Modified Robbins Device Research Articles

Assessment of urinary tract biomaterial encrustation using a modified Robbins device continuous flow model.

Encrustation of biomaterials employed in the urinary tract remains a major problem resulting in obstruction or blockage of catheters and stents. Therefore, resistance to encrustation is a desirable feature of biomaterials employed in such devices. The novel assessment of biomaterial encrustation employing a continuous flow model based on a modified Robbins device is described. Artificial urine was used in conjunction with 5% CO2 to simulate the physiological environment within the upper urinary tract. The widely used urinary device biomaterials, silicone and polyurethane, were investigated in the model for hydroxyapatite and struvite encrustation. Scanning electron microscopy, energy dispersive X-ray analysis, and atomic absorption spectroscopy all showed that silicone was less prone to encrustation than polyurethane and that hydroxyapatite deposition was predominant on both surfaces. The model has the advantage that a large number of biomaterials may be investigated simultaneously because several Robbins devices may be placed in parallel. The model is recommended for comparative evaluation of biomaterial candidates for use in urinary tract devices.

A dual fluorescence technique for visualization of Staphylococcus epidermidis biofilm using scanning confocal laser microscopy.

A new dual fluorescence technique is described which, when combined with scanning confocal laser microscopy (SCLM), can be used to visualize the components of biofilm produced by Staphylococcus epidermidis. Chemostat cultures of RP62A (a well-characterized slime-producing strain of S. epidermidis) were used to produce mature biofilm on polyvinylchloride (PVC) disks immobilized in a modified Robbins device using a 'seed' and 'feed' model system. Serial horizontal and vertical optical thin sections, as well as three-dimensional computer reconstructions, were obtained on in situ biofilm using the dual fluorescence procedure. Bacteria were visualized by green autofluorescence excited at 488 nm with an Argon laser. Cell-associated and exocellular matrix material (slime) was visualized by red fluorescence excited at 568 nm with a Krypton laser after interaction of the biofilm with Texas Red-labeled wheat germ agglutinin which is a slime-specific lectin marker. Structural analysis revealed that the cocci grew in slime-embedded cell clusters forming distinct conical-shaped microcolonies. Interspersed open channels served to connect the bulk liquid with the deepest layers of the mature, hydrated biofilm which increased overall surface area and likely facilitated the exchange of nutrients and waste products throughout the biofilm. The combined dual fluorescence technique and SCLM is potentially useful as a specific noninvasive tool for studying the effect of antimicrobial agents on the process of biofilm formation and for the characterization of the architecture of S. epidermidis biofilm formed in in vivo and in vitro on medical grade virgin or modified inert polymer surfaces.

Biofilm formation in laminar flow usingPseudomonas fluorescens EX101

The relationship between biofilm formation and Reynolds number in laminar flow has been investigated usingPseudomonas fluorescens EX101. It was shown using a Modified Robbins Device that in laminar flow, numbers of viable cells in a developed biofilm increased with Reynolds number (Re 2, 17 and 51.5), as would be expected in a system where molecular transport to the wall is limited by diffusion. By monitoring fluorescent beads in a flowcell with a scanning confocal laser microscope at similar low Reynolds numbers, the velocity profile close to the solid surface was determined. It was shown that the presence of a thin bacterial film (up to 12 μm) displaced the flow profile away from the wall by a distance equivalent to the film thickness. Total cell counts from the Modified Robbins Device samples were not significantly different at the different flow rates but were higher than viable counts. Interruption of the flow had no significant effect on colonisation by the bacteria through the Modified Robbins Device in the first few hours. However, viable numbers were reduced when the flow was stopped at 7 h after initial colonisation.

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 μrel, 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.

