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Research Article

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Use of a new, simple, laboratory method for screening the antimicrobial and antiviral properties of hand sanitizers BABAK BABAN, PHD, JUN YAO LIU, BS, FRANKLIN R. TAY, BDSC (HONS), PHD & DAVID H. PASHLEY, DMD, PHD ABSTRACT: Purpose: To develop a simple, laboratory method for screening the antimicrobial/antiviral activity of hand sanitizers, to replace the more time consuming use of human volunteers. Methods: A Rapid Agar Plate Assay (RAPA) was developed that uses sterile agar plates to simulate skin surfaces. After treating the agar plates with putative hand sanitizers, the plates were inoculated with gram-positive S. aureus or gram-negative E. coli. Untreated agar plates served as controls. After incubation for 48 hours, the bacteria were recovered and stained with fluorescent dyes. The number of live/dead bacteria was quantitated by flow cytometry. For anti-viral activity, mammalian cell lines were grown to confluency and infected with noroviruses (murine norovirus or feline calicivirus), and the number of dead cells was quantitated as the log10 of number of cells killed. A liquid hand soap without any antibacterial activity (LHS) was used as the control. A popular ethanol-based hand sanitizer (GHS) was compared to a new quaternary ammonium-containing bactericidal hand cream (ABC). Results: The liquid soap was not effective against either gram-positive or gram-negative bacteria, or viruses. Both GHS and ABC were very effective against S. aureus, but much less so against E. coli. Both GHS and ABC were even more effective against the two noroviruses that cause gastrointestinal diseases, than they were against gram-positive bacteria. These results support the use of RAPA as an effective laboratory screening test to evaluate the antibacterial/antiviral activity of hand sanitizers or other antimicrobial products. (Am J Dent 2012;25:327-331). CLINICAL SIGNIFICANCE: This laboratory study showed that some no-rinse anti-bacterial hand sanitizers can inactivate viruses better than they can kill bacteria. Hand sanitizers can contribute to universal precautions used in dental offices. : Dr. David H. Pashley, Department of Oral Biology, College of Dental Medicine, Georgia Health Sciences University, 1120 15th Street, CL-2112, Augusta, GA 30912-1129, USA. E- : [email protected]

Introduction Proper hand hygiene in dental offices can significantly reduce the risk and spread of infection.1 A recent survey of hand hygiene procedures of dental practitioners concluded that there is lack of knowledge among dentists regarding proper hand hygiene.2 When selecting hand hygiene techniques, one must determine the type of procedure to be performed (whether blood will contaminate gloves or skin), the persistence of the decontamination (whether the antimicrobial in the hand disinfectant adhere to the skin or easily rinse off), and the potential risk of spreading infection (if the patient is likely to carry hepatitis or HIV viruses).1 Examination gloves contain microscopic imperfections but are adequate for routine, noninvasive procedures. Surgical sterile gloves are preferred in high risk patients or procedures. Wearing gloves reduces the risk of contamination 70-80%.3 However, wearing gloves creates a warm, moist environment where bacteria can multiply. This increases the number of microfora on skin under gloves. Thus, hand hygiene is essential before donning new gloves. Although 69-93% of general dentists use soap and water for hand hygiene,1 most do not wash for at least 15 seconds.3 Other studies have shown that alcohol-based hand disinfectants are more effective than soap and water,4 especially those that contain agents with persistent anti-microbial activity.1 For invasive surgery in high risk patients, clinicians are advised to wash hands with soap and water, followed by an alcohol-based hand sanitizer with persistent activity, before donning sterile gloves. Dentists who come into hand contact with many patients each day can inadvertently become contaminated with patho-

gens from one patient that can be transferred to the next patient. Between patient hand washing or hand sanitizer use is known to significantly lower the risk of such cross-contamination.5-8 It is not always possible to wash hands if a sink and running water is unavailable. In such cases, good disinfection may be accomplished by using no-rinse hand sanitizers such as ethanolbased gels.9 Alcohol-based hand sanitizers are popular and have been reported to be an effective means for increasing hand hygiene compliance and reducing infection rates.10 Although alcohols used in hand sanitizers are generally regarded as nonallergic,11 there is concern that using 20-30 applications of ethanol-based hand sanitizers each day may extract important lipids from the skin that serve as a protective chemical barrier to water-borne pathogens12 and cause microabrasions in skin.13 There is also concern that inadvertent or intentional ingestion of ethanol-based hand sanitizers may result in elevation of blood alcohol levels, leading to acute toxicity.14 If hands were treated with an antibacterial/antiviral cream that was hydrophobic, the cream could coat the hands with antimicrobial agents such as chlorhexidine or quaternary ammonium compounds. These would not only disinfect skin but increase its barrier properties, using an external hydrophobic layer of silicones, for instance. Such products would not extract lipids from skin, would be nonirritating, and would offer persistent protection.15,16 Dentists and dental staff must protect themselves and their patients from office-acquired infections.2,17 While all dental instruments, burs and handpieces are sterilized and patients are protected by fresh examination gloves and face masks, the disinfection of hands and surfaces remains an important part of taking universal precautions .5 Prior to marketing, antimicrobial and antiviral hand sanitiz-

