DESC:MICROBICIDAL SURFACES
TECHNICAL FIELD

[001] The Subject matter relates to the field of microbicidal surfaces that are created by immobilising certain substances on the surface of devices. More particularly, but not exclusively, the subject matter relates to medical devices that are used in and on the skin of humans and animals.

BACKGROUND
[002] Today Hospital acquired infections have become a major cause of concern worldwide especially to the patients that are hospitalised for a long period of time and the caregivers as well .Infections that are a source cause like the Foley’s catheter or that are spread by the contaminated surfaces or handling gloves that are acting as Fomites are a leading cause of death among patients.
In general medical devices are packed very clean and sterilised before use. However as soon as the packet is open and put to use, the device itself starts accumulating infection due to its very nature of use like the Foley’s catheter or Central IV cannula or the urine bags or the examination and operation gloves or even the sutures. There is very strong need to keep the device sterile for a long period of time during its usage so that the microbial load is not built on during its use or even after discarding it. Some of the solutions that are existing at present have depleting active ingredient that keeps on losing efficacy over time or stops performing completely after the effective concentration is reduced beyond minimum inhibitory concentration due to its consumption in usage.
Current invention teaches methods to make the surfaces sterile by non eluting method such that the surface of medical device retains microbicidal activity for a long Period of time , and also studies the efficacy of the method in different cases.
In certain cases there is need of tackling infections around the use area like the Foley’s catheter especially when the bacterial flora is seeping through the skin. Certain biofilm inhibiting combinations are needed in such crucial areas .In such cases judicious combination of eluting and non eluting microbicidal is needed. The eluting component here is not performing the function of preservative of device and hence the effective concentration of the eluting component can be kept far lower to have less cell toxicity. This would mean some of the highly active eluting components which are not finding use at present due to the cell toxicity can find its use on account of its lower effective usage quantity. Certain biofilm inhibiting combinations are needed in such crucial areas
BRIEF DESCRIPTION OF DRAWINGS
[003] Fig – 1 Schematic diagram of manufacturing of Antimicrobial Gloves
[004] Fig – 2 Dipping of formers in latex bath
[005] Fig – 3 Antimicrobial Efficacy at different concentrations if Antimicrobial on Gloves
[006] Fig – 4 Spectral Graph of Polar solvent Leachate
[007] Fig – 5 Spectral Graph of Nonpolar solvent Leachate
[008] Fig – 6 Schematic Diagram of Mode of action of AMB
[009] Fig – 7 Tensile Strength and Modulus of Gloves before and after Ageing
[0010] Fig – 8 Elongation properties of Gloves before and after Ageing
[0011] Fig – 9 Foleys Catheter
[0012] Fig – 10 Electron Micrograph of Bacterial load on a Fileys Catheter
[0013] Fig – 11 Antimicrobial Activity by AATCC147 A: 0.5% monocytogenes ATCC 19115 B: 0.5 % E coli ATCC 25922 , C: 1.0 % monocytogenes ATCC 19115 D : 1.0 % E coli ATCC 25922, E: 1.5 % monocytogenes ATCC 19115, , F: 1.5 % E coli ATCC 25922
[0014] Fig – 12 Bactericidal Efficacy by ASTM D7907 on Catheters
[0015] Fig – 13 Leachability Analysis over time

DETAILED DESCRIPTION
[0016] In recent times Hospital acquired infections have become a major cause of concern worldwide to the patients that are hospitalised for a long period of time and the caregivers who spend large amount of time in the contaminated environment. The risk is multiplied manifold due to the fact that newer and newer versions of strains are getting evolved and with each passing of days we are facing with acute threat of drug resistant pathogens.

[0017] There are many things in hospital that are used to treat patient or used as tools to treat the patients including consumables like catheters, gloves, body fluid collection bags etc .These things have high likelihood of getting contaminated and act as secondary source of infections. Such things are called Fomites.

[0018] Today Fomites are becoming increasing cause of concerns in areas like Intensive Care units (ICU) and Neonatal ICU where immune compromised patients are more likely to get the infections which can lead to deaths of patients.

