Determining the efficacy of hand sanitizers against virulent nosocomial infections

The goal of this study was to determine the effectiveness of 0.13% benzalkonium chloride (BAC) (Steirolotion™), 100% ethanol, and 70% ethyl alcohol (Purell™) hand sanitizer in subduing the growth of nosocomial bacteria – methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant Enterococcus (VRE), and Pseudomonas aeruginosa (P. aeruginosa) - when plated on culture media over an extended period of time. In addition, our objective was to quantify the length of time these hand sanitizer agents remained effective, and to extrapolate their efficacy in decreasing the transmission of hospital-acquired infections. 50 microliters of either BAC, 100% ethanol, or 70% ethyl alcohol hand gel sanitizer were pipetted onto Trypticase soy agar with 5% sheep blood plates that were cultured with either MRSA, VRE, or P. aeruginosa. The plates were then incubated at 37.0?. The zone of inhibition (ZOI) was measured daily for 5 days and additionally zones were noted whether or not regrowth recurred in areas where previous growth had initially been inhibited. BAC was found to be superior to both 100% ethanol and 70% ethyl alcohol in the inhibition of MRSA over all time points (p values < .05). BAC was found to be superior to 70% ethyl alcohol in the inhibition of VRE over all time points (p values < .05), but not statistically superior to 100% ethanol against the inhibition of VRE over any time points. BAC was found to be superior to 70% ethyl alcohol and 100% ethanol in the inhibition of pseudomonas over 72 and 24 hours, respectively (p values < .05). The results of this study demonstrate in vitro efficacy of BAC of preventing regrowth of common nosocomial bacteria over a prolonged period of time, especially when compared to ethyl alcohol. Further study is warranted to determine in vivo effectiveness of this formulation of BAC as well as the appropriate time frame of application for effectiveness against P. aeruginosa.


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Hospital-acquired or healthcare-associated infections (HAIs) are the most common 54 complication in hospitalized patients [1]. They occur with an estimated incidence of 4.5 HAIs 55 per 100 hospital admissions, and amount to an additional burden of $35 to $45 billion dollars on 56 the healthcare system [2]. They are responsible for significant hardship accounting for more than 57 90,000 deaths each year, putting HAIs among the top 5 leading causes of death in the United 58 States [3][4][5]. Transmission of pathogens from healthcare staff serves as an important source of 59 HAIs. Personal hygiene is a crucial aspect of reducing transmission, and hand washing, or 60 sanitizing is required with every patient contact [6].

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Both alcohol-based and alcohol-free hand sanitizers are available options when hand 63 washing is not available or efficient. Alcohol-based sanitizers containing 60-95% alcohol are 64 most often used in hospitals. Benzalkonium chloride (BAC) is the active ingredient contained in 65 most alcohol-free hand sanitizer products available today. It has been theorized that BAC 66 possesses an extended killing time of bacteria when the solution has dried compared to alcohol-67 based agents [7-10].

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The goal of this study was to determine the duration of efficacy of .13% BAC, 70% ethyl 70 alcohol, and 100% ethanol in decreasing methicillin-resistant Staphylococcus aureus (MRSA), 71 vancomycin-resistant Enterococcus (VRE), and Pseudomonas aeruginosa (P. aeruginosa) 72 colonization and regrowth over an extended period of time when plated on culture media. 73 74 Materials and Methods

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BD BBL™ Trypticase™ soy agar slants prepared media of nosocomial bacteria MRSA, 76 VRE, and P. aeruginosa were grown on agar plates for 24 hours at 37 degrees Celsius and used 77 to establish a reservoir. 78 79 A 0.5 McFarland standard solution was created for the MRSA, VRE, and P. aeruginosa 80 bacteria strains, by using a calibrated inoculating loop to transfer bacteria from the incubated 81 blood agar plates to a vial of saline with 0% absorbance until the absorbance of the vial solution 82 was between 0.08 and 0.1%.

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A bacteria lawn was created by using a cotton applicator to evenly distribute an aliquot of 85 the 0.5 McFarland standard MRSA solution across the surface of Trypticase™ soy agar with 5% 86 sheep blood plates. This process was repeated using the 0.5 Mcfarland standard for MRSA, 87 VRE, and P. aeruginosa solutions until eight bacteria lawns of each solution were created. 88 89 50 microliters of 0.13% benzalkonium chloride (BAC) (Steirolotion TM , Germcure, Houma 90 Louisiana), 100% ethanol (Sigma-Aldrich Inc., St. Louis, Missouri) and ethyl alcohol 70% 91 (Purell™, Gojo, Akron, Ohio) solution were pipetted onto each of the eight 5% sheep blood agar 92 plates. Reverse pipetting was used to ensure accurate amounts of viscous solution were pipetted 93 onto the plates. Plates were left for one hour to dry.