MODULATION OF THE BIOFILM OF PSEUDOMONAS AERUGINOSA BY QUINOLONES IN AN IN-VITRO MODEL OF VASCULAR CATHETER COLONIZATON

The effect of quinolones ciprofloxacin (CPX), norfloxacin (NOR), pefloxacin (PFX), and ofloxacin (OFX) on the adherence and on the preformed biofilms of P. aeruginosa to vascular catheters (VC) was studied in an in-vitro model using modified Robbins device. The presence of adherent viable bacteria was determined by culture on blood agar plates and quantitation of CFUS after dislodging the adherent cells into normal saline by sonication. The glycocalyx formation and adherence were also studied by scanning electron microscopy (SEM). In the presence of subinhibitory concentration of quinolones (0.5-, 0.25-, & 0.125-MIC) no or slight growth was observed around the segments and the number of the adherent cells was reduced to 21-23%, 40-46%, and 55-70% of the controls, respectively. Significant reduction in the glycocalyx formation and adherent bacteria was observed by SEM. In a separate set of experiments, the catheter segments were precolonized with P. aeruginosa for 24h and exposed to the quinolones in concentration 50 µg/ml (5 MIC) and 100 µg/ml for 2hrs. Slight or no growth was observed around the treated segments, while a heavy growth was reduced to less than 5% of the controls in VC treated with 50µg/ml of the quinolones and complete removal of the adherent cells was achieved with 100 µg/ml. By SEM a very small amount of glycocalyx and a few scattered microcolonies were seen after treatment with 50 µg/ml, no organisms were seen in the presence of 100 µg/ml. These data show that the subinhibitory concentrations of CPX, NOR, PFX, and OFX sgnificantly inhibit the adherence of P. aeruginosa to vascular catheters. Clinically achievable concentration of quinolones were able to eradicate the preformed biofilm on the VC.

Combination effect of fosfomycin and ofloxacin against Pseudomonas aeruginosa growing in a biofilm

We examined the combined effect of fosfomycin and ofloxacin against Pseudomonas aeruginosa in biofilms by using an in vitro experimental system with a modified Robbins device. Sessile cells in a mature or immature biofilm, developed on a silicon disk, were used, and an ATP bioluminescence assay was employed to assess antibacterial effects. A synergistic effect of fosfomycin and ofloxacin was clearly detected when concentrations at which each drug independently produced no detectable decrease in the bioactivity of sessile cells were used. Exposure of the cells in a mature biofilm to fosfomycin at concentrations of one-eighth of the MIC to 10 times the MIC (6.25 to 500 micrograms/ml) and ofloxacin at three or 10 times the MIC (18.75 or 62.5 micrograms/ml) resulted in reduction of the bioactivity to 1.5 to 4.5% after 72 h. Young sessile cells in an immature biofilm were more susceptible to this combination therapy. With a combination of fosfomycin at three times the MIC and ofloxacin at three times the MIC, complete eradication was confirmed by both ATP assay and scanning electron microscopy.

Lytic infection ofEscherichia colibiofilms by bacteriophage T4

Escherichia coli 3000 XIII formed biofilms on the surface of polyvinylchloride coupons in a modified Robbins device. Bacteriophage T4D+ infected cells in the biofilm and replicated. It is commonly held that bacteriophage cannot infect surface-attached bacteria (biofilms) because such bacteria are protected by an exopolymeric matrix that binds macromolecules and prevents their diffusion into the biofilm. To our knowledge this is the first observation that a bacteriophage can infect and multiply within cells growing as a biofilm.

Disinfection tests with intact biofilms: combined use of the Modified Robbins Device with impedance detection

Many disinfectants are currently evaluated using suspensions of pure bacterial cultures. These tests may be misleading since studies have shown that bacteria colonising surfaces (biofilms) are more resistant to disinfectants than the same cells released into suspension. This demonstrates the importance of maintaining the intact biofilm structure during biocide testing. Using a Malthus 2000 instrument, biofilm activity was measured in situ (without disrupting the biofilm structure) following disinfection of surfaces. These in situ measurements showed a higher number of surviving cells than cell recovery by swabbing or vortexing. This demonstrates the importance of the structural integrity of the biofilm during both disinfection and detection of survivors. A procedure was required for growing biofilms of a standard cell number on multiple discs, which could be placed directly into Malthus tubes following disinfection. The Modified Robbins Device was evaluated for this purpose, and was found to be suitable for growing 25 biofilm discs of a uniform cell number. Finally the use of the Modified Robbins Device and the Malthus were combined in a series of disinfection studies, which proved to give a simple, reliable and realistic method for studying the decontamination of biofilms.

Development of a flow method for susceptibility testing of oral biofilms in vitro.

Bacteria in biofilms are known to be more resistant than bacteria in batch cultures to antimicrobial agents. The purpose of the present study was to develop a flow method for formation of oral biofilms permitting susceptibility testing of plaque bacteria. A brain heart infusion (BHI) Streptococcus sanguis 804 culture was pumped through a modified Robbins Device (MRD) with 25 exchangeable silicone disks at 40 ml/h. After 24-48 h disks were removed and biofilm cells dispersed by vortex mixing and low-output ultrasonication. Colony forming units (cfu)/cm2 were determined after aerobic incubation on blood agar plates. Optimal biofilm formation was found after growth for 48 h at 37 degrees C in BHI + 1% sucrose, using saliva-coated silicone disks in inverted MRDs, yielding on average 4.4 x 10(5) cfu/cm2. Similar results were obtained for S. sanguis ATCC 10556 and five clinical isolates. Testing the susceptibility of S. sanguis to chlorhexidine gluconate showed increased resistance of biofilms compared to batch culture. Thus an appropriate biofilm model for susceptibility testing of oral microorganisms has been established.