[0019] Fomites are also acting as sources that contaminate the care-givers hands of clothes and cause them to be secondary carriers of infections across the hospitals.

[0020] Many modern ways of reducing the contamination load in hospital are being used today like sanitation with effective pathogen cleaners, wiping with alcohol, frequent mopping of devices and sterilization of the consumables.

[0021] Many medical devices are sterilised before use. However as soon as the packet of device is open and put to use, it loses the sterility, the device itself starts accumulating infection due to its very nature of use. Very common among such devices are Foleys catheter, Central IV cannula, the urine bags, the examination and operation gloves or even the sutures.

[0022] There is very strong need to keep the device sterile for a long period of time during its usage so that the microbial load is not built on during its use or even after discarding it.

[0023] Thus the devices are expected to retain sterility during the course of its use. In Other words long lasting antimicrobial activity is needed to be incorporated in the device.

[0024] Some of the solutions to these problems that are existing at present are the use of bactericidal or bacteriostatic agents applied or sprayed over the device. They are called local disinfectants.

[0025] The substances that are used on the device as local disinfectants have depleting active ingredient that keeps on losing efficacy over time or stops performing completely after the effective concentration is reduced beyond minimum inhibitory concentration due to its consumption in usage.

[0026] In such situations its vital to use long lasting, non depleting antimicrobial agents that are not mobilised on the devices surface but are immobilised on the device and still they retain the microbicidal activity. The device thus becomes forever sterile and does not act as Fomite. The microbicidal substance thus now act as preservative of the material as well.

[0027] There are no leaching of any antimicrobial substance from the device as the device has immobilised antimicrobial on the surface. Such surfaces are widely needed in applications like gloves or barrier films used during surgical procedures.

[0028] In certain cases there is need of tackling infections around the use area along with the device itself. In such cases only non leaching, immobilised microbicidal substances are not serving the purpose. Latest examples are the Foley’s catheter, especially when the bacterial flora is seeping through the skin. In cases of long usage a biofilm laden with bacteria are present and certain biofilm inhibiting combinations are needed in such crucial areas.

[0029] In such cases judicious combination of eluting and non eluting microbicidal is needed. The non eluting part is working as preservative of the device and acts only on the surfaces of the device when a pathogen comes in contact. Whereas the eluting component here is not performing the function of preservative of device and acts only on the free bacterial colonies in the vicinity of device and hence the effective concentration of the eluting component can be kept far lower than otherwise needed when both the functions are to be done by single antimicrobial .

[0030] Less amount of eluting antimicrobial means less cell toxicity. This would also mean some of the highly active eluting components which are not finding use at present due to the higher cell toxicity can find its use on account of its lower effective usage quantity.

[0031] Current invention discusses method and studies products that are made as per the stated principal and measures the efficacy of them in different cases.

[0032] The non eluting compound that is used in the study is represented by general formula

Formula - I
wherein:
R1 = hydrogen and/or C1 to C4 alkyl;
R2 = divalent hydrocarbon radical with C1 to C8 carbon atoms;
R3 = hydrogen or C1 to C4 alkyl;
R4 = hydrogen or C1 to C10 alkyl;
R5 = C8 to C22 saturated or unsaturated hydrocarbon radical; and
X = alkyl halide.

[0033] In an implementation, the microbicidal agent is one of a 3-(trimethoxysilyl)propyl-N-octadecyl-N,N-dimethyl ammonium chloride, 3-(trimethoxysilyl)propyl-N-tetradecyl-N,N-dimethyl ammonium chloride, 3-(trimethoxysilyl) propyl-N,N-didecyl-N-methyl ammonium chloride, 3-(trihydroxysilyl) propyl-N-octadecyl-N,N-dimethyl ammonium chloride. In an example, the microbicidal agent is preferably 3-(trimethoxysilyl) propyl-N-tetradecyl-N,N-dimethyl ammonium chloride.
[0034] The Schematic manufacturing diagram of the plant where commercial gloves are prepared is shown as below.

