use of a modified Robbins device to directly compare the adhesion of Staphylococcus epidermidis RP62A to surfaces

Staphylococcus epidermidis is a frequent cause of infection associated with the use of biomedical devices. Flow cell studies of the interaction between bacteria and surfaces do not generally allow direct comparison of different materials using the same bacterial suspension. The use of a modified Robbins Device (MRD) to compare the adhesion to different surfaces of Staph. epidermidis RP62A grown in continuous culture was investigated. Adhesion to glass was compared with siliconized glass, plasma-conditioned glass, titanium, stainless steel and Teflon. Attachment to siliconized glass was also compared with glass under differing ionic strength, and divalent cation concentrations. Both the differences in numbers adhering and changes in adhesion (slope) through the MRD were compared. There was a trend towards higher numbers adhering to the discs at the in-flow end of the MRD than at the outflow end, probably reflecting depletion of adherent bacteria in the interacting stream. Adhesion of Staph. epidermidis RP62A to siliconized glass and Teflon was reduced when compared to glass with increasing flow rates. Adhesion to stainless steel was not affected by flow rate and titanium gave a different slope of adhesion through the MRD when compared with glass, suggesting an interaction with different sub-populations within the interacting stream. Differences between siliconized glass and glass at flow rates of 300 ml h-1 were abolished by the addition of calcium or EDTA and reduced by the addition of magnesium. Increasing ionic strength reduced the statistical significance of the differences between glass and siliconized glass. Pre-conditioning of glass with pooled human plasma reduced adhesion compared with untreated glass and again gave a different slope to glass. The MRD linked to a chemostat can be used to compare directly bacterial adhesion to potential biomaterials. Variable depletion of the interacting stream should be taken into account in the interpretation of results. Divalent cation concentration, substrate properties and flow rate were important determinants of the comparative adhesion of Staph. epidermidis RP62A to surfaces.     

Impact of Lactococcus lactis spp. lactis Bio Adhesion on Pathogenic Bacillus cereus Biofilm on Silicone Flowing System

Bacillus cereus is a food pathogen that can attach on most of the surfaces and form biofilms, which facilitate the persistence and resistance toward antimicrobials. The aims of this study were (i) to characterize the structural dynamics of B. cereus sessile growth in two nutritional environments (with or without a nutrient flow), and (ii) to evaluate the impact of bio adhesion of Lactococcus lactis on B. cereus biofilm. Significantly greater biofilm volume and thickness were observed under dynamic conditions than under static conditions after 48 h and B. cereus biofilm was highly organized. The variation of physico-chemical characteristics of silicone by B. cereus bio adhesion favours the adhesion of hydrophilic Lc. lactis on the surface adhered by biofilm. Lc. lactis was able to adhere to silicone surface and produce biofilm obviously exhibited a significant reduction of B. cereus adhered cells up to nine orders of magnitude after 48 h of contact with competitive activity for nutrient and oxygen. This study constitutes a step ahead in developing strategies to prevent microbial colonization of silicone with lactococcal protective biofilm.     

Use of the modified robbins device and fluorescent staining to screen plant extracts for the inhibition of S. mutans biofilm formation

Streptococcus mutans plays an important role in the formation of dental plaque. To study biofilm growth on hydroxyapatite (HA) in vitro, a flow system based on a Modified Robbins Device (MRD) and a method for the quantification of the biomass using fluorescent staining with SYTO(R) 9 were developed. The combined approach was used to assess the inhibitory effect of plant extracts on biofilm formation in concentrations below their minimal inhibitory concentrations.     

Application of chimeric glucanase comprising mutanase and dextranase for prevention of dental biofilm formation

Water‐insoluble glucan (WIG) produced by mutans streptococci, an important cariogenic pathogen, plays an important role in the formation of dental biofilm and adhesion of biofilm to tooth surfaces. Glucanohydrolases, such as mutanase (α‐1,3‐glucanase) and dextranase (α‐1,6‐glucanase), are able to hydrolyze WIG. The purposes of this study were to construct bi‐functional chimeric glucanase, composed of mutanase and dextranase, and to examine the effects of this chimeric glucanase on the formation and decomposition of biofilm. The mutanase gene from Paenibacillus humicus NA1123 and the dextranase gene from Streptococcus mutans ATCC 25175 were cloned and ligated into a pE‐SUMOstar Amp plasmid vector. The resultant his‐tagged fusion chimeric glucanase was expressed in Escherichia coli BL21 (DE3) and partially purified. The effects of chimeric glucanase on the formation and decomposition of biofilm formed on a glass surface by Streptococcus sobrinus 6715 glucosyltransferases were then examined. This biofilm was fractionated into firmly adherent, loosely adherent, and non‐adherent WIG fractions. Amounts of WIG in each fraction were determined by a phenol‐sulfuric acid method, and reducing sugars were quantified by the Somogyi–Nelson method. Chimeric glucanase reduced the formation of the total amount of WIG in a dose‐dependent manner, and significant reductions of WIG in the adherent fraction were observed. Moreover, the chimeric glucanase was able to decompose biofilm, being 4.1 times more effective at glucan inhibition of biofilm formation than a mixture of dextranase and mutanase. These results suggest that the chimeric glucanase is useful for prevention of dental biofilm formation.     

