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Research: Bactericidal Activity

 

FINAL REPORT

 

EVALUATION OF THE BACTERICIDAL ACTIVITY OF AN ANTISEPTIC FORMULATION USING THE EX VIVO HUMAN SKIN METHOD

 

Syed A. Sattar, Ph.D.
Professor of Microbiology
Faculty of Medicine, University of Ottawa
Ottawa, Ontario, Canada K1H 8M5

 

July, 1997 

 

INTRODUCTION


We were initially contacted by Mr. John Duffy of Bio-Safe Skin Products Inc. (formerly Biotech-Transfer) about the possibility of evaluating a barrier lotion for its bactericidal activity using the ex vivo human skin method developed in our laboratory (Graham et al., 1996). The details of such a study were negotiated toward the end of 1996 and a contractual agreement to conduct it was finalized in February, 1997. The preparation and experimental work of this project were commenced at the beginning of March, 1997. A progress report was provided to the sponsor on May 23, 1997.


MATERIALS AND METHODS


The Product: Samples from three separate lots of each of two different formulations of the product Bio-Safe® Skin Shield™ Barrier Lotion were received in our laboratory on March 10, 1997. Soon upon their arrival here they were stored at room temperature in a place accessible only to authorized personnel. As far as we are aware, one formulation contained 0.3% of Triclosan and the other 0.6% of the same chemical. According to the literature supplied by the sponsor, SSBN also contains a polymer which can protect the skin against harsh chemicals. The protective effect can last for at least four hours and can withstand perspiration and repeated washings during that period. Among the listed regular users of the product are healthcare personnel, food handlers, daycare workers, industrial hygienists and veterinarians.


The Bacteria: The bacteria tested in this study were:
(1)   Methicillin-resistant Staphylococcus aureus (MRSA)
(2)   Vancomycin-resistant Enterococcus faecalis (VRE)
(3)   Enterotoxigenic Escherichia coli (ETEC)
These bacteria were selected for this study because of (a) their importance as nosocomial and foodborne pathogens, (b) their ability to spread through contaminated hands and (c) the fact that they are unsafe for experimental contamination of the hands of human volunteers and that skin care products against them could only be tested using either an in vitro or an ex vivo  method.

 

Seed cultures of all three of the above-mentioned bacteria were obtained by us as clinical isolates from the Bacteriology Laboratory of the Children's Hospital of Eastern Ontario (CHEO) in Ottawa. In our laboratory these bacteria were cultured and maintained on plates of trypticase soy agar (Difco). They were grown overnight in tubes of trypticase soy broth (Difco) to prepare the inocula for the experiments described below.

 

Skin Samples: All skin samples used were those removed from normal adult females by a local plastic surgeon (Dr. M. Bell) during breast or abdominal reductions. Permission for the collection and use of such skin was first obtained from the Ethics Committee of the Ottawa Civic Hospital where all the surgeries were performed. Informed and written consent was obtained from each donor prior to the collection of the tissue samples. Sterile cell culture maintenance medium, without serum, was used as the collection and transportation fluid for the tissue samples. All tissues specimens were processed within four hours of removal from the donor. The tissues were prepared by removing any excess subcutaneous fat and then secured onto a dissecting board. Patches (approximately 2.3 cm in diameter) were then punched out of the skin.

 

After mounting, the tissue holder was inverted with the exposed (subcutaneous) side kept immersed in cell culture medium. Thus, the skin serves as the bottom of a well, such that the viral inoculum and then the eluent can be placed into the well. Using previously described biochemical (measurement of lactate production) and histopathological methods with freshly excised human skin bathed in tissue culture medium, we have shown that we can maintain the skin viable in this system for at least 8 hrs.

 

The Ex Vivo Method: We have previously reported the use of fingerpads of adult volunteers to assess quantitatively the germicidal activity of skin care products against viruses and bacteria (Ansari et al., 1989). Since ethics and safety considerations limit the application of the method to use with relatively safe infectious agents, we used here an ex vivo method (Graham et al., 1996).

 

After the removal of subcutaneous adiposa tissue from the epithelial layer skin was stretched onto a dissecting board to be punch out approximately 2-3 cm circles
The circular patches were mounted on 1cm diameter cylindrical metal chambers. and tightened with rubber bands around them against the metal chamber wall, by keeping the aquamous epithelial inside of the chamber wall, and orienting the basal lamina as an external surface.

 

The skin carrying chambers were than transferred into 12 wells plates and each aquomous epithelial patches were inoculated with 10 ul pathogenic bacterial suspension except the first well was kept as a blank.
The experiment carried on as triplicate depending on the size of a skin specimen was obtain after the surgery.

