An Antimicrobial Efficacy of Different Hand Sanitizers

Hand hygiene, hand sanitizing, is essential in reducing the transmission of infectious disease.The outbreaks of any infectious diseases increased public awareness of the practice of hand sanitizing and resulted in the introduction of new products to the markets. This study assesses the antibacterial activity of selectedhand sanitizer products against some of the clinical isolates using diffusion susceptibility tests methods. The mean diameters of zones of inhibition (in mm) observed in Group 1, Group 2, Group 3, Group 4 and Group 5 were (21.4 ± 6), (08.0 ± 0.5), (11.9 ± 1.5), (11.4± 0.5) and (20.6 ± 1), respectively. Maximum inhibition was found with Group 1 against all the tested organisms. Data were statistically analyzed using analysis of variance and also were statistically significant at P < 0.001.Results showed higher inhibitory activity of the products to Staphylococcus aureus than Escherichia coli and Pseudomonas Aeruginosa All tested sanitizers showed higher zones of inhibition, indicating their overall effectiveness.

and has been reported to give better results than hand washing (Pickering et al., 2010). Scientific studies have shown that after hand washing, as many as 80% of individuals retain some pathogenic bacteria on their hands (Tambekar DH, et al, 2007). The outbreak of diseases led to an increased awareness of the role of hand sanitizers in infection control (Olalekan and Adeola 2014;Nwabuezeet al., 2016).Most of these products have made numerous claims, notably their ability to eliminate 99.9% of microorganisms (Odebisi-Omokanye 2015). Therefore, pathogenic organisms were selected to assessment the antimicrobial efficacy of different hand sanitizers.

2-Material and Methods
The present study is an in vitro study conducted at the Department of Clinical Laboratory Science-Mohammad Almanaa College. Ethical clearance for the study was obtained from the scientific research unit.

2.1-Different brands of hand sanitizers
were selected out of many available in the market based on their popularity usage.

2.2-Tested microorganisms
Mueller-Hinton, nutrient broth agar and nutrient agar were used for agar diffusion for bacterial isolate preservation. The clinical isolates of Staphylococcus aureus,, Escherichia coli and Psuedomonasaeruginosa were obtained from the culture plates of the respective microorganisms preserved on the nutrient agar slants and were stored at 4°C in the laboratory of Microbiology in Clinical Laboratory Science Department.

2.3-Susceptibility test
Disk agar diffusion technique was used for the evaluationof antimicrobial efficacy of hand sanitizers using previously described methods (Otokunefor and Dappa 2017;Magaldi et al., 2004;CLSI, 2012). The agar well diffusion testwas carried out as a preliminary screen to assess the antimicrobial activities of the various products. This involved the use of an inoculum corresponding to 0.5 McFarland.

2.4-Minimum Inhibitory Concentration (MIC):
MIC testing was carried out to determine the minimum concentration of test substances which could cause an inhibition of the growth of the test isolates. Following 24 hours incubation at 37˚C, the MIC was determined as the lowest concentration of test substance which caused an inhibition of the growth of the test organisms. The test inoculums was swab inoculated to a Mueller Hinton agar plate and allowed to stand at room temperature for 15 minutes. Following this, 4 wells were created on the plates using a 6 mm cork borer and 0.2 ml of differing concentrations (100%, 50% and 25%) with three replicates of the test substance added to individual wells. After 24hoursincubation at 37˚C, the zones of inhibition were then measured (Otokunefor and Dappa 2017).

2.5-Minimum Bactericidal Concentration (MBC):
Todetermine the MBC of each test substrate, against each test isolate, the three lowest concentrations which resulted in an inhibition of the test organism were subcultured into nutrient agar plates, incubated at 37˚C for 24 hours and observed for growth. The MBC was taken as the least concentration which did not result in the growth of the organism (Otokunefor and Dappa 2017).

2.6-Statistical analysis
Data were statistically analyzed with analysis ofvariance followed by post hoc test for group-wisecomparisons. All statistical procedures were performedusing Statistical Package for Social Sciences (SPSS) version 21.0 software (IBM, Armonk, NY, USA). zone of inhibition (in mm) wasanalyzed using the mean of all the readings obtained,and the difference in the values of different handsanitizers was statistically significant at P < 0.001.

3-Results and Discussion
All tested organisms were affected by Hand sanitizers. the zone of inhibition against the particular test organismwas performed to assess the antimicrobial effectiveness was assessed by measuring. Maximum inhibition (in mm) was seen in Group 1 i.e., 21.4 ± 1.414 and minimum in Group 2 i.e., 8 ± 0.95. The difference in the values of the different sanitizers was statistically significant [P < 0.001, Table 1.
Group 1 showed the highest antimicrobial effectiveness followed by Group 2, 3, 4and Group 5, against all the different test organisms used in the study. Group 1 & 5 could inhibit all of the tested bacteria, whereas other group of sanitizers showed a limited action (Table 1).
It was found statistically high significant difference was observed against all the bacterial isolates when Group 1 was compared with any other group. However, there was no statistically significant difference when Group 2, Group3, and Group 4 were compared with each other against all test organisms. The mean difference is statistically significant at the 0.05 level. The highest inhibition zoon was register for group 1 against S. aureus (24.9mm) while the lowest was for group 2 against E. coli (6.6mm) ( Table 1, 2) All hand wash products exhibited inhibitory activity against the test isolates (Table 2), with zones of inhibition ranging from 14.2 mm to 24.2 mm at concentrations of 100%, while it was ranging from (6.0 to 16.2) mm at concentrations of 50%. A general reduction in inhibitory activity was associated with a reduction in product concentration, and inhibition was still observed at concentrations as low as 25%, in some cases ( Table 2). The widest variation was observed with group 1 and 5 which showed a high % inhibition in growth of against all tested microbes as 8-24.2 mm inhibition of S. aureus growth. Group 2 was the least effective. This product showed low activity at all against al microbes comparing to others. An MIC of 25% (Table 2), Group 1 and 5 appeared to be the more effective hand wash as it showed an MIC less than 50% in 75% of cases as opposed to the other products which showed an MIC of >50% only in 50% of cases (Table 3).while MBC was varied among microbes and groups and ranged from 25 -100% asshowed in table 3.

