Does the effectiveness of hand sanitizer reduce over time - Essay Example

Hand hygiene is one of the simplest and effective ways to prevent the spread of microbial infections. Using alcohol-based hand sanitizers is highly recommended in hospitals and other professional industries where water and hand soaps are not readily available. Although research indicates that alcohol-based hand sanitizer sufficiently reduces the numbers of disease causing microorganisms on hands, the effectiveness of expired or hand sanitizers whose expiration dates are close have not been not studied. In this research, the effectiveness of alcohol-based hand sanitizers against Escherichieae coli and Staphylococcus aureus was studied by using agar-well and disk diffusion methods. The numbers of the bacteria on hands before and after applying the hand sanitizers were compared, with the dates printed on the hand sanitizers being crucial in determining their storage/shelf lives. Zone of inhibition was measured in agar-well and disk diffusion methods to determine the antibacterial effectiveness of the hand sanitizers. Concentration plating technique was also used to quantify the numbers of bacteria colonies on hands before and after applying the sanitizers. The result showed that agar-well method was not suitable for testing an effectiveness of a hand sanitizer. For disk diffusion test, E.coli was susceptible to a hand sanitizer, while S. aureus was resistant to a hand sanitizer. A hand sanitizer with 31 months to the expiration dates was found to be more effective against E.coli than expired hand sanitizers as the zone of inhibition was comparatively larger. Comparing hands before and after the application of hand sanitizers, the use of a hand sanitizer with 31 months before expiration and 6 months after expiration showed 99.15% and 87.68% decrease respectively in the numbers of bacteria on hands. Hand sanitizers with more than 12 months before expired were, therefore, recommended, as their antibacterial effectiveness was found to be greater. Introduction Hand hygiene is one of the simplest and effective ways to prevent the spread of microbial infections. A hand sanitizer is a supplement or an alternative to hand washing with soap. The purpose of a hand sanitizer is to eliminate germs on hands that could cause diseases. Although a proper hand washing with soap is more effective in killing bacteria and viruses than hand sanitizers (Grayson et al., 2009), the latter is widely used in most hospitals as fast and efficient antiseptic tool when water and hand soaps are not readily available. Accessible dispensers with an alcohol-based, waterless hand sanitizer have been demonstrated to significantly increase hand hygiene among health care workers (Bischoff et al., 2000). Notably, a hand sanitizer is not only effective against bacteria and viruses, but also on other microorganisms such as fungi. During a flu season, for instance, alcohol-based hand sanitizers have proven effective in getting rid of such disease causing microorganisms commonly associated with respiratory and/or gastrointestinal infections (Sandora et al., 2005). There are two main types of hand sanitizer: alcohol-based and non-alcoholic based sanitizers. Non-alcohol based hand sanitizer consists of non-toxic and non-flammable ingredients which are friendly to users and the environment. However, such hand sanitizers may cause germs to develop resistance to the sanitizing agent, and so, they merely reduce their growth rather than kill them (Todd et al., 2010). In contrast, alcohol-based hand sanitizers contain ethanol concentrations of between 60–95%, with excellent chances of killing gram positive, gram negative and fungal organisms. Ethanol functions to denature proteins in the infectious microorganisms and no resistance to this type of hand sanitizers have been identified, in effect, making it more effective at killing germs than those with a lower alcohol concentration or non alcohol-based hand sanitizers (Kampf and Kramer, 2004). However, both types of hand sanitizers may not work well with heavily soiled or greasy hands from handling of fatty foods and/or playing sport (Charbonneau et al., 2000). Therefore, this study focused only on alcohol based hand sanitizers. The main active ingredient in most hand sanitizers is ethyl alcohol, which kills germs within seconds by basically destroying/dissolving their lipid cell membranes and denaturing proteins within them. Other ingredients are caprylyl glycol, isopropyl myristate, tocopheryl acetate, and fragrance. Caprylyl glycol is one of medium chain triglyceride family that helps increase the shelf life of a product by increasing the antimicrobial activity of other preservatives (Traul et al. 2000). Isopropyl myristate is synthetic oil widely used in the cosmetics and pharmaceutical industries as a lubricant or emollient. Given the rapidness and the physical nature by which the alcohol based hand sanitizers act, there is no mechanism by which the germs can become resistant to it. They also evaporate from the hands within seconds, giving the microorganisms no opportunity for adaptation. As Fendler et al. (2002) notes in their studuy, alcohol hand sanitizer is but highly effective against a broad spectrum of bacteria, including antibiotic-resistant species such as vancomycin-resistant Enterococcus faecalis (VRE) and kills them in less than 30-seconds. In addition, it was also highly effective against fungi and some viruses such as Influenza virus and Rhinovirus. However, an alcohol-based hand sanitizer has its