 

The experimental procedure was set up under three different controls in order to compare to anti-germicidal effect of the Bio-Safe® Skin Shield™ Barrier lotion on the pathogenic bacteria inoculated skin patches,
The two controls were inoculated with the pathogenic bacteria except one inoculation was made on agar, the second control was inoculated into aqueous media (PBS or PB)

 

For the sterility test of a skin one of the skin patch was kept as a blank, without any specific bacterial inoculum in order to determine any bacterial contamination
The skin patch inside the metal chamber ,of the blank was washed by flashing the surface of the skin with 1 ml of PBS or PB The eluent from the chamber was collected and diluted under serial concentrations and inoculated on the agar plates to determine the sterility of the skin.

 

For the second control the skin pace was inoculated with 10 ul of pathogenic bacterial suspension. (Staphylococcus aureus, Metacyline resistant Staphylococus areaus, and E.Coli # 0.157 and Vancomycin Resistant Enterococcus ) After the air dry of the inoculated area the eluent was collected under the same procedure as mention above before under the any treatment of the bacteria inoculated skin patch with Biotech-Transfer Skin Shield Barrier Lotion

 

The third control was represented again by the inoculation of the 10ul of a pathogenic bacterial suspension into 0.890 ml of PBS or PB buffer in a 2mls tube and incubating the inoculum for 1 min. with 100ul of Bio-Safe® Skin Shield™ Barrier lotion in this aqueous buffer solution.

 

The two different concentration of the product was tested during the experimental procedure.(0.3% and 0.6%).The test of the Bio-Safe® Skin Shield™ lotion was carried on inoculation of the 10 ul of pathogenic bacteria (Staphylococcus aureus, Metacyline resistant Staphylococus areaus,  and E.Coli # 0.157 and Vancomycin Resistant Enterococcus) on skin patches individually for the each experiments

 

Following bacterial inoculum of the stratum corneum patches the inocula were air dried and they were treated with 100 uL of Bio-Safe® Skin Shield™ Barrier lotion under different concentration of the product material (%O.3 and %O.6) keeping one minute incubation at the room temperature

 

The stratum corneum patches were eluted with 0.890 ml Earle balance salt solution, or Phosphate buffer by flashing the surface of the each skin patches in the each carrier chambers .The eluent from each patch was collected into individual ependorff tube.

 

The serial dilutions were done from every individual tube as mention above and 100 uL of sample from the each tube were spreaded on bacterial TSA agar plates to determine the CFU values from each sample.and valued the inhibitory growth effect of the product on the human pathogens by testing on the ex vivo skin samples.
The results showed with the 6% concentration value of the product did effect the growth of the bacteria almost close to 3 log values. (See the charts )

 

ACKNOWLEDGMENTS

 

This project was conducted with the technical assistance of Ms. Oya Hill, M.Sc.
We are most grateful to Dr. Michael Bell, Plastic Surgeon, for supplying us with the skin samples used in this study. The assistance of Dr. Frank Chan in providing us with the bacterial isolates is gratefully acknowledged. The help of Mr. Gilles Morier of our university's Research Services was most valuable in dealing with all the contractual aspects of this project. We also wish to thank Ms. Mary Louise Graham for the initial technical training provided to Ms. Oya Hill.
Mr. John Duffy of Bio-Safe Skin Products Inc. (formerly Biotech-Transfer) was very helpful and understanding throughout the duration of this study and we thank him for it.

 

LITERATURE CITED


Ansari, S.A., S.A. Sattar, V.S. Springthorpe, G.A. Wells and W. Tostowaryk (1989). In vivo protocol for testing efficacy of handwash agents against viruses and bacteria: Experiments with rotavirus and Escherichia coli. Appl. Environ. Microbiol. 55: 3113-3118.
Graham, M.L., V.S. Springthorpe and S.A. Sattar (1996). Ex vivo protocol for testing virus survival on human skin: Experiments with herpesvirus 2. Appl. Environ. Microbiol. 62: 4252-4255.

 

 


 

TABLE 1. THE BACTERICIDAL ACTIVITY OF SKIN SHIELD
AND BIO-SAFE ANTIBACTERIAL POLYMER LOTIONS*

 

* from S.A. Sattar, Final Report to Biotech-Transfer, July 1997, p.9.
Notes:

1. Skin Shield is the 0.6% formula, Bio-Safe is the 0.3% formula
2. Biotech-Transfer incorporated Bio-Safe Skin Products Inc. in 1998

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