共焦点レーザー走査顕微鏡による緑膿菌バイオフィルムの観察

Confocal laser scanning microscope (CLSM) is a new type of microscope, which permits non-invasive optical sectioning of biomaterials by reducing out-of-focus haze. A biofilm of Pseudomonas aeruginosa was developed on the silicon disks using a modified Robbins device and used to visualize hydrated living biofilm with CLSM. In order to examine optimum staining agents and conditions, acridine orange, fluorescein isothiocyanate (FITC), FITC-conjugated concanavalin A (FITC-ConA), Evans blue, safranine and rhodamine-conjugated concanavalin A were used in three different medium (pH 4.5, pH 7.5, pH 9.5). Only extracellular matrix was stained as a net-like structure for FITC-ConA and only bacteria for acridine orange and safranine. Although the staining patterns with FITC and Evans blue were affected markedly by the medium pH, those with other staining agents were not affected significantly. It was considered that the staining characteristics specific for each agent and changes of staining pattern by pH were probably due to relative differences among matrix, bacteria and staining agents in their electrostatic charges. In addition to sagittal sectioning images of P. aeruginosa biofilm, clear doubles staining images, which differentiates bacteria from matrix in the identical material, could be obtained with the combination of safranine and FITC-ConA.

Testing antimicrobial susceptibilities of adherent bacteria by a method that incorporates guidelines of the National Committee for Clinical Laboratory Standards.

Membrane filters (0.22-microns pore size) were colonized with 10(4) CFU of logarithmic-phase bacteria per filter under laminar flow conditions in a Modified Robbins Device. Colonized filters were then placed upon agar dilution plates for MIC determinations. Subsequently, filters were transferred to control plates and incubated to obtain MBCs.

An ATP bioluminescence assay for the analysis of bacterial biofilms

To analyze the bio-activity of the bacterial biofilm, the ATP content of the biofilm was assayed using the ATP bioluminescence assay. A linear relationship between the ATP content and the number of the planktonic Pseudomonas aeruginosa cells was observed. An in vitro experiment was set up using a modified Robbins device (MRD). A biofilm of P. aeruginosa was developed on the silicon discs of this device by recirculating artificial urine medium through the MRD. The activity of the biofilm, assayed by ATP luminescence, increased rapidly during the first 4 hrs of circulation, and essentially remained unchanged for 32 hrs during medium circulation. After the biofilm developed, the medium was exchanged to the new artificial urine with piperacillin or clarythromycin or ofloxacin and circulated for 8 hrs. The biofilm changed its bio-activity by the antibiotics, and the change could be detected by measuring the ATP content. These changes of ATP content studied were correlated well with the findings detected by scanning electron microscopy. Finally, ATP measurement of the bacterial biofilm was useful in analyzing the bio-activity of bacterial biofilm.

In Vitro ingrowth of yeasts into medical grade silicone rubber

Abstract Shunt-valves made of silicone rubber are placed between the trachea and the oesophagus in order to allow patients after laryngectomy to produce voice again. The oesophageal side of the shunt-valve, however, becomes rapidly colonized by a biofilm, consisting of bacteria and yeasts. Electron microscopy on explanted shunt-valves demonstrates that the yeast show ingrowth into the silicone rubber. Surface treatment of the silicone rubber is often suggested to be a pathway to reduce adhesion and ingrowth of yeasts to prostheses. An effective development of such a treatment requires an in vitro system by which similar defects as found in vivo can be created under laboratory conditions. Candida tropicalis , isolated from an explanted prosthesis, could be made to grow into the silicone rubber under laboratory conditions by the removal of all nutrients using a modified Robbins device. After the removal of all nutrients, the yeast cells adapted a filamentous growth form. Only filamentously growing yeasts appeared to be able to penetrate the silicone rubber.

Effect of Subinhibitory Concentrations of Clindamycin and Trospectomycin on the Adherence of Staphylococcus epidermidis in an In Vitro Model of Vascular Catheter Colonization

Septicemia, often due to Staphylococcus epidermidis, is a life-threatening complication associated with indwelling vascular catheters. An important factor in the development of such infections is glycocalix, or slime. An in vitro model that mimics intravenous delivery systems in humans was developed. It consisted of a modified Robbins device containing slices of silicone catheters in the removable ports, through which S. epidermidis diluted in 5% dextrose-normal saline with 10% heat-inactivated normal human serum was run, with and without clindamycin and trospectomycin. S. epidermidis was recovered from all catheters in the absence of antibiotics; no growth was detected with antibiotics. Scanning electron microscopy demonstrated significant reduction in glycocalix and no visible organisms with all concentrations except 0.5 micrograms/ml trospectomycin and 1 microgram/ml clindamycin; for those, a moderate amount of glycocalix and a few bacteria were seen. Thus, subinhibitory levels of trospectomycin and clindamycin may have a role in the prevention of microbial adherence to vascular catheters.