Adhesion of the marine fouling diatom amphora coffeaeformis to non‐solid gel surfaces

Diatom adhesion to different gel surfaces was tested under different shear conditions, using the fouling marine diatom Amphora coffeaeformis as test organism. Four polymers were selected to obtain a test matrix containing gels with different surface charge as well as different surface energies, viz. agarose, alginate, chitosan and chemically modified polyvinylalcohol (PVA‐SbQ). Three experimental systems were applied to obtain different shear rates. Experimental system 1 consisted of gels cast in a cell culturing well plate for comparing initial adhesion as well as long term biofilm development in the absence of shear. In experimental system 2, microscope slide based test surfaces were tested in aquaria under low shear conditions. A rotating annular biofilm reactor was used to obtain high and controlled shear rates. At high shear rates A. coffeaeformis cells adhered better to the charged polymer gels (alginate and chitosan) than to the low charged polymer gels (agarose and PVA‐SbQ). In the system where shear was absent A. coffeaeformis cells developed a biofilm on agarose equivalent to the charged polymer gels, while adhesion to PVA‐SbQ remained low at all shear rates. It is concluded that non‐solid surfaces did not represent an obstacle to settling and growth of this organism. As observed for solid surfaces, low charge density led to reduced attachment, particularly at high shear.     

Holo and apo-transferrins interfere with adherence to abiotic surfaces and with adhesion/invasion to HeLa cells in Staphylococcus spp.

Staphylococcus aureus and Staphylococcus epidermidis are the major cause of infections associated with implanted medical devices. Colonization on abiotic and biotic surfaces is often sustained by biofilm forming strains. Human natural defenses can interfere with this virulence factor. We investigated the effect of human apo-transferrin (apo-Tf, the iron-free form of transferrin, Tf) and holo-transferrin (holo-Tf, the iron-saturated form) on biofilm formation by CA-MRSA S. aureus USA300 type (ST8-IV) and S. epidermidis (a clinical isolate and ATCC 35984 strain). Furthermore S. aureus adhesion and invasion assays were performed in a eukaryotic cell line. A strong reduction in biofilm formation with both Tfs was obtained albeit at very different concentrations. In particular, the reduction in biofilm formation was higher with apo-Tf rather than obtained with holo-Tf. Furthermore, while S. aureus adhesion to eukaryotic cells was not appreciably affected, their invasion was highly inhibited in the presence of holo-Tf, and partially inhibited by the apo form. Our results suggest that Tfs could be used as antibacterial adjuvant therapy in infection sustained by staphylococci to strongly reduce their virulence related to adhesion and cellular invasion.     

A novel in vitro flat‐bed perfusion biofilm model for determining the potential antimicrobial efficacy of topical wound treatments

Aims: To develop an in vitro flat‐bed perfusion biofilm model that could be used to determine the antimicrobial efficacy of topically applied treatments. Methods and Results: Pseudomonas aeruginosa and Staphylococcus aureus biofilms were grown within continuously perfused cellulose matrices. Enumeration of the biofilm density and eluate was performed at various sampling times, enabling determination of the biofilm growth rate. Two antimicrobial wound dressings were applied to the surface of mature biofilms and periodically sampled. To enable real‐time imaging of biofilm growth and potential antimicrobial kinetics, a bioluminescent Ps. aeruginosa biofilm was monitored using low‐light photometry. Target species produced reproducible steady‐state biofilms at a density of c. 10⁷ per biofilm support matrix, after 24‐h perfusion. Test dressings elicited significant antimicrobial effects, producing differing kill kinetic profiles. There was a good correlation between photon and viable count data. Conclusions: The model enables determination of the antimicrobial profile of topically applied treatments against target species biofilms, accurately differentiating bactericidal from bacteriostatic effects. Moreover, these effects could be monitored in real time using bioluminescence. Significance and Impact of the Study: This is the first in vitro biofilm model which can assess the antimicrobial potential of topical therapies in a dynamic growth environment.     

