In-vitro antibacterial activity of commercially available hand sanitizers

Hands are the prime means of transmission of microbes and the subsequent cause of nosocomial infections. To overcome the negative effect of microbial burden in health care settings, hand sanitizers are recommended for hand disinfection. Keeping this view, the present study was designed to demonstrate the antibacterial activity of fifteen hand sanitizers available in the market against the standard bacterial isolates (Staphylococcus aureus, Escherichia coli, Staphylococcus epidermidis and Pseudomonas aeruginosa) by using Kirby Bauer disc diffusion method. Their efficacy was checked at two concentrations (100% and 50%). Among the fifteen sanitizers tested, three of them Biotol=75%, Antibacterial hand gel=75% and Hiclean=62.5% were found to be the most effective. Other sanitizers such as Purell , Enliven, Lifebuoy, Rivaj were seen in the efficacy range of 12.5%-25% while no activity (0%) was observed in antiseptics Carex, Safeguard, Blue+King, Dial, Dettol, Cool and cool, Infectiguard and Newlife against all the tested bacterial isolates. Our study concluded that sanitizer Biotol, Antibacterial hand gel and Hiclean exhibited maximum efficacy at both concentrations and thus fulfilled the claim of their manufacturer as germicidal.


Introduction
Hands are the prime means of transmission of pathogens [1]. That are the subsequent cause of nosocomial infections and food borne illness [2]. Hand hygiene usually refers to different procedures of eliminating or killing microorganisms present on hands, by either hand washing or sanitizing [3,4]. It is renowned as an efficient, cost-effective and easy to perform practice that not only reduce the cross transmission of nosocomial infections but also the burden related with these infections [3,5].
Hand sanitizers (HS) can be termed as "an alternative to hand washing [6]. When opportunities are not available to access portable water or to wash hands with soap and warm water" [7]. HS are available in diversity of forms (gel or liquid) and formulations such as alcoholic and nonalcoholic [8,9]. Antimicrobial property of the antiseptics is due to their active ingredients. Among alcoholic sanitizers, ethyl alcohol and isopropyl are the common active ingredients while propylene glycol, glycerin, polyacrylic acid are the some inactive ingredients [8]. Beside these, Nonalcoholic hand rubs are also available based on benzalkonium chloride or povidoneiodine chlorinated aromatic compound triclosan and pyroglutamic acids, but studies estimating these are limited. Benzalkonium chloride is well known to have limited activity against gram-negative bacteria as compared to alcoholic hand sanitizers and is susceptible to contamination by these bacteria [10]. The mechanism of action of the sanitizers is more or less similar to denaturing the protein of microbes. Lipids are also denatured by alcohol and causes dehydration in bacteria. On the other hand, Benzalkonium Chloride has the ability to denature cell membrane and protein, and effective against wide range of organisms [11]. The present study was conducted to evaluate the efficacy of different brands of hand rubs against some skin colonizing bacteria. In order to provide data to the public, surgeons, physicians and nursing staff for the selection of better and protective hand antiseptics against disease causing microbes because all the products advertised to the community as germicidal are not efficient in decreasing microbial concentration on hands. Although, the companies claim their products are effective against "germs and harmful bacteria" by 99.9%. However, there is a dire need of study for the verification of these claims.

Preparation of dilution
The efficacy of sanitizers was checked at 100% and 50% concentrations. For the preparation of 50% concentration of sanitizers, 2ml of distilled water was added into a test tubes followed by 2ml sanitizers. Then all the tubes were vortex for proper mixing of water and sanitizers. Each sample was then put into separate petri plates labelled with their specific name.

Isolation and identification of organisms
The products were tested against the following isolates E. coli, P. aeruginosa, S. aureus and S. epidermidis. Sterile cotton swabs damped in normal saline were used to collect sample. For S. aureus and S. epidermidis, samples were collected from skin of arms and hands while transient bacteria (P. aeruginosa and E. coli) were isolated from contaminated hands of volunteer students and wound of patient. Samples were cultured on MacConkey agar and nutrient agar and then incubated for 24 hours at 37℃. Gram staining for each isolate was performed to categorize the bacteria as gram positive or gram negative. For the confirmation and studying of their biochemical characteristics, these cultures were subjected to a series of biochemical tests (Catalase, and Coagulase, Oxidase and IMVIC). S. epidermidis was further confirmed by checking its sensitivity against Novobiocin antibiotic. These isolates were maintained in nutrient broth by adding 20% glycerol and stored it at -80℃ for further use. The antibacterial activity of sanitizers checked by using Kirby Bauer disc diffusion method [12]. Sterile filter paper discs (9mm) were soaked in sanitizer for 2hrs in order to get full saturation. Sterile swab sticks were used to carpet spread the standardized culture (0.5 Mc Farland) on to the surface of already prepared Mueller Hinton agar plate and allowed to dry for 10 to 15 minutes. Discs were kept on the surface of lawn culture. Discs soaked in distilled water were used as a negative control and ethyl alcohol as positive control. Incubation was carried out for 24hrs at 37 C. Each experiment was repeated in triplicates. The antibacterial activity was analyzed by measuring the diameter of inhibition zone and recorded in millimeters (mm). Table 1 shows the susceptibility pattern of microorganisms against highly effective and least effective sanitizers with their zone of inhibition and standard error at 100% and 50% concentration.

