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Antimicrobial silver oxide incorporated urinary catheters for infection resistance.


The advances in medical technology have resulted in an increase in the use of devices and implants to improve the quality of life and replace diseased organs. One of the frequent and serious complications of the use of these devices has been the development of implant associated infections. As per statistics both in the US and the European National prevalence studies approximately 00% of all nosocomial infections are UTI (Urinary tract infection). About 80% of Nosocomial urinary tract infections are due to instrumentation of the urinary tract, mainly urinary catheterization. Each year urinary catheters are used in more than 0 million patients in acute care hospitals and extended-care facilities in the United States alone making nosocomial catheter associated urinary tract infection (CAUTI), a major cause of increasing length of hospital stay and treatment costs. Multiple antibiotic resistant organisms [1-4] cause most of these infections. When the economic burden is considered a study in UK reported that Hospital acquired infection (HAI) resulted in extra 14 days in hospital, 10% chance of death, and an extra 3104.00[pounds sterling] spent on health care and extra six days of work. The study concluded that the economic burden of HAI was about 1[pounds sterling] billion a year for NHS in England and that it affects 1 in 10 patients [0]. CAUTI may be symptomatic or asymptomatic. Asymptomatic catheter associated bacteriuria creates a huge reservoir of antibiotic resistant organisms in the hospital setting specially in the Intensive care units (ICU) [6]. In addition, when infection persists, it leads to complications such as prostatitis, epididymitis, cystitis, pyelonephritis and bacteremia, which is associated with significant mortality [6, 7].

Pathogenesis of CAUTI depends on duration of catheter insertion and the causative agents are from the patients' own colonic and perineal flora or from hands of health care personnel. Pathogens from these areas and from the urine collection bag can enter the urinary bladder through the extra luminal surface i.e outer surface of the catheter and through the intraluminal surface i.e. through the internal surface of the catheter lumen [3]. As the micro-organism migrates along the external and internal luminal surface of the catheter, a number of interactions take place that determines the development of infection. The initial step in medical device related infection is the adherence of bacteria to the biomaterial surface. Following adherence, they form colonies, synthesize exopolymeric slime and form biofilm. At the material surface interface, properties of the implanted biomaterial and the local hydrodynamic environment, govern bacterial attachment. Surface energy, wettability/hydrophobicity influence initial bacterial attachment to surfaces [8]. Bacterial adhesion followed by biofilm formation is a prerequisite for CAUTI. The biofilm so formed acts as a nidus of infection.

Various surface modifications were tried with the understanding that, bacteria forms biofilm on surfaces, which acts as the source of infection. Several strategies have been attempted to control or prevent biofilm formation like making the catheter surface more hydrophilic with hydrogel coatings, use of antimicrobial ointments or lubricants on the catheter surface, coating or impregnation of catheters with antimicrobial agents like antibiotics nitrofurazone, minocyclin and rifampicin, metal ions like silver etc [9, 10].

Silver as an antimicrobial agent has many advantages like its broad-spectrum antimicrobial activity, low toxicity to the human body, and long lasting biocidal activity with high thermal stability [11]. The oligodynamic property of silver ions binding to both microbial DNA preventing bacterial replication and to sulfhydryl groups of metabolic enzymes of bacterial electron transport chain, causing their inactivation [12] is responsible for its broad spectrum antimicrobial activity. A large number of medical devices including urinary catheters, vascular catheters, peritoneal catheters, prosthetic heart-valve sewing rings, sutures etc. have been coated with Silver or silver compounds. However, many of these commercially available silver-coated catheters are only marginally effective as the hydrophobic polymer matrix limits silver ion concentration near surface. In the method described in this paper, silver as silver oxide is impregnated and released as silver ions, making it a very effective antimicrobial agent. This is assayed using different microbiological techniques. The antimicrobial activity is dependant on the amount of silver ions leaching out of the catheter and this was estimated by spectrophotometric method.

As urinary catheters are used for urine drainage, mechanical properties and surface properties of these catheters are important for insertion and retrieval of catheters in patients. Catheters having less friction minimize injury or inflammation of urethra during catheterization and its retrieval. After catheterization of longer duration, the safe retrieval depends on the mechanical properties of the catheter. So the mechanical properties of the Foleys latex catheters subsequent to AgO impregnation and aging were assayed.