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328 Baban et al ers require proof of efficacy through clinical studies involving human subjects. To date, evaluation of the antimicrobial and/or antiviral efficacy of hand sanitizers involves the application of these agents on the hands of human volunteers.19-23 Although clinical use of human volunteers remains the gold standard for US Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC) approval, the cost of such studies is high for initial screening purposes. This study introduces the use of a new laboratory method to evaluate the disinfection properties of three different types of hand sanitizers, to reduce the cost of such screening studies by eliminating the need and expense of using large numbers of human volunteers. The test null hypotheses were that there was no difference in the antibacterial effects of the three tested hand sanitizers, and that there was no difference in the anti-viral activity of the three hand sanitizers.

Materials and Methods Antibacterial activity RAPA Assay - After reviewing a wide range of pre-clinical studies and ex vivo methods, we employed a novel method Rapid Agar Plate Assay (RAPA), that was first reported by Ansari et al.24 It is a simple and rapid method that uses the bacteriological agar surface as a surrogate substrate for skin, and combines elements of two widely used clinical methods (Agar Patch25 and Cup Scrub26,27). Bacterial strains - Staphylococcus aureus, ATCC #6538 and Escherichia coli, ATCC #11229 were purchased from Fisher Scientific.a Culture media and reagents - EZ-CFU One-Step ready-to-use bacterial preparations were re-suspended according to the supplier s instruction (S. aureus cat# 23-001-904 and E. coli cat# 23-001-51a). After achieving logarithmic (log) growth, the bacterial cells were harvested by centrifugation at 1500 g. The bacterial pellets were re-suspended in phosphate buffered saline, (PBS) and centrifuged once more. Finally, the pellets were re-suspended in PBS to achieve a spectrophotometric density of A = 0.1 at 620 nm in PBS (representing about 107 CFU/mL). Bacterial challenge dose preparation - Bacterial stock preparations of 107 CFU/mL were serially diluted to 10-5 in PBS. A bacterial challenge dose (100 µL) was derived from the 10-4 dilution, which contained about 200-300 colony forming units (CFU) of bacteria. Substrate - Trypticase soy agar plates (TSA) were used as substitute for human skin for evaluating the test products. TSA plates were prepared using 20 mL of sterile molten TSA (per manufacturer s recommendation) medium per plate. Test products - A liquid soap containing no antibacterial or antiviral agents (Soft Soap, LHS),b an ethanol-based hand sanitizer (Purell, GHS),c and an antibacterial hand cream (FiteBac, ABC)d were the test products compared in this study. GHS,b serving as the gold standard sanitizer in this study, contained 62% ethanol (vol/vol) as the active ingredient. ABCd contained benzalkonium chloride and octadecyldimethyl trimethoxysilylpropyl ammonium chloride in cyclopentasiloxane. Antibacterial evaluation The agar surface of the TSA plate was briefly rinsed in running sterile water, to ensure that the agar surface was hy-