Table 1. Susceptibility pattern of the microorganisms using sanitizers at 100% and 50% concentration by disc diffusion method
Key; SE= Standard Errors Susceptibility pattern of microorganisms against highly effective sanitizers at 100% concentration According to the results, three out of fifteen hand sanitizers that were the subject of this study showed the maximum efficacy profiles as shown in (Figure 1). It shows that sanitizer Biotol, Antibacterial hand gel and Hiclean were the most potent having highest efficacy against the three isolates:

Susceptibility of microorganisms against sanitizers at 50% concentration
Our findings indicate that, when the product efficacy was checked at 50% concentration, the same inhibitory activity

Susceptibility percentage of isolates to the sanitizers at 100% concentration
Susceptibility percentage of isolates were also calculated shown in the (Figure 3). At 100% concentration, the results indicated that among gram-positive organism, S. aureus was resistant to 40% and susceptible to 46.6% of hand antiseptics and S. epidermidis was resistant to 66.6% and susceptible to 20% of the sanitizers evaluated. Among gram-negative organisms, E. coli was resistant to 40% and susceptible to 20% of the sanitizers and P. aeruginosa was resistant to 66.6% and susceptible to 6.66% of all the antiseptics used in the study.

Figure 3. Susceptibility percentage of microorganisms to different hand sanitizers at 100% concentration
Susceptibility percentage of isolates to the sanitizers at 50% concentration Susceptibility percentage was also calculated at 50% concentration shown in (Figure 4). The results indicated that among gram-positive organisms, S. aureus was resistant to 73.3% and susceptible to 20% of the sanitizers, S. epidermidis was resistant to 80% and susceptible to 20% of all the antiseptics that are evaluated in the study. Among gram-negative organisms, E. coli was resistant to 80% and susceptible to 13.3% and P. aeruginosa was resistant to 86.6% and not susceptible to any of the hand sanitizers used in the study.   analysed the effectiveness of a number of hand sanitizers and found that among the tested products only one was efficient against 6.5% of the isolates tested. Another recent study carried out in Kenya [19] revealed that 25% of tested products were showing their activity against only 33% of the tested organisms and an indefinite number of the antiseptics were not effective at all against any of the test organism. Results checked at 50% concentration in which almost not all the sanitizers except Sanitizer Hiclean, Biotol and Antibacterial hand gel exhibited any activity against any tested organism, which supports the findings of [17] who showed that antibacterial activity of most of the hand sanitizers decreases with increase in dilution or decrease in its concentration.
Our study had few limitations as well that we did not test all the known hand sanitizers at District Abbottabad for the reason that they were not available in the market at that time. To our certain knowledge, we collected all the sanitizers, which were available at that time. One of the shortcomings of our study was that we had no knowledge of the active ingredient that are used in different hand sanitizers. Our study recommends that effectiveness of different products should be checked properly before it is commercialized and sent to the market. Manufacturer and regulatory authorities should uphold strict control through the quality and effectiveness of these products and the second most important thing is that consumer must not blindly trust the manufacturer claims as some companies' claims that their products are 99.9% germicidal but they are not so efficient. Therefore, we recommend that consumers should have prior knowledge of the products effectiveness.

Conclusion
After testing the activity of fifteen brands of hand sanitizers (alcoholic and nonalcoholic), the results showed that two alcoholic antiseptics (Biotol and Antibacterial hand gel) possessed the highest antibacterial activity, as all the tested isolates were sensitive to them. Among non-alcoholic sanitizers, Hi clean showed the best results while the other sanitizers showed the variable degree of activity. Apart from these findings, these products do not fulfill the claim of their manufacturer as having 99.99% germicidal.