Fig – 1 Schematic diagram of manufacturing of Antimicrobial Gloves

[0035] The Schematic flow shows a stripping , wet washing , former cleaning and oven dry stage , where the formers are cleaned and then dipped in coagulation batch. The formers are then dried and dipped in latex bath and dried in first passage to form a gel on formers. These are then pre washed and dipped in the antimicrobial solution batch followed by beading and drying in the vulcanization oven. After this the formed gloves are post leach washed dried and stripped. Depending on the type of glove it is either powdered or non powdered. The time from dipping to stripping is regulated to 15 minutes. The working parameters of the trials are listed in below table 1.

Sr.No. Batch Name Recipe Name Process Speed Formers/minute Temperature of Curing Chamber oC Dwell Time in Minutes No of units produced Consumption of AMB Solution in L
1 AMBL01 Recipe1 56 130 15.31 min 4949 33.6
2 AMBL02 Recipe2 56 131 15.35 min 4949 33.6
3 AMBL03 Recipe3 56 132 15.33 min 4949 33.6
Table – 1 Parameters of trails

Fig – 2 Dipping of formers in latex bath
[0036] Antimicrobial solution is prepared in the trough which contains Ph balancers like acetic acid, binding primers like amino acids . In case of the study the antimicrobial that was immobilized on the surface was 3-(trimethoxysilyl) propyl-N-tetradecyl-N,N-dimethyl ammonium chloride. It will be referred to as AMB hence forth in the article. Three different recipes were used as listed below in table 2.

Recipe name Concentration on Weight of Glove Batch Concentration Chemical Qty in 1000 litre bath Additional Chemical Solution
Recipe 1 0.35% 5.85gpl 5.85kg 5.85kg (L1)
Recipe 2 0.5% 8.33gpl 8.33kg 2.48kg (L2)
Recipe 3 0.75% 12.5gpl 12.5kg 4.17kg (L3)
Table – 2 Batches with different concentrations

[0037] The concentrations of the bath were maintained by topping additional quantity of the chemical.

[0038] The following detailed description includes references to the accompanying drawings, which form part of the detailed description. The drawings show illustrations in accordance with example embodiments. These example embodiments are described in enough detail to enable those skilled in the art to practice the present subject matter. However, it will be apparent to one of ordinary skill in the art that the present subject matter may be practiced without these specific details. The embodiments can be combined, other embodiments can be utilized or structural and logical changes can be made without departing from the scope of the subject matter. The following detailed

[0039] Physical parameters of the gloves were also checked, and they did not show any marked difference in the physical parameters of the gloves. Refer to the table and chart.

[0040] Antibacterial activity for quickness to kill was evaluated by ASTM 6329-98 Standard protocol and the results of the findings are listed below. It can be seen that the mechanism of kill is almost instantaneous with more than 99 % Kill happening within first 1 minute of the contact time. Table 3 summarises the outcome.

TEST STRAINS INOCULUM STRENGTH TEST MATERIAL Area 1 min 10 min 30 min 1 Hrs 4 Hrs 8 Hrs
Staphylococcus Aureus 1.06 x 106 Cfu/0.5 ml 1 sq inch 99.50% 99.97% 99.99% 99.99% 99.99% 99.99%
Escherichia Coli 1.06 x 106 Cfu/0.5 ml 1 sq inch 99.49% 99.98% 99.99% 99.99% 99.99% 99.99%
Methicillin Resistant Staphylococcus Aureus (MRSA) 1.02 x 106 Cfu/0.5 ml 1 sq inch 99.52% 99.98% 99.99% 99.99% 99.99% 99.99%
Table – 3 Antimicrobial activity – Time to kill by ASTM 6329-98