Materials and Methods

Citrate phosphate buffer (CPB), Phosphate buffered saline (PBS), Glutarldehyde, Isopropanol, Malachite green stain, Acridine orange stain (Himedia), Peptone water, Nutrient broth (NB) (Himedia, India), Nutrient agar (NA) (Himedia), Tryptone soya broth (TSB) (Himedia), Mueller Hinton Agar (MHA) (Himedia). Ampicillin, Gentamicin, Amikacin, Nalidixic acid, Norfloxacin, Ciprofloxacin--(Himedia Labs, India). Latex Foley's Urinary catheters (Brand A, B & C commercially available) were used for AgO coating. For all microbiological testing brand A catheter impregnated with Silver Oxide was used. Bacterial strains:--ATCC (American Type culture collection) strains and Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, isolated from used catheters retrieved from patients in the Neurosurgery ward of our hospital.

Antimicrobial Silver oxide incorporation in to Latex Urinary catheter

The methodology described is a patented technology. In brief, Urinary catheters, commercially available were used. It was swollen by immersing in an organic solvent like toluene, then placed in a solution of Silver nitrate in diethyl acetamide/ formamide. The solution was heated to 70 to 80 [degrees]C for incorporation of AgO. Further the catheters were washed in excess of water to remove the diethyl acetamide /formamide, and dried in vacuum. Polar solvents such as water or solvents like ethanol can be used with slight modifications in the process, for the reaction to be carried out in cold. This is a very simple and cost effective method for development of active anti-microbial catheters.

Surface property of catheter (Contact Angle measurement)

Hydrophobicity/ Hydrophilicity of catheter material surface was studied by measuring the water contact angle using NRL contact angle Goniometer, Rame'-Hart Inc, New Jersey, USA; Model: 100-00. The optical axis and specimen platform were leveled after switching on and test samples were placed on the observation platform and base line was adjusted to achieve co-incidence. A droplet of water was deposited on the sample surface and contact angle was measured directly from the measuring reticule after adjusting to view extreme right or left of the drop and adjusting the cross section to be tangent with the base line of the drop [13]. Three experiments were done and each experiment was repeated three times.

Mechanical properties of the catheters

The tensile strength and elongation at break of unmodified and modified catheters were determined as per ASTM D 412-80. Catheter samples size 7.0cm was used. Aging was carried out by initially drying in hot air oven at 70 [degrees]C for 7 days and then in synthetic urine at 37 [degrees]C for 7 days. (Synthetic urine composition: Urea 2.0gm, Sodium chloride 0.9 gm, Disodium orthophosphate anhydrous 0.20 gm. ammonium chloride 0.3gm, creatinine 0.2 gm and sodium sulphite hydrated in 100 mL distilled water. Universal testing machine (Instron model 1011) was used. The gauge length was 00mm and the rate of grip separation was 000 mm/min [14]. Three separate evaluation was done.

Evaluation of Silver in the catheters

A known weight (1gm) of Silver Oxide impregnated catheter was placed in 20ml of distilled deionised water for 24 hours at 37 [degrees] C at 100rpm. The solution was then analysed for the presence of silver ions spectrophotometrically using the Silver Test kit from M/s Merck. A Cary Bio 100 UV-Visible spectrophotometer was used for the measurement. The experiment was repeated thrice and average taken.

Cytotoxicity studies of Silver oxide modified catheters: L929 fibroblast cell line was used for the study and ISO 10993-0 procedures were followed [10]. The test was performed by both direct contact method and indirect contact method. For direct contact method, the test sample was placed on sub-confluent monolayer of L-929 mouse fibroblast cells and incubated. Following incubation, the cell culture was examined microscopically for cellular response around the samples. For indirect method extracts of in culture medium were added to the sub confluent layer of L929 cells incubated and examined microscopically for cellular response. The extract wa's added at 100% of concentration and at 60% concentration. Minimum essential medium with 10%fetal bovine serum was the medium used.