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The MRSA, VRE, and P. aeruginosa inoculated plates were incubated at 37 degrees 96 Celsius overnight. They were all grown in aerobic conditions. The plates were removed from the 97 incubator every 24 hours for a growth period of 120 hours to take photographs and quantitative 98 measurements of the zone of inhibition (ZOI) of each antiseptic. Measurements were performed 99 for a total of 120 hours for the MRSA, VRE, and P. aeruginosa plates. 100 101 One methodology was utilized to perform quantitative measurements of the ZOI for each 102 antiseptic. The methodology used to perform digital measurements of the ZOI was the free 103 internet software program, ImageJ. ImageJ utilizes the pixels of the digital photographs taken 104 and the known standard diameters of the agar plates to quantitatively measure the ZOI. Four 105 researchers made the digital measurements independently to increase validity of the 106 measurements. 107 108 Statistical analysis was performed by measuring the difference in area of inhibition 109 between ethyl alcohol, ethanol, and benzalkonium chloride for P. aeruginosa, VRE, and MRSA 110 each. P-values were obtained using t-tests comparing each solution independently. 111 112 Results

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Ethyl alcohol and Ethanol showed significant regrowth of bacteria within 24 hours 114 against P. aeruginosa, VRE, and MRSA (Figs 1-3). This regrowth of bacteria continued the full 115 5 days, or 120 hours, that the study was conducted. BAC showed regrowth of only P. aeruginosa 116 after the initial 24-hour period had passed. For BAC, no regrowth was noted throughout the 120 117 hours in MRSA or VRE after the initial ZOI had been established (Figs 1 and 2, 4 and 5). For P. aeruginosa, BAC showed larger zones of inhibition on day 2, 3 and 5, but ethanol 131 showed larger zones of inhibition on days 1 and 4. This is shown in Table 1. Aside from days 1 132 and 4 of pseudomonas, Table 1, Ethyl alcohol did not demonstrate a clear zone of inhibition for 133 any other plates and thus was labeled as 0 due to significant regrowth of bacteria beginning at 24 134 hours and lasting the full 120 hours (Figures 1-6) The ratio of percent loss of initial zone vs each subsequent zone revealed that ethyl 153 alcohol did not inhibit growth of MRSA and VRE throughout the study period (Figs 7 and 8). 154 BAC was most effective at preventing regrowth of all three bacteria. All three solutions had 155 significant regrowth of P. aeruginosa at day 5; BAC had a regrowth of 95.5%, Ethanol 98.0% 156 and ethyl alcohol 100% (Fig 9). For VRE by day 5, BAC showed to have a 7.6% regrowth of the 157 initial zone vs Ethanol's 53.0% and ethyl alcohol's 100% regrowth (Fig 8). The day 5 regrowth 158 of MRSA vs BAC was shown to be only 5.7% while it was 21.9% and 100% for ethanol and 159 ethyl alcohol, respectively (Fig 7). Overall, BAC did not exhibit any regrowth within the initial 160 zone of inhibition vs MRSA and VRE (Figs 1 and 2, 4 and 5). Causative agents of HAIs and the routes by which they spread have been well 171 documented for over a century. Despite knowledge of the factors that influence the risks of HAIs 172 and means to prevent or control them [11], patients in the healthcare setting continue to acquire 173 HAIs [8]. These infections result in increased morbidity and mortality for the patient, and 174 ultimately higher health care costs for both the patient and the health care system [2, 12]. As one 175 example, it's estimated that preventing a single postoperative surgical site infection could 176 potentially save the healthcare system upwards of $60,000 [9]. 177 178 The overlying goal of this study was to test the effectiveness and duration of effect of 179 hand sanitizers that could be used in the healthcare setting to potentially decrease the incidence 180 of HAIs. This study demonstrates the anti-microbial activity of alcohol-based hand sanitizing 181 agents (70% ethyl alcohol and 100% ethanol) and alcohol-free agents (BAC) against common 182 virulent antibiotic-resistant micro-organisms MRSA, VRE, and P. aeruginosa in vitro over a