[0041] Antimicrobial Efficacy of the gloves was measured with AATCC 147 Method. The outcome of the test confirms a very good zone of inhibition seen around the test item. The zone is seemed to be wider with increasing concentration of the AMB immobilized on the surface of the glove. The essential steps of the method are as listed below ;
A. Prepare nutrient agar plates prepare nutrient broth tubes
B. Transfer a loop full of test organisms in to tube containing 5 ml of sterile nutrient broth marked with date, name of the bacterium, strain number. Be left one UN-inoculated tubes as control tubes
C. Incubate all the tubes incubate at 37°C for 24 hours
D. Prepare the test specimen by cutting the specimen as square shapes.
E. Prepare test inoculums by transferring 1.0± 0.1 mL of 24 hours old bacterial culture into tubes contain 9.0 ± 0.1 mL of sterile distilled water and mix it with the help of vortex mixer.
F. Mark the nutrient agar plates, such as name of the test date, organisms, sample particulars, and individual signature.
G. Using a 4 mm inoculating loop, transfer a loop full of test innoculum (which prepared in step
E) on respectively marked sterile nutrient agar plates by means of 5 parallel streaks without refilling the test innoculum.
H. Incinerate the loop immediately.
I. Gently press the test specimen transversely across the five innoculum streaks with the help of sterile forceps.
J. Place sterile glass slides on the ends of the specimen If the sample are unable the contact.
K. Incubate at 37 ± 2 °C for 18 – 24 hours.
L. After 18 – 24 hours incubation examine the plates for results.
M. The test results are updated in table and photos.
N. The average width of zones of inhibition4 along a streak on either side of the test specimen may be calculated using the following equation
W = (T-D)/2

Where
W = Width of clear zone of inhibition in mm
T = Total diameter of test specimen and clear zone in mm
D = Diameter of the test specimen in mm

[0042] The table below gives details of the study as per the AATCC 147 method along with the micrographs of the plates.

0.35 % AMB on Glove 0.5 % AMB on Glove

0.75 % AMB on Glove Positive Control

Fig – 3 Antimicrobial Efficacy at different concentrations if AMB on Gloves .
Organism Staphylococcus aureus2 ATCC 6538 (Concentration of 106 CFU/ml) under in-vitro condition shows the quantitative details as below

Culture concentration CFU/Ml AMB Treated % Total Zone Glove width Difference observed Total activity in %
106 0.35% 35 mm 28 mm 10 mm 38.8%
106 0.5% 38 mm 24 mm 14 mm 42.2%
106 0.75% 37 mm 21 mm 16 mm 41.1%

106 Positive control 37 mm 26 mm 11 mm 41.1%
Table – 4 Evaluation of Antimicrobial Efficacy at different Concentrations of AMB

[0043] Quantitative evaluation of bactericidal efficacy of the gloves was done by American Society for Testing and Material (ASTM ) D 7907 - Standard .This test method is designed specifically for examination gloves. The test confirms the antibacterial activity of the treatment wherein the treated gloves are excellent with increasing concentration of the AMB concentration immobilized on the surface of the gloves. The essential steps of the test are listed below;
Test Organisms -24 hours old Staphylococcus aureus2 ATCC 6538
A. Prepare nutrient broth tubes.
B. Transfer a loop full of test organisms in to tube containing 5 ml of sterile nutrient broth marked with date, name of the bacterium, strain number. Keep one UN-inoculated tubes as control tubes
C. Incubate all the tubes incubate at 37°C for 24 hours
D. Prepare the test specimen by cutting the specimen as round/square shapes with size of 2x2 cm.
E. Prepare test innoculum by transferring 1.0 ± 0.1 mL of 24 hours old bacterial culture into tubes contain 9.0 ± 0.1 mL of sterile distilled water and mix it with the help of vortex mixer. Make sure that the test culture should be in the concentration of 108 CFU/ml.
F. 108 CFU/mL bacterial suspensions (challenge innoculum) is prepared in sterile saline or phosphate buffered saline with 5% bovine serum albumin (BSA).
G. 20µL of the innoculum is transferred to test specimen, dispersed and held in place by a sterile cover-slip. The contact periods for Test specimen and innoculum are 5 min, 10 min, 15 min.
H. After the specified contact time, the test specimen is transferred with its cover-slip into a 50 mL sterile conical centrifuge tube containing 10 mL of neutralizing solution.
I. The solution is vortexes for 15 seconds.
J. Serial dilution is performed on the neutralizer and plated.
K. Incubate at 37 ± 2 °C for 18 – 24 hours.
L. After 18 – 24 hours incubation examine the plates for bacterial colonies grown.
M. The test results are updated in table 1.