drated. Then, with a sterile gloved hand, ABC cream, GHS and LHS (ca. 45 µg/cm2) were directly applied on the entire agar surface for 10-15 seconds. The plates were air dried under a level 2 safety cabinet in the inverted position without lid for 45 minutes. Subsequently, 100 µL of bacterial challenge dose (containing 200-300 CFU) was applied and evenly distributed using a sterile metal spreader. Plates were incubated at 37°C for 24 hours in 5% CO2 air. The bacterial colonies were counted and analyzed using appropriate statistical tools. Each product was tested in replicates of 3-6 TSA plates. Antibacterial susceptibility testing To assess bacterial membrane integrity as one of the mechanisms by which test products in this study might act against bacterial growth, the LIVE/DEAD membrane permeability methode was performed according to the manufacturer s guidelines and our previously described flow cytometry protocols.28 In brief, bacterial fluorescent stain dyes (usually red and green) were used for detecting and quantitating dead vs. live bacteria, respectively. Bacteria stained with these nonnucleic acid labeling stains exhibit bright fluorescence, and can be resolved using the appropriate flow cytometric channels. These fluorescent stains efficiently label a variety of different bacteria species. The intensity of the staining appears to depend on several factors, including gram character, outer membrane composition and overall membrane integrity. In the species tested, gram-positive bacteria generally exhibited brighter fluorescence than gram-negative bacteria, and cells with compromised membranes accumulated more dye than intact cells. Bacterial stain experimental protocol All procedures were done according to the manufacturer s instructions, and are summarized as follows: Preparation of stock solution of the fluorescent bacterial stains - A vial of the bacterial stain was warmed to room temperature before opening. A 1 mM stock solution of dye was prepared by dissolving the vial contents in 74 µL DMSO for the green bacterial stain or 69 µL for the red bacterial stain. Preparation of working solution of the fluorescent bacterial stains - A 100 µM working solution of the fluorescent bacterial stains was prepared by adding 2 µL of each of the 1 mM stock solutions prepared above to 18 µL of DMSO in a microcentrifuge tube and mixed well. Staining protocol - Bacterial colonies were harvested from culture plates as described above for RAPA, centrifuged and diluted in PBS. Then, bacterial cells were stained by adding 2 µL of the working dye solution as prepared above to 1 mL of bacterial samples, that were incubated for 15 minutes at room temperature (25°C). Flow cytometry measurements - These measurements were performed on a flow cytometer (BD FACS Caliburf) according to our previously described flow cytometry protocols.28 Briefly, red fluorescence was measured above 630 nm (FL2) and green fluorescence was measured at 488 nm (FL1). The trigger was set for the green fluorescence channel FL1. Cells were gated based on forward and side scatter properties of total bacterial cell population stained with bacterial fluorochromes as described above. Dead bacteria stained red and live bacteria stained green.29 All experiments were repeated 3-4 times.

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Antiviral activity Both viruses used in these studies, feline calicivirus (FCV) and murine norovirus (MNV),30-33 were propagated in their allocated cell lines. The FCV was used to infect CrFK cells (ATCC CCL-94) and MNV was used with RAW 267.4 cells (ATCC TIB-71). Cells were grown in Dulbecco s Modified Eagle s Medium, complemented with 10% heat-inactivated fetal calf serum, 2% penicillin (5000 U/ml) and streptomycin (5000 mg/ml), and 1% HEPES buffer. Monolayers of the respective cells were prepared in 25 cm2 flasks (5 x 106 cells) at an m.o.i. (multiplicity of infection) of 10. Infectivity and plaque assays - In order to determine if cells were infected by MNV or FCV, titers of each virus production were determined by conventional plaque assay, as previously described.33,34 Briefly, the appropriate mammalian cells were dispensed in culture plates at a density of 2 x 106 cells per plate, and grown to 80 to 90% confluency in 5 mL of complete minimum essential medium at 37°C. Then, the mammalian cells were infected with MNV or FCV for 1 hour, with gentle 15-second shaking every 15 minutes to allow viral absorption. After 1 hour, the inoculum was removed, the cells were overlaid with 5 mL of medium containing 0.5% agarose, and incubated for 48 hours to allow for viral replication and cell death. A second agarose overlay, which included 0.75% neutral red solution to identify living cells, was then added (3 mL). Plaques of dead cells were counted 8 hours later. Plates with 5 to 50 plaques were used to determine the virus titer in plaque forming units (PFU). Plaques are areas in a cell monolayer which appear as white spots when visualized by eye, caused by virus-induced lysis. Suspension viral assays - Suspension tests for antiviral activity were performed according to the protocol previously described by Macinga et al,21 with the following modification. Briefly, 4.5 mL of test substances were dispensed into a sterile 15 mL falcon tube, and mixed with 0.5 mL of the virus suspension. The mixture was vortexed for 10 seconds, and held for the remainder of the 45-second exposure time. Immediately following the exposure period, a 0.1 mL aliquot was removed from the tubes, and tested for the presence of viable virus by infectivity assays and the plaque assay described above. Statistical analyses Because the LHS controls gave reduction factors (mean log10) that were always lower than the GHS and ABC values, the data were not normally distributed. Thus, the data were analyzed using Kruskal-Wallis ANOVA. Where the corresponding omnibus test indicated statistically significant results, Dunn s multiple comparisons post-hoc analyses were used for comparing the different formulations. Statistical significance was preset at = 0.05.