Characterisation and in vitro activities of surface attached dihydropyrrol-2-ones against Gram-negative and Gram-positive bacteria

Bacterial infection of biomedical devices is still a major barrier to their use. This is compounded by increasing antibiotic resistance. Here, the specific covalent attachment of a series of dihydropyrrol-2-one (DHP), analogues of bacterial quorum sensing inhibitors, to surfaces via a Michael-type addition reaction is described. Differences in efficiency of attachment related to the substituent groups were found by X-ray photoelectron spectroscopy. The physical characteristics of the surfaces were further explored by atomic force microscopy and contact angle measurements. The ability of these coatings to prevent the formation of a biofilm by Pseudomonas aeruginosa and Staphylococcus aureus was examined using confocal laser scanning microscopy and image analysis. The DHP-treated surfaces showed significant reductions in bacterial adhesion without increased killing for both strains of bacteria (p < 0.001). 5-Methylene-1-(prop-2-enoyl)-4-phenyl-dihydropyrrol-2-one was identified as having broad spectrum activity and consequently represents an excellent candidate for the development of novel surfaces for the prevention of biomedical device infections.     

In vitro model systems for exploring oral biofilms: From single-species populations to complex multi-species communities

Numerous in vitro biofilm model systems are available to study oral biofilms. Over the past several decades, increased understanding of oral biology and advances in technology have facilitated more accurate simulation of intraoral conditions and have allowed for the increased generalizability of in vitro oral biofilm studies. The integration of contemporary systems with confocal microscopy and 16S rRNA community profiling has enhanced the capabilities of in vitro biofilm model systems to quantify biofilm architecture and analyse microbial community composition. In this review, we describe several model systems relevant to modern in vitro oral biofilm studies: the constant depth film fermenter, Sorbarod perfusion system, drip–flow reactor, modified Robbins device, flowcells and microfluidic systems. We highlight how combining these systems with confocal microscopy and community composition analysis tools aids exploration of oral biofilm development under different conditions and in response to antimicrobial/anti-biofilm agents. The review closes with a discussion of future directions for the field of in vitro oral biofilm imaging and analysis.     

In vitro actinomycete biofilm development and inhibition by the polyene antibiotic,nystatin, on IUD copper surfaces

The presence of intrauterine contraceptive devices (IUDs) gives a solid surface for attachment and an ideal niche for biofilm to form and flourish. Pelvic actinomycosis is often associated with the use of IUDs. Treatment of IUD-associated pelvic actinomycosis requires the immediate removal of the IUD. Therefore, this article presents in vitro evidence to support the use of novel antibiotics in the treatment of actinomycete biofilms. Twenty one clinical actinomycetes isolates from endocervical swabs of IUD wearers were assessed for their biofilm forming ability. An in vitro biofilm model with three isolates, Streptomyces strain A4, Nocardia strain C15 and Nocardia strain C17 was subjected to treatment with nystatin. Inhibition of biofilm formation by nystatin was found to be concentration dependent, with MBIC₅₀ values in the range 0.08–0.16 mg ml^?1. Furthermore, at a concentration of 0.16 mg ml^?1, nystatin inhibited the twitching motility of the isolates, providing evidence for a possible mechanism of biofilm inhibition.     

Multiple drug resistance and strength of attachment to surfaces in Pseudomonas aeruginosa isolates

Aims: To investigate the presence of a relationship between the strength of attachment of Pseudomonas aeruginosa to stainless steel surfaces and their observed multiple drug resistance. Methods and Results: Multiple drug resistance of clinical and environmental isolates of Ps. aeruginosa was evaluated using disc diffusion method. The blot succession technique was used to quantify the strength of attachment of Ps. aeruginosa isolates. Different multiple drug–resistant Ps. aeruginosa isolates exhibited variable attachment strength. Although the highest multiple drug–resistant clinical isolate was shown to have the least attachment strength among clinical isolates, a weak correlation was found between attachment strength and multiple resistance among our investigated Ps. aeruginosa isolates. Conclusions: There is a weak correlation between multiple drug resistance and strength of attachment to stainless steel surfaces. Significance and Impact of the Study: Even low‐resistant Ps. aeruginosa could have the potential of attaching firmly to surfaces and forming biofilm.