`Contact period D (0.35%) E (0.50%) F (0.75%) Negative control
5 min 15 CFU/ml 10 CFU/ml 8 CFU/ml 38 CFU/ml
10 min 12 CFU/ml 8 CFU/ml 4 CFU/ml 49 CFU/ml
15 min 03 CFU/ml 2 CFU/ml 0 CFU/ml 69 CFU/ml
Table – 5 Evaluation of Bactericidal Efficacy by ASTM D7907

[0044] Leachability analysis was done with the help of UV Vis Spectrophotometer by employing one Polar solvent – water and one non polar solvent –Acetone. Exhaustive leaching conditions were employed as tabulated below.

S.No Sample particulars Extraction volume (ml) Incubation Conditions
Time (h) Temperature Agitation
(° C) (rpm)
1 0.75% AMB solution + Distilled Water 3 72 37 100
2 0.75% CM5050 + Acetone
3 1gm treated glove + Distilled Water 3 72 37 100
4 1gm treated glove + Acetone
5 1gm untreated glove + Distilled Water 3 72 37 100
6 1gm untreated glove + Acetone
Table – 6 Parameters of Exhaustive Leaching Conditions

[0045] Analysis of the leaching data shows that the AMB solution is attached and immobilized on the surface of the gloves and does not leach under exhaustive conditions that the glove is expected to face during its usage in any solution ranging from polar to nonpolar solvent.

Fig – 4 Spectral Graph of Polar solvent Leachate

Fig – 5 Spectral Graph of Nonpolar solvent Leachate

[0046] The analysis of non leach ability stresses to lay importance to the fact that the zone of inhibition is seen on the plate method in spite of non leaching of the AMB. The reason for the same can be understood by the fact that primary mode of the AMB referred here is the kill that happens by physical lyses of the cell wall of the pathogens.

[0047] Most of the pathogens carry negative charge on the surfaces and they are attracted towards the positively charged aliphatic chain riding on the nitrogen base. The positive charge not only leads to electrocution of the pathogens but also to the physical rupture of the cell wall of the pathogen. The long aliphatic chains act as swords that are present on the surface of the device. It is because of this physical mechanism of kill, the AMB is not consumed in the operation and remains intact on the surface of device and continues to be active for a very long period of time. Figure Below shows the schematic diagram of the mechanism of the kill. The presences of electric charge create a zone around the device where a bacterium in the incubation plates senses and keep a safe distance from it. It is due to the presence of this electrical charge field around the device, a clear zone of influence around the device is seen during the qualitative testing of the device for antimicrobial effcicay.This zone is called zone of influence.

Fig – 6 Schematic Diagram of Mode of action of AMB

[0048] Physical properties of the gloves were analyzed before and after accelerated ageing. The gloves were aged at 50 OC for 90 days .The Tensile strength, Elongation at break and Modulus were measured by Instron and the results are as tabulated below.

Before Ageing(BA) After Ageing (AA)
Trial Details TS EL MS TS EL MS
Without AMB 30.9 866.9 3.6 22.9 672.2 4.9
0.35 % Trial 29.7 811.9 4.5 21.1 657.5 5.2
0.5 % Trial 29.3 796.3 4.5 20.7 631.9 6.4
0.75 % Trial 28.4 790.0 4.2 20.6 625.6 6.9
TS= Tensile Strength (Mpa), EL= Elongation at break (%), MS=Modulus ( Mpa)

Table – 7 Tensile properties of the Gloves

[0049] From the graphs below it can be seen that there were slight drop in the tensile strength of the treated gloves. The Elongation and Modulus increased slightly. Statistical analysis showed that these changes were not statistically significant to conclude the adverse effect of the treatments.

Fig – 7 Tensile Strength and Modulus of Gloves before and after Ageing

Fig – 8 Elongation properties of Gloves before and after Ageing

[0050] Invitro Cytotoxicity of the test item was performed by qualitative evaluation by examining the cells under the microscope to access the general morphology of the cells and graded. Qualitative Evaluation using neutral red uptake assay for the test item Antimicrobial wound dressing Showed a viability of 94.5 % when compared with the negative control. Based on the results obtained in the study, it is concluded that the treated gloves are considered Non Cytotoxicity under the condition of the present test carried out using Balb/c 3T3 cells line.