Antibacterial property of Silver oxide (AgO) impregnated latex catheter

Antibacterial property of the Silver Oxide incorporated Latex catheter was assayed by modified Kirby--Bauer Disc Diffusion method [16]. The-Muller-Hinton agar used was prepared from dehydrated media as per manufacturer's instructions. Approximately 20 to 30 ml of the medium (pH 7.2 to 7.4) was poured onto 9 cm petri-dishes and stored at 2-8 [degrees]C. The inoculum density of the tested strains were adjusted to give a concentration of 2- 0 x[10.sup.5] cfu/ml using McFarland standard. Both standard ATCC strains and isolates from Foley's catheters retrieved from patients randomly selected were used. The isolates used were

Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa. Standard strains Gram negative Pseudomonas aeruginosa ATCC 27803 and gram positive Staphylococcus aureus ATCC 20923 were also tested. The test and control 0.0cm long catheter pieces were cut from AgO incorporated and unincorporated Foleys catheters. These were placed on the inoculated plates along with the antibiotic discs using sterile forceps, keeping a distance of at least 24 mm apart and incubated at 30 + 2.0[degrees] C for 18 to 24 hours. The unmodified catheter pieces were the controls. The zone of inhibition was compared to standard antibiotic discs used for antibiotic sensitivity assay in cases of UTI (urinary tract infections). The standard antibiotic discs used in the study were Ampicillin, Gentamicin, Amikacin, Nalidixic acid, Norfloxacin, Ciprofloxacin. The zones of inhibition formed were measured to the nearest millimeter (inclusive of the disc diameter).

Acridine orange staining of catheter pieces exposed to biofilm forming bacterial strains [17]

Silver oxide coated and uncoated 1cm long latex catheter pieces were exposed to the bacterial strains for 7 days in tryptone soya broth (TSB) for biofilm formation. The catheter pieces were rinsed in fresh sterile PBS and transferred to 0% glutarldehyde solution for fixing at 22 [+ or -] 2 [degrees] C, 1hr and washed thrice in CPB. The washed catheter pieces were stained with Acridine orange staining solution for 10 min., washed off with CPB, air dried and treated with 0 ml Isopropanol for 0 min. It was then counter stained with malachite green staining solution for 10 minute, washed in CPB, air dried and examined under Fluorescent microscope (Leica DMR).


The specific method we used for the incorporation of AgO is solution based and ensured that both the external and internal surfaces of Foley's latex catheters were uniformly impregnated with AgO as seen in Fig 1 and 2. We had used commercially available catheters. The method of coating is not brand specific as any type of catheter may be coated by this method. Additionally it is simple, less expensive and easy to perform, does not need any sophisticated or expensive equipments and does not alter the manufacturing procedure but can be done after the final step. This is the uniqueness of our methodology.

Catheter material characterization

Water contact angle of the catheter material surface was done for noting the hydrophobicity/ hydrophilicity of the Silver oxide impregnated catheter. Both the outer surface and inner lumen of the Silver oxide impregnated and unmodified catheter pieces were tested. The results in Table 1, showed that after surface modification of the latex catheter with Silver oxide, the contact angle values increased, increasing surface hydrophobicity of the coated vis-a-vis the unmodified catheter. The layered AgO on the surface may be the reason for the increased hydrophobicity since AgO is insoluble in water and discourage wetting of the surface.

Mechanical features of the catheters

Three different brands of commercially available catheters were used to determine the effect of AgO incorporation on the integrity of the material of the catheters. The tensile strength and elongation at break of unmodified and modified catheters were determined after aging in synthetic urine using Universal testing machine --Instron. The mechanical property due to aging on AgO impregnated and unmodified catheters of different brands of commercially available catheters (coded as A, B and C) were tested. The results are tabulated in Table 2.

The general reduction in mechanical parameters may be due to the incorporation of AgO into the latex matrix. The AgO particles can prevent the alignment of the polymer chains on stretching leading to rupture. Other than catheter coded B which showed 60% reduction in mechanical properties on AgO incorporation, the ultimate stress--stain parameters of the modified catheter indicate that they still possess the required mechanical integrity for the intended application [14].

Evaluation of Silver in the catheters

The antimicrobial property is due to the presence of silver ions migrating out of the catheter when in situ and at the same time the released silver ions should be at levels nontoxic to users. The experiment was repeated thrice and it was found that on an average the amount of Silver released from one gram of the material was 0.0299ppm, which is way below the toxicity limit for human beings which as per United States environmental protection Agency guidelines (1991) is 0 ug/kg body weight/day for a person.