Results Quantitation of the efficacy of antimicrobial agents is generally done by expressing the results in a reduction factor (RF)15 i.e., how well a test product decreases the amount of bacteria or viruses on an agar plate.15 It is calculated as the control or baseline count in log10 minus the post-treatment number using a logarithmic scale to the base of 10. That is, if a treatment reduced the number of microbes 302-fold, the mean log10 reduction of those bacteria would be 2.48 (Table 1). Any treatment that yields a reduction of more than 2 (i.e. reduced microbi-

Laboratory method for screening hand sanitizers 329 Table 1. Antibacterial activity of hand sanitizers.

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Log10 reduction (Log10 CFU/mL; mean ± SD) ____________________________________________________________ Test products

S. aureus

E. coli

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LHS (non-antibacterial) GHS (antibacterial ethanol) ABC (antibacterial QACs)

A

0.06 ± 0.03 2.48 ± 0.47 B 2.12 ± 0.55 B

0.02 ± 0.01 a 0.18 ± 0.05 b 0.11 ± 0.07 b

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LHS liquid soap hand sanitizer containing no antibacterial activity; GHS ethanol-based antibacterial hand sanitizer; ABC antibacterial hand cream containing 0.1% benzalkonium chloride and 0.5 wt% octadecyldimethyltrimethoxysilylpropyl ammonium chloride, a quaternary ammonium compound (QAC). For S. aureus (first data column), groups with the same upper case letter superscripts are not statistically significant (P> 0.05). For E. coli (second data column), groups with the same lower case letter superscripts are not statistically significant (P> 0.05).

al population 100-fold) would be considered a good disinfectant. Table 1 shows the efficacy of three hand sanitizers against two bacterial strains. S. aureus was used as an example of a gram-positive bacterium that is regarded as susceptible to common antimicrobial agents. The liquid soap (LHS) that contained no antimicrobial active ingredient only reduced the number of S. aureus by 1.15-fold (i.e. 0.06 log10). The slight efficacy is a result of dilution of the bacteria by the water in the soap. The ethanol-based GHS antibacterial hand sanitizer reduced the S. aureus count by 302-fold, while the antibacterial hand cream (ABC) reduced it by 131.8-fold. The ABC cream contained two quaternary ammonium antimicrobial agents, benzalkonium chloride and octadecyldimethyl-trimethylsilylpropyl ammonium chloride, in a hydrophobic cyclopenta-siloxane vehicle. Both GHS and AMC were effective in killing grampositive bacteria. When these hand sanitizers were tested against a gramnegative bacteria strain, E. coli, none of the hand sanitizers were very effective. The liquid soap (LHS), without any antimicrobial properties, only reduced the E. coli count 1.05-fold (i.e. 0.02 log10; Table 1). The ethanol-based hand sanitizer (GHS) reduced the E. coli count 1.5-fold (i.e. 0.18 log10), while the hydrophobic hand sanitizer (ABC) only reduced the E. coli count 1.3-fold (i.e. 0.11 log10). This indicates how difficult it is to kill gram-negative bacteria using hand sanitizers. Flow cytometry histograms showing the fluorescence of live vs. dead gram-positive S. aureus are shown in Fig. 1A, while those of gram-negative E. coli are shown in Fig. 1B. In Fig. 1A, the area under the green fluorescence peak shows the proportion of measured bacteria that had intact membrane integrity, and were not damaged by LHS, the liquid soap. The broad area under the right red curve indicates that both GHS and AMC were very effective in killing most of the grampositive S. aureus, because they disrupted the membrane integrity of those bacteria, which resulted in their death. Figure 1B shows that far more of the E. coli resisted the hand sanitizers because the area under the green curve is almost equal to the area under the red curve. This means that the hand sanitizers only killed about half of the gram-negative cells, although both GHS and AMC were more effective than LHS. Table 2 compares the antiviral activity of LHS (the hand soap), GHS (ethanol-based) and ABC (quaternary ammoniumbased) hand sanitizers against two different viruses. The LHS had no antiviral effect. Surprisingly, both GHS and AMC were more effective at killing viruses than at killing gram-positive bacteria. For the suspension virus assay, GHS hand sanitizer

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330 Baban et al

Fig. 1. Flow cytometry histograms showing fluorescence of live and dead grampositive (S. aureus), left panel, and gram-negative bacteria (E. coli), right panel, stained with two fluorescent bacterial stains. Green shaded histogram shows high bacterial membrane integrity and less damage to the cell structure, compared to the blue and red histograms, which indicate less bacterial membrane integrity and more damage to the cell structure as a result of GHS or ABC application. Table 2. Antiviral activity of hand sanitizers against noroviruses.

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Log reduction (Log PFU/mL; mean ± SD)

10 10 ___________________________________________________________________

Test products

LHS

GHS

ABC

0.8 ± 0.6A 1.0 ± 0.5a

3.20 ± 0.62B 4.85 ± 0.97b

2.35 ± 0.53B 3.18 ± 0.74b

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Feline calcivirus Murine norovirus

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LHS = liquid hand soap with no antiviral activity; GHS = ethanol-based hand sanitizer; ABC = antibacterial quaternary ammonium hand sanitizer. For feline calcivirus (first data row), groups with the same upper case letter superscripts are not statistically significant (P> 0.05). For murine norovirus (second data row), groups with the same lower case letter superscripts are not statistically significant (P> 0.05).

reduced the viral count of feline calicivirus more than 1585-fold. It was even more effective against murine norovirus, reducing the virus count 70,795-fold. The ABC hand sanitizer reduced the feline calicivirus 224-fold and that of murine norovirus by 1513fold. For each virus, the difference between the GHS and ABS hand sanitizers is statistically significant (P< 0.05). Similar results were obtained from the plaque assays, although the difference between the GHS and ABS hand sanitizers are not statistically significant (P> 0.05). The results of the two hand sanitizers and the LHS control in inhibiting plaque formation by murine norovirus are summarized in Fig. 2. A similar tendency could be seen for the feline calcivirus (not shown). In Fig. 2, three plates of mammalian cells infected with murine norovirus are illustrated. The plate treated with LHS exhibits multiple clear areas (virus- induced lytic zones called plaques). The bar graph below indicates the cumulative area of cell lysis in the LHStreated plate. Both ABC- and GHS-treated plates exhibited significantly fewer plaques than the LHS-treated plates (P< 0.05), indicating that ABC and GHS treatments were very effective in inactivating noroviruses. No difference could be detected between the two hand sanitizers.

Discussion Since the liquid soap had no antibacterial or antiviral activity, while GHS and ABC hand sanitizers had good antiviral and antibacterial activity against gram-positive bacteria, the results of this study require rejection of both test null hypotheses that there are no differences in the antibacterial or antiviral activities of the three tested hand sanitizers.

Fig. 2. Effects of different hand sanitizers murine norovirus on viral replication. Plaque assay (shown in images of plates above bar graphs) showed that treatment with either ABC or GHS decreased the plaque numbers compared to cells treated with LHS. The cumulative pixel areas of the norovirus-induced plaques were statistically compared. No significant difference was observed between ABC and GHS in terms of effectiveness. Groups identified with different lower case letters are significantly different at P< 0.05.

The results of this study support the use of the RAPA (rapid agar plate assay) technique as a screening tool to test the efficacy of various hand sanitizers against gram-positive versus gram-negative bacteria, and viruses. The use of fluorescent bacterial stains and flow cytometry saves much time and gives rapid, quantitative results. The higher efficacy of GSH and AMC at inactivating viruses compared to bacteria is probably due to the simpler exterior surface of viruses. Because of the lack of a cell culture system for human norovirus, feline calicivirus has been widely used as a surrogate to predict the efficacy of disinfectants and sanitizers against human norovirus. Recently, murine norovirus has been used as an alternative surrogate for human norovirus. However, studies on alcohol-based hand sanitizers indicated that they have variable virucidal effects of the two surrogate viruses used in the current study.35,36 Antiviral hand sanitizers may interfere with infectivity or viral replication or both processes. Alcohols are known to denature proteins.12 Quaternary ammonium compounds in ABC have fixed positive charges that change the charge density of proteins.36 They may alter the binding of viral particles to mammalian cell membranes. Noroviruses are responsible for gastroenteritis in humans. If GHS and ABC can inactivate human noroviruses as well as they inactivate both feline and murine noroviruses, they should be useful in preventing the spread of such viruses. The utility of the RAPA technique is that it can be adapted to other uses. For instance, if one wants to evaluate the substantivity of hand sanitizer ingredients, one can treat the sterile agar plates with standard “doses” of hand sanitizers and then rinse those treated surfaces with increasing amounts of water to simulate multiple hand washings. Then the washed agar surfaces could be challenged with a standard number of bacteria to determine how effective the residual antimicrobial

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agents were after multiple hand washings. This method uses equipment and techniques that are commonly found in microbiological laboratories. It saves time and money and is more precise than the glove-contamination method used with human volunteers. The RAPA technique easily identified the lack of efficacy of liquid soap hand sanitizer (LHS) and the good efficacy of both the ethanol-based GHS and the hydrophobic quaternary ammonium-based AMC. Preliminary results using ABC