Growth of Bacterial Biofilms on Tenckhoff Catheter Discs in vitro after Simulated Touch Contamination of the Y-Connecting Set in Continuous Ambulatory Peritoneal Dialysis

We simulated touch contamination of peritoneal dialysis fluids perfused through an in vitro system with a modified Robbins' device (MRD) and Y-connecting tubings, to study the pathogenesis of bacterial biofilm (BB) growth on Tenckhoff catheter (TC) discs. The spike ends of Y-connecting sets were dipped in a suspension of freshly cultured cells of Staphylococcus epidermidis (3 X 10(8) cfu/ml), and connected to 2 litres of 0.5% dianeal solution which was perfused through the MRD with plugs containing TC discs. Four simulated clinical exchanges were performed with or without prior flushing and/or bleach treatment of the Y sets. Control experiments were done with fresh Dianeal solution with no contamination, flushing, or bleach treatment. BB growth on the TC discs was examined by scanning electron microscopy (SEM) and transmission electron microscopy and quantitated by routine culture of scrapings from the discs. We noted that touch contamination of dialysis fluids via the spike ends of the connecting sets can generate dense BB growth on TC discs in this experimental system (62 +/- 8% by SEM and 10.2 +/- 8.3 X 10(3) cfu/ml by culture). This growth of BB was significantly reduced by flushing the Y set with sterile Dianeal solution (24.3 +/- 3% by SEM and 5.7 +/- 3.5 X 10(1) cfu/ml by culture) and was absent by bleach treatment. We conclude that although bleach treatment of Y sets can prevent BB growth, the 'flushing' procedure alone can significantly reduce BB growth on TC from touch contamination of dialysate fluid.

Significance of biofilm-adherent bacterial microcolonies on Tenckhoff catheters of CAPD patients.

The phenomenon of biofilm bacterial adherence to bioprosthetic devices and their relationship to various human infections are now well established. We have recently demonstrated that biofilm bacterial colonization of Tenckhoff catheters (TC) is very common in patients undergoing continuous ambulatory peritoneal dialysis (CAPD) treatment. Based on our current work related to isolation of biofilm bacteria from peritoneal effluents of CAPD patients, we describe a novel way of diagnosis of this form of infection by using a modified Robbins device. Additionally, we describe the possible interaction of host defense factors with biofilm bacteria and ways to eliminate their colonization of TC for developing an overall strategy to prevent CAPD-associated peritonitis.

Colonization of n-butyl-2-cyanoacrylate tissue adhesive by Staphylococcus epidermidis.

In vitro adhesion of Staphylococcus epidermidis to cyanoacrylate tissue adhesive surfaces was investigated by employing a modified Robbins device. Bacterial colonization of n-butyl-2-cyanoacrylate tissue adhesive polymer surfaces was demonstrated by employing scanning electron microscopy, epifluorescence technique, and aerobic plate counts. The S. epidermidis was found to rapidly colonize the n-butyl-2-cyanoacrylate polymer surface producing a biofilm composed of embedded bacteria in an extensive amorphous matrix that totally occludes the material surface. The results indicate that the bacteriocidal properties of the polymer to this bacteria are weak or absent.

Antibiotic resistance of Pseudomonas aeruginosa colonizing a urinary catheter in vitro.

A modified Robbins Device was used to establish coherent biofilms of Pseudomonas aeruginosa on the surface of catheter material in an artificial urine milieu and the ability of an antibiotic to penetrate the biofilm and kill the enclosed bacteria was assessed. The Pseudomonas aeruginosa strain used had been isolated from a patient with urinary tract infection. Although planktonic (floating) cells of the Pseudomonas aeruginosa strain were inhibited by less than 1 mg/l of tobramycin and killed by 50 mg/l, contact with 1,000 mg/l of tobramycin for 12 h failed to kill all the sessile (adherent) bacteria in the biofilms on the surface of the catheter material. Surviving sessile bacteria recovered directly from the exposure to 1,000 mg/l of tobramycin were inhibited by 0.4 mg/l of this agent when tested as dispersed planktonic cells by standard MIC methods. It is suggested that growth within thick adherent biofilms confers upon cells of Pseudomonas aeruginosa a large measure of resistance to aminoglycosides and other antibiotics that may help to explain the frequent failure of antibiotic chemotherapy in catheter-associated urinary tract infections.