limitation; it is not as effective as antimicrobial hand soap in killing some of the bacteria and/or viruses. It is ineffective, for example, against Norwalk virus on contaminated hands (Liu et al., 2010). This study concentrated on S. aureus and E.coli since both bacterial species were found in high level on bare and gloved hands with poor hand hygiene (Ayçiçek et al., 2004). Although many researches show that an alcohol-based hand sanitizer sufficiently reduces the number of germs on hands, the effectiveness of expired and/or almost expired hand sanitizers has never been examined. Most hand sanitizer brands have the expiration periods or shelf lives of two to three years. Noteworthy, many expired hand sanitizers have been donated to third world countries because of over-storage. The effectiveness of such sanitizers in killing bacteria may well have reduced over time as alcohol content decreased below 60% (Kampf and Kramer 2004). some of the external factors such as light, heat, and mishandling, all may reduce alcohol content and degrade other antimicrobial chemicals. This study aims to address the question that the effectiveness of hand sanitizers reduces over time or not. Materials and Methods Collections of hand sanitizers and bacteria of interest 10 bottles of one or two fl oz. Purell® alcohol-based hand sanitizers with different expiration dates that varied from three years before expiration dates to six months after the expiration date were collected. All hand sanitizers were stored at room temperature with an exposure to light. The alcohol content was 70% v/v as printed in all bottles. Differences in colors and scents were ignored since they were assumed to have no effect on the antimicrobial effectiveness. Culture of Escherichieae coli and Staphylococcus aureus were prepared in nutrient broth in order to determine antibacterial effect of hand sanitizers against the two species. Using agar-well and disk diffusion methods. Preparation of wells on nutrient agar plates was modified from Bell and Grundy (1968). Sterile pen cap with 5 millimeters was used to punch four wells in each agar plate as four replicates. E.coli or S.aureus were inoculated onto the nutrient plates by using sterile cotton bud. Then, 0.5-1 ml of hand sanitizer was placed into each well. The cultures were incubated overnight at 37 °c in order to measure the zone of inhibition. Application of 1 ml of a hand sanitizer directly on cultured nutrient agars was used as a control. For the preparation of disk diffusion, blank disks were placed on emptied petri disk. The diameter of a blank disk was 5 mm. One ml of hand sanitizer was placed onto each disk. Diffused disks were left in a closed petri disk for one hour. E.coli or S.aureus were inoculated onto the nutrient plates by using sterile cotton bud. Four diffused disks were then placed onto each nutrient agar plate. The cultures were incubated overnight at 37 °c in order to measure the zone of inhibition. Test of antibacterial effectiveness of hand sanitizers on hands Four hand sanitizers, which were 31 months before expiration date, 12 months before expiration date, one month after expiration date, and six months after expiration date, were studied. Hands before applying a hand sanitizer were swopped and the sample was cultured in culture broth as the control. Then, three ml of a hand sanitizer was applied onto the hands. The hands were then dried, hand sanitizer applied, swopped and the sample cultured in a culture broth overnight. The culture broth was then transferred and diluted by using a simple concentration plating technique. The total volumes of all dilution tubes were 1000 μL. The serial dilutions were 10-4, 10-5, 10-6, and 10-7. 100 μL of each serial dilution tube was then placed and spread onto a nutrient agar plate by using a simple spread plate technique in order to get 10-5, 10-6, 10-7, and 10-8 mL of the original sample. All culture plates were incubated in an oven at 37 °C overnight before quantifying numbers of bacterial colonies on each culture plate. One spot in a culture plate was considered one bacterial colony. Numbers of bacterial colonies were counted and calculated to get the original cell density (OCD) as colony forming units per mL. The original cell density was obtained by dividing the actual number of colonies counted by the dilution of the dilution tube that multiplies by the volume transferred to the plate (mL). Result For agar-well method, zone of inhibition were not observed. Applying a hand sanitizer directly on a nutrient agar as a control also showed the same result. For disk diffusion method with S. aureus culture, no zone of inhibition were observed on nutrient agars using hand sanitizers that expired 5 months and 1 month earlier, and another that had 8 months to its expiration date. The others showed very small zone of inhibition for S. aureus. For disk diffusion method with E.coli culture, a hand sanitizer with 21 months to expiration date showed the largest average zone of inhibition; 13 mm. Hand sanitizers that had more than 15 months to their actual dates of expiration showed 10 mm. of zone inhibition or higher (Table 2). Comparing numbers of bacterial colonies on hands both before and after the application of hand sanitizers with different expiration dates at 10-7 dilution, the hand sanitizer with the furthest expiration date, 31 months to expiration, showed the highest decrease in numbers of bacteria colonies, with that of 1-month expired hand sanitizer showing the lowest decrease in numbers of bacterial colonies (Table 1). At the