[0051] Similar study was done on another embodiment i.e the Foleys catheter. Here the device is expected to stay inside the body and help in guiding the urine through the urinary track and collect it in urine collection bag. Catheter is inserted in the body through the penis for men and through urethra for women.

[0052] Indications for using a catheter include providing relief when there is urinary retention, monitoring urine output for critically ill persons, managing urination during surgery, and providing end-of-life care. This may include comatose patients, before and after hysterectomies, before and after caesarean sections, patients who had genital injury, patients whose prostate is enlarged , patients with acute urinary retention, patients who are unable due to paralysis or patients who cannot control their bladder. It is estimated that around 15-20% of the hospitalised patients use catheter. Figure below shows a Foleys catheter .

Fig – 9 Foleys Catheter

[0053] The main complications that arise from the use of catheter are tissue trauma and infection. It is recorded in many studies that after 48 hours of catheterization, most catheters are colonized with bacteria, thus leading to possible Bacteraemia and its complications. Catheters can also cause renal inflammation, nephro-cysto-lithiasis, and pyelonephritis if left in for prolonged periods. The most common short term complications are inability to insert catheter, and causation of tissue trauma during the insertion. Urinary tract infection (UTI) causes over 40% of hospital-acquired infections Most infections due to urinary catheters Leading to increased morbidity and costs. There is a big risk of biofilm formation in the area of insertion. Biofilm is a extracellular Polymers wherein pathogens attach to and grow on a surface and produce extracellular polymers. This covers the colonies of pathogens and safeguards them from any external threats. A normal antiseptic that would be effective on a sort of pathogen would not work on the same sort of pathogens when they form a biofilm. Thus Biofilm are hard to remove and hard to treat.

[0054] Bacterial Infections build on very fast to catheters which are either made from latex or Silicon. The micrograph given below will highlight the speed of growth.

Fig – 10 Electron Micrograph of Bacterial load on a Foleys Catheter

[0055] Catheters have a unique problem of not only becoming the site of infections to the patients during usage but also facilitate the passage of infection by acting as passage to pathogens. In other words, pathogens are seeped inside the body and may reach the internal organs through the skin flora of the patient wearing it or by the cross contaminations.

[0056] There is need to keep the catheter sterile for longer duration of time and there are products in market that have active drugs loaded in the catheter which slowly leach after insertion in the body and keep the catheter and nearby sterile.

[0057] However like any other leachable device the drug has half life period so after certain duration of usage the effective concentration of the antibiotic drug falls below the minimum inhibitory concentration[MIC], which cause a strong resistant bacteria strain to evolve, making it difficult to treat the infections.

[0058] Thus there is need felt to have a permanently immobilized antimicrobial on the catheter and at the same time also have a component that is leached purposefully so that the vicinity around the catheter inside the body is also protected from the seeping microbial danger, which leads to formation of biofilm as the duration increases.

[0059] In the study, latex catheter was used and treated with AMB solution as explained elsewhere in the article. Embodiments were also made by treating the catheter with eluding drug called Chlorhexidine Gluconate. One more set was prepared by treating the catheters with combination of AMB and Chlorhexidine Gluconate. Thus, three sets were prepared which had one set with non leaching AMB immobilized on it, one set with leaching Chlorhexidine Gluconate coated and one set with combination of Immobilized AMB and Chlorhexidine Gluconate.

[0060] The brief description of the method that was adopted for treatment is described here. First all the latex Catheter were given acid wash with 0.1% of HCL bath for 2 minutes and dried in hot air oven at temperature of 100oC. AMB formulations were prepared as discussed in prior part of the article .The formulation solution is mixed to homogenize the solution. Catheter were then dipped in formulation bath for 2 minutes and samples were stirred to get uniform coating. Samples were cured by placing them in hot air oven at temperature of 100oc for 3 min. After curing, the samples are rinsed with cold water to remove any non-mobilized AMB. Washing is done for 2 minutes and then the catheter is dried in hot air oven at temperature of 100oc for 3 min. AMB solutions for effective concentration of 0.5 %, 1 % and 1.5 % are prepared.