4-Discussion
The Human body and especially normal human skin always harbor bacteria (10 2 and 10 6 CFU/cm 2 ). As main source of transfer infections, hand considered to play main role in transfer of bacteria from the hands to food, so that people plays an important role in the spread of many communicable diseases. (Kimura AC, et al., 2004). Microbe transferring from hands and causing infections, depends on many factors such as; density of microorganisms on the hands, the type and duration of contact, the type of micro-organism, resident flora, and their colonization resistance ( To overcome the negative impact of microbial contamination, hand sanitizers are recommended as a hygiene technique to keep hands clean. (Boyce JM, and Pittet D, 2002) Most commonly and available hand sanitizers in the market were selected for the study. Among the four hand sanitizers used in this study, group 1, 3, 4 and, 5 were alcoholbased and group 2 was herbal, i.e., nonalcohol-based hand sanitizer. Many studies have been conducted to assess the antimicrobial effectiveness of hand sanitizers alone, but very few literatures are available to assess the difference between various disinfectants and hand sanitizers. Disinfectants are chemical agents with an immediate and sustained activity which destroys micro-organisms to such a level mandated for hygienic and surgical indications. On the other, handsanitizers are agents with an activity to reduce micro-organismsnumbers meet the public health requirements. Alcohol is the better form as disinfectant (propanol) to reduce bacteria as compared to ethanol sanitizers in 15-30 s.
One of the previous research studied the impact of hand hygiene in disease prevention, they found that hand sanitizing has more recently been the proscribed method of hygiene, possibly due to the higher compliance rates associated with it and its particular usefulness in areas lacking adequate water supply (Nwabuezeet al., 2016). Over the years, S. aureus and E. coli have been documented as the two most common pathogens isolated in the clinical microbiology laboratory. These organisms are notorious for their ability to cause a wide variety of diseases, exhibit a wide repertoire of virulence factors and a high level of antibiotic resistance. Additionally, along with K.pneumoniae and P. aeruginosa, these organisms canbe spread via the hands. This study found variable efficacy of the hand sanitizers assessed. While similar levels of inhibition were noted against S. aureus and K.pneumonia. Based on the results, the variable level of activity of hand sanitizers were reported, by Sharif and Ansari, and found that the efficacy of many types hand sanitizing, that only one product was effective against 6.5% of the isolates tested (Sharif and Ansari 2015).  (2017) found that 25% of tested products were effective against only 33% of the test isolates. While study reported a possible link of efficacy to composition and noted that the ethanol-based products resulted in a higher efficacy than the isopropyl based products.For most alcohol-based hand sanitizers, the alcohol components are the major active ingredients. These act by disrupting tissue membranes, denaturing proteins and dissolving lipids (Oke et al., 2013). It therefore follows that products with higher alcohol concentrations (up to 90%) would be more effective than products with lower alcohol concentrations (below 60%). Similar to a previous report (Odebisi-Omokanyeetal., 2015), our study noteda lower level ofsusceptibility to all the tested products in 2 of the 3 Gram negative organisms tested. Both S. aureus and K. pneumoniae however, showed similar levels ofsusceptibility. The results of this study were totally agreed with both (Oke et al., 2013;Odebisi-Omokanye et al., 2015). And unlike both studies which reported a total lack of bacteriocidal activity possibly due to improper storage, all hand sanitizing products in this study exhibited bactericidal activity this point at the general effectiveness of the products assayed in this study.

5-Conclusion:
While the results of this study show thatthe products assayed have higher efficacy and higher inhibitory activity to S. aureus than E. coli andP.Aeruginosa. All the sanitizers showed good activities, with inhibition of bacteria noted at concentrations as low as 25% and MBC was varied among microbes and groups and ranged from 25 -100% . More stringent checks of products introduced into the market may therefore be necessary to ensure that they meet set international standards both in the composition of inhibitory substance and texture to ensure uniformity in activity against pathogens.

6-AKNOWLEDGMENT
Research stared by the supporting of Research Unit in Mohammed Al-Mana College for Medical Sciences. Ethical approval number ( Scientific Research Unit is pleased to inform you that your proposal entitled " An in vitro assessment of antimicrobial efficacy of different hand sanitizers " is approved from IRB with the approval number "SR/RP/46".