very 10-7 dilution, numbers of bacterial colonies decreased from 71 to none for the hand sanitizer with 31 months before expiration. Whereas numbers of bacterial colonies decreased from 168 to 91 for the 1 month expired hand sanitizer. There was a small smear portion on 10-8 dilution agar plates of hands after applying hand sanitizers with 12 months before expiration date and 1 month after the expiration date. At 10-8 dilution, numbers of bacterial colonies decreased from 3 to none for the hand sanitizer with 31 months before expiration date. However, numbers of bacterial colonies increased from 10 to 29 for the hand sanitizer with 12 months before expiration. Among four hand sanitizers with different expiration dates that were used to quantify the numbers of bacterial colonies and to calculate the original count density (CFU per ml), the hand sanitizer with the furthest expiration date, 31 months before expiration, showed a significant decrease from 7.1x109 CFU per ml to 6x107 CFU per ml which was 99.15% decrease in bacterial count density after applying the hand sanitizer on hands (Figure 1). The hand sanitizer with the furthest expiration date, 12 months before expiration, showed a decrease from 2.12x1010 CFU per ml to 5.5x108 CFU per ml, which was 97.41% decrease of bacterial count density after applying the hand sanitizer on hands. After applying the 6-month and 1-month expired hand sanitizer, the results showed 87.68% and 82.68% decrease in original count density respectively. Discussion One of the advantages of agar-well and disk diffusion techniques was that specific types of bacteria could be selected to study their resistance to a hand sanitizer. Moreover, agar-well and disk diffusion techniques were less time-consuming compared to concentration plating technique. In the agar-well experiment, no zone of inhibition was observed on both S.aureus and E.coli culture. Since alcohol content in a hand sanitizer could easily evaporate in an incubator at 37 °c, well-agar technique was not an appropriate technique to deal with chemicals that had low boiling point. Evidence from available studies indicates that methanol extracted from medicinal plants has higher antibacterial activity when using disk diffusion technique compared to agar-well technique (Parekh et al., 2005). Therefore, media such as diffusion disks were required in order to absorb alcohol-based substances and to prevent evaporation. In disk diffusion experiment against E.coli, the zone of inhibition slightly decreased as numbers of months before expiration date decreased. The decrease in zone of inhibition indicated that the effectiveness of hand sanitizer against E.coli reduced over time. Nonetheless, E.coli was susceptible to an alcohol-based hand sanitizer, with Purell hand sanitizer having approximately 99% effectiveness against enterobacteria, including E.coli (Hammond et al., 2000). However, no or small zone of inhibition was observed in disk diffusion experiment against S.aureus. Small zone of inhibition indicated that S.aureus was resistant to a hand sanitizer. Similarly, a study by David et al. (2013) showed that only 3 out of 10 alcohol-wipe brands showed antibacterial effects against S.aureus. Since S.aureus are commonly found on human skin, it is possible that they developed a resistance to antibacterial agents applied directly on human skin over time. Among four hand sanitizers with different expiration dates, a hand sanitizer with 31 months to expiration date was the most effective in reducing the numbers of bacteria on actual hands. However, it did not kill 99.99% of germs as usually stated on the bottles destined for sale. A hand sanitizer with 12 months to expiration, for instance, reduced 97.41% of bacteria on hands, which was a slight decrease in the antibacterial effectiveness compared to a hand sanitizer with 31 months to expiration date. However, none of the expired hand sanitizers tested on hands showed a significant decrease in the antibacterial effectiveness. Although the percentage decrease of unexpired and expired hand sanitizers did not have a big difference, numbers of bacteria on hands that were not killed by expired hand sanitizer were more than 2.9x109 CFU per ml. The reduction of the effectiveness of a hand sanitizer may be caused by the reduction of alcohol content over time; an observation well affirmed by Kramer et al. (2002) that suggested that antimicrobial gel that have at least 80% ethanol (v/v) and ethanol content less than 80% would have diminished antimicrobial effectiveness. The effectiveness of hand sanitizer does reduce over time. Hand sanitizers that had more than 12 months before expiration date killed more than 97% of germs, and thus, were considered effective. However, according to disk diffusion test, S.aureus, which is frequently found on human skin, was not susceptible to a hand sanitizer. As such, a hand sanitizer may not be effective against other bacteria that reside on human skin frequently exposed to antibacterial agents. That although hand sanitizers that had more than 12 months before expiration date are recommended when water and soap are not readily available, a proper hand washing with soap is more effective against a variety of bacteria, and thus, sanitizers should not be considered absolute substitutes to the hand washing with soap. As indicated in a number of studies, Gram-negative bacilli and staphylococci, including S. aureus that are not easily removed by hand sanitizers, are but easily removed by hand washing and disinfection methods (Ojajärvi, 1980). Read More