[0061] To prepare the catheters with Chlorhexidine Gluconate similar preparations as above were followed. Instead of AMB solution, the leachable drug solution of Chlorhexidine with effective solution of 2 % , 3 % and 4 % were used. Instead of curing, only drying cycle at temperate not above 80 degree centigrade was followed.

[0062] To prepare the catheters with combination of AMB and Chlorhexidine solutions, method 1 and method 2 described above were followed sequentially. Table below gives the details of the treatments carried out on the Catheters.

Trial Details Trial 1 Trial 2 Trial 3
AMB % 0.5 1.0 1.5
CHG % 2.0 3.0 4.0
AMB % +CHG % 0.5+2 1+3 1.5+4
Table – 8 Parameters of trails on Catheters Treatments

[0063] Evaluation of antimicrobial activity by AATCC 147 of all the iterations gave good antimicrobial activity as can be seen in the plates below. Even at trail 1 level the results were showing a good zone of inhibition.

TEST ITEM A TEST ITEM B

TEST ITEM C TEST ITEM D

TEST ITEM E TEST ITEM F

Fig – 11. Antimicrobial Activity AATCC147. TEST ITEM A: 0.5% monocytogenes ATCC 19115, TEST ITEM B: 0.5 % E coli ATCC 25922 , TEST ITEM C: 1.0 % monocytogenes ATCC 19115, TEST ITEM D : 1.0 % E coli ATCC 25922, TEST ITEM E: 1.5 % monocytogenes ATCC 19115, TEST ITEM F: 1.5 % E coli ATCC 25922

[0064] Evaluation of bactericidal efficacy by ASTM D7907 method for the catheters gave following results as shown in below table.

Contact Period 0.5% AMB 1% AMB 1.5% AMB
5 min 50 CFU/ml 39 CFU/ml 34CFU/ml
10 min 20 CFU/ml 12 CFU/ml 10 CFU/ml
15 min 04 CFU/ml 02 CFU/ml 0 CFU/ml
Contact Period 2 % CHG 3 % CHG 4 % CHG
5 min 48 CFU/ml 30 CFU/ml 29 CFU/ml
10 min 08 CFU/ml 07 CFU/ml 07 CFU/ml
15 min 03 CFU/ml 0 CFU/ml 0 CFU/ml
Contact Period 0.5% AMB + 2 % CHG 1% AMB + 3 % CHG 1.5% AMB + 4 % CHG
5 min 19 CFU/ml 07 CFU/ml 8 CFU/ml
10 min 05 CFU/ml 02 CFU/ml 0 CFU/ml
15 min 0 CFU/ml 0 CFU/ml 0 CFU/ml
Table – 9 Bactericidal Efficacy by ASTM D7907 on Catheters

[0065] The plot of the efficacy below clearly shows that the activity is remarkably higher when the combination of leaching and non leaching technology is used as compared to only one of them considered individually.

Fig – 12 Bactericidal Efficacy by ASTM D7907 on Catheters

[0066] Cytotoxicity analysis showed almost similar results as the gloves and it was found to be noncytotoxic.

[0067] Analysis of leachate was done by method of invitro release kinematics. In this solution of phosphate buffer was used as leachable medium and the leachate was analysed from time to time for the presence of the AMB solution and the drug Chlorhexidine Gluconate.

Contact Period 0.5% AMB 1% AMB 1.5% AMB
1 Hr 0 0 0
12 Hr 0 0 0
24 Hr 0 0 0
36 Hr 0 0 0
48 Hr 0 0 0
60 Hr 0 0 0
Contact Period 2 % CHG 3 % CHG 4 % CHG
1 Hr 15 20 34
12 Hr 45 50 60
24 Hr 56 64 72
36 Hr 62 74 85
48 Hr 67 79 92
60 Hr 72 88 99
Contact Period 0.5% AMB + 2 % CHG 1% AMB + 3 % CHG 1.5% AMB + 4 % CHG
1 Hr 12 23 31
12 Hr 44 51 58
24 Hr 58 66 73
36 Hr 62 72 85
48 Hr 66 81 90
60 Hr 69 85 96
Table – 10 Leachability analysis over time by Chromatography

[0068] The analysis of the presence of the chemicals was done by LCMS Chromatography. The analysis shows that with time the amount of compound released in case of CHG increases for over 60 hrs and then gradually flattens out. AMB solution that is immobilized on the surface does not leach out.