Cytotoxicity Studies

Cytotoxic analysis was carried out by both Direct and indirect contact method as per ISO 10993-5 [15].



By Direct contact method both the unmodified and AgO impregnated catheters did not show any cytotoxic effect (Fig 0&6). The normal morphology of the fibroblast cells was clearly seen.

But, in the indirect contact method where an extract is used we observed that higher concentration i.e 100% of of unmodified catheter showed mild toxicity while the AgO impregnated catheter was non-cytotoxic. At 60% concentration of the extract, both catheters AgO coated and uncoated were non-cytotoxic.

Antimicrobial activity studies

Table 3 lists the zone of inhibition to antibiotics and Silver oxide coated and uncoated Foley's catheters for the bacterial strains isolated from urinary catheters retrieved from patients. These bacterial strains, which had formed biofilms on the retrieved catheters, are capable of causing CAUTI (catheter associated urinary tract infections). These results show that the bacterial strains were resistant to all the antibiotics tested but sensitive to [Ag.sup.+] in AgO impregnated Foleys catheters. The uncoated catheter did not show any zone of inhibition. These observations indicate that inspite of possessing resistance to known antibiotics the tested bacterial strains were sensitive to silver ions. Figure 4 is a representative picture of the antibiogram tests with the AgO coated catheter showing a clear zone of inhibition in comparison to the uncoated catheter.

Figure 3 is fluorescent microscopic image of bacterial adhesion and biofilm formation on to coated and uncoated catheters. In fig 3 A, Silver oxide coated catheter pieces after exposure to bacterial cultures for 7 days were stained with Acridine orange and showed very few or no bacilli while the uncoated catheter pieces (Fig 3 B) had thick bacterial biofilm, observed as groups of yellow to orange colored bacilli. The observations were similar for all the brands of catheter. At all times bacterial adhesion and biofilm formation on AgO modified catheters had been prevented while the tested bacteria adhered and formed biofilms on unmodified catheters.




The fifth leading cause of death in United States after cancer, heart attack, stroke and pneumonia are Nosocomial infections. The large increase in number and occurrence of antibiotic resistant strains has prompted renewed interest in use of silver as an antibacterial agent [11]. Silver is a metal known for its broad spectrum antimicrobial activity against gram--positive and gram negative bacteria, fungi, protozoa and certain viruses [3], including antibiotic resistant strains [2-4,11]. The success of infection resistant medical device will depend on the ability of the device surface to prevent bacterial adhesion and biofilm formation. Bacterial biofilm is formed by initial bacterial attachment which is reversible and is guided by physical forces like Brownian movements, Vander Waals forces, gravitational forces, hydrophobic interactions etc. and subsequent irreversible adhesion mediated by molecular reactions between bacterial surface structures and substratum leading to the formation of stable biofilms [18]. Of the number of approaches adopted for development of infection resistant medical devices, a successful device will prevent bacterial adhesion and biofilm formation as biofilms on devices are the nidus of infection. So in effect, an effective antimicrobial surface will not only prevent initial bacterial attachment but also reduce the number of bacteria in the surrounding milieu so that the possibility of further attachment and biofilm formation is curtailed.

Silver is an effective anti-microbial agent, which is active in a wide variety of materials including glass, titanium and polymers [22]. Published data show that silver when impregnated is more effective than when present as surface coating. In surface coating silver can be readily deactivated by protein anions [2,19,20]. Here we present a method for impregnating latex Foley's catheters so that both the internal and external surfaces are uniformly coated with silver as seen in figure 1& 2. The method is simple and cost effective and does not affect the manufacturing process, as it can be included as the last step. Bacteria usually migrate along the internal and external surfaces of the catheter from the urine collection bag or meatal region causing adhesion and biofilm formation. Therefore, this method of impregnation of silver essentially protects both inner and outer catheter surfaces [2, 11].


The mechanism of antibacterial activity of silver is due to the silver ions [3,19,20], which bind strongly to electron donor groups in biological molecules. This causes defects in bacterial cell wall. Silver ions also bind to bacterial proteins and DNA effecting permeability and bacterial metabolism at all levels. Hertrick etal [19] has shown that polymers that release silver have strong antibacterial property, act as reservoirs of silver and are capable of releasing silver ions for extended periods and for this the production of an effective zone of inhibition is essential. It serves to prevent adhesion of microorganisms to the medical device and prevents adhesion of a variety of host derived adhesins such as fibronectin, laminin, fibrinogen, fibrin etc which also promotes microbial adhesion and biofilm formation [21, 22]. In this paper we report a method of incorporation of AgO which gives an effective zone of inhibition (Fig 4 & and table 3) against both gram positive and gram negative organisms, is non-cytotoxic (Fig 5 &6) and the coated catheter retains the necessary mechanical properties (Table 2), retaining its suitability for use after AgO incorporation.


The AgO impregnated catheters gave an effective zone of inhibition as seen in table 2 and that too against organisms that were resistant to the battery of antibiotics commonly used in clinical practice both in treatment against UTI. The fact that these strains had been isolated from Foley's catheters retrieved from patients increases the significance of this method of AgO modification of Foley's catheter. AgO is present through out the catheter and that too in a diffusible form ensuring Ag+ ion diffusion in sufficient concentration and this points to its possible effectiveness when challenged in the clinical setting.

Bacterial adhesion to materials depends upon the bacterial nature and material surface characteristics. Generally, bacteria with hydrophobic properties prefer hydrophobic material surfaces. Hydrophilic materials are more resistant to bacterial adhesion than hydrophobic material [18] i.e. hydrophobic surfaces encourage bacterial adhesion [21]. Surface characterization of Silver oxide (AgO) impregnated catheter by measurement of contact angle, indicated that the Silver oxide coating increased the hydrophobic nature of the material as seen in Table 2. Bacterial surfaces are highly hydrophobic and defined by bacterial capsule constituents. This results in hydrophobic attraction with hydrophobic material surfaces resulting in increased bacterial adhesion and biofilm formation.

Contrary to this general behavior, in this case, adhesion of bacteria was extremely low indicating the release of antibacterial Ag ions. This gains significance when we consider Harkes etal [23] suggestion on strategies to reduce CAUTI, by inhibition of bacterial growth on surfaces, rather than by modifying the physiochemical properties of the catheter surface.

The mechanical property of three different catheters available in the Indian market were tested after AgO incorporation and analyzed after aging in simulated urine. Two of the three catheters both modified and unmodified did not show any appreciable variation on aging. The third catheter coded B showed deterioration of mechanical properties and this may be due to the catheter material composition which is inherent to the material nature and not due to adverse effect of the AgO impregnation procedure.

The toxicity of silver towards prokaryotic and eukaryotic cells was determined and a selective toxicity towards prokaryotic cells has been reported. Silver toxicity has been reported to occur at serum levels as low as 0.3 ug/mL and manifests as argyria, leucopenia and alterations in renal and neural tissues [11,12]. Our initial cytotoxic screening both by direct contact method and by indirect contact method using extract of the material clearly shows that there is no cytotoxicity.


Although with improved techniques in medical care, we have been able to contain the increasing percentage of prosthetic device related infections the sheer volume of their usage and the consequences of infection associated with device usage remains a major challenge for Physicians and biomedical researchers. In our work, we have described a method of incorporating Silver oxide into finished Latex Foley's catheters with out substantially altering its properties. The method is easy to perform, does not need special equipments and can be easily incorporated into the manufacturing process. It has effective antimicrobial activity which prevents both bacterial adhesion and subsequent biofilm formation and is non-cytotoxic, thus fulfilling its promise of an effective anti-microbial surface.


We thank the Neurosurgery Department of SCTIMST for the help in collection of used Foley's catheters from patients. We Thank Dr TV Kumari for help in performing the Invitro cytotoxicity assays.


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A. Maya Nandkumar * ([dagger]), M.C. Ranjit ([dagger]), S.S. Pradeep Kumar T, P.R. Hari([section]), P. Ramesh ([double dagger]) and K Sreenivasan ([section])

([dagger]) Division of Microbiology, ([section]) Laboratory of Polymer Analysis, ([double dagger]) Polymer processing laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram 690012

* Corresponding author: A Maya Nandkumar (anmaya(S)

Received 7 May 2010; Accepted 3 August 2010
Table 1: Contact angle measurement in triplicate of both
unmodified and AgO impregnated Foley's catheters. Each
value is average of three readings.

Experiment          Contact angles

                  Unmodified catheter

             Outer surface   Inner lumen

1                 52             48
2                 39             37
3                 43             51

Experiment           Contact angles

              AgO impregnated catheter

             Outer surface   Inner lumen

1                 63             62
2                 59             60
3                 59             63

Table 2: Mechanical property of Silver oxide impregnated
and unmodified catheters. Three commercially available
brands were used in the study

Brand     Unmodified      Silver oxide         % variation
          (After aging    impregnated (After
          Stress at       aging Stress at
          maximum load)   maximum load)

Brand A   3.72            2.1                  31
Brand B   4.29            1.7                  60
Brand C   2.24            2.1                  8

Table 3: Antibiogram of the different strains of bacteria tested.
Each antibiotic has a standard zone diameter with reference to a
particular bacterial strain for terming as sensitive, resistant
and moderately sensitive.

Microbial strain    Antimicrobial used     Zone of      Sensitive/
used                                       inhibition    Resistant

E.coli-            Ampicillin-10mcg       0 mm
  (isolate from    Gentamicin-10mcg       10 mm
  biofilms         Amikacin-30mcg         12 mm
  on Foley's       Nalidixic acid-30mcg   0 mm          Resistant
  catheters        Norfloxacin-10mcg      0 mm
  retrieved from   Ciprofloxacin-5mcg     8 mm
  patients)        Unmodified Foleys      0 mm
                   catheter disc
                   Silver oxide           8 mm          Sensitive
                   Foleys catheter disc
Proteus            Ampicillin-10mcg       0 mm
  mirabilis        Gentamicin-10mcg       10 mm
  (isolate from    Amikacin-30mcg         13 mm
  biofilms on      Nalidixic acid-30mcg   0 mm          Resistant
  Foley's          Norfloxacin-10mcg      12 mm
  catheters        Ciprofloxacin-5mcg     12 mm
  retrieved        Unmodified Foleys      0 mm
  patients l       catheter disc
  isolate)         Silver oxide           10 mm         Sensitive
                   Foleys catheter disc
Pseudomonas        Ampicillin-10mcg       0 mm
  aeruginosa-      Gentamicin-10mcg       0 mm
  (isolate from    Amikacin-30mcg         0 mm          Resistant
  biofilms         Nalidixic acid-30mcg   0 mm
  on Foley's       Norfloxacin- 10mcg     0 mm
  catheters        Ciprofloxacin-5mcg     0 mm
  retrieved from   Unmodified Foleys      0 mm
  patients         catheter disc
  isolate)         Silver oxide           17 mm         Sensitive
                   Foleys catheter disc
S.aureus ATCC      Ampicillin-10mcg       27 mm
  25923            Gentamicin-10mcg       20 mm
                   Amikacin-30mcg         22 mm         sensitive
                   Nalidixic acid-30mcg   0 mm
                   Norfloxacin-10mcg      17 mm
                   Ciprofloxacin-5mcg     24 mm         resistant
                   catheter disc          0 mm
                   Silver oxide                         Sensitive
                   Foleys catheter disc   11 mm
Pseudomonas        Ampicillin-10mcg       0 mm          resistant
  aeruginosa       Gentamicin-10mcg       18 mm
  ATCC 27853       Amikacin-30mcg         19 mm
                   Nalidixic acid-30mcg   0 mm          sensitive
                   Norfloxacin-10mcg      22 mm
                   Ciprofloxacin-5mcg     25 mm         resistant
                   Unmodified Foleys      0 mm
                   catheter disc
                   Silver oxide           12 mm         sensitive
                   impregnated Foleys
                   catheter disc
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Author:Nandkumar, A. Maya; Ranjit, M.C.; Kumar, S.S. Pradeep; Hari, P.R.; Ramesh, P.; Sreenivasan, K.
Publication:Trends in Biomaterials and Artificial Organs
Date:Oct 1, 2010
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