Fig – 13 Leachability Analysis over time

[0069] The results thus show that not only is the antimicrobial activity of the immobilized AMB present for long duration without leaching and depletion, but the effective combination of coating the catheter with Chlorhexidine Gluconate ensures the area around the catheter is kept sterile. Thus while immobilized AMB act as preservative of the catheter preventing the build-up of infection load on the catheter, the diffused antimicrobial in the body around the catheter ensures the pathogenic load that may seep inside the body is kept under control.

[0070] Thus in any medical device that is likely to be used on or inside the body, a judicious choice of leaching and non-leaching technology ensures the load of leaching component is kept far lesser than otherwise required due to the presence of non-leaching component that acts as the preservative of the device. This would mean some of the highly active eluting components which are not finding use at present due to the cell toxicity can find its use on account of its lower effective usage quantity and low cell toxicity.

[0071] It shall be noted that some of the processes described above are described as sequence of steps; this was done solely for the sake of illustration. Accordingly, it is contemplated that some steps may be added, some steps may be omitted, the order of the steps may be re-arranged, or some steps may be performed simultaneously.

[0072] Although embodiments have been described with reference to specific example embodiments, it will be evident that various modifications and changes may be made to these embodiments without departing from the broader spirit and scope of the system and method described herein. Accordingly, the specification and drawings are to be regarded in an illustrative rather than a restrictive sense.

[0073] Many alterations and modifications of the present subject matter will no doubt become apparent to a person of ordinary skill in the art after having read the foregoing description. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. It is to be understood that the description above contains many specifications; these should not be construed as limiting the scope of the present subject matter but as merely providing illustrations of some of the personally preferred embodiments of this present subject matter.

[0074] Thus the scope of the present subject matter should be determined by the appended claims and their legal equivalents rather than by the examples given.

,CLAIMS:1] A medical device used internally and/or externally with body, made as an intrinsic microbicidal product ,wherein the microbicidal chemicals are immobilised on the device by surface bonding or complexed thereto, to make them noneluting and last longer and the process is achieved by either of, use of surface treatment or surface modification, plasma treatment, polymer grafting, superhydrophobicity, silane or siloxane cross linking or any method which does not cause the elution of the microbicidal components or the combination thereof.
2] ] The composition according to claim 1 that are immobilised, further comprises at least one active agent selected from the group consisting of a biocide, a disinfectant, an antiseptic, an antibiotic, an antimicrobial peptide, a lytic bacteriophage, a surfactant; an adhesion blocker; an oligonucleotide, quaternary ammonium compound and a chelator or the combination thereof .
3]The device as claimed in claim 1 may contain, in addition, an eluting chemicals which act as microbicidal agents that go in the body and act on the pathogens.
4] ] The eluting ingredients as claimed in claim 3, has said antimicrobial agent selected from the group of antiseptics consisting of (a-terpineol, methylisothiazolone, cetylpyridinium chloride, chloroxyleneol, hexachlorophene, chlorhexidine and other cationic biguanides, methylene chloride, iodine and iodophores, triclosan, taurinamides, nitrofurantoin, methenamine, aldehydes, azylic acid, silver, benzyl peroxide, alcohols, and carboxylic acids and salts.
5]The device as claimed in claim 1 may be the surface of any medical device in form of a membrane like a glove or a pipeline a catheter or a bag like urinary bag or a thread like suture or mesh like hernia mesh or an implant or a wound dressing or similar items.
6] The composition as claimed in claim 3 is formed by mixing ingredients as claimed in claim 1 from 0.1 % to 10 % and more preferably 0.2 % to 4 % on weight of the device basis wherein the eluting part may be in the range of 0.5 to 15%.
7] The device as claimed in claim 1 may be used internally or externally with the body as a invasive or non-invasive medical device.
8]The device as claimed in claim 1 may be made from Latex, Silicon, Nitrile, polyurethane or any polymeric material like nylon, polyethylene ,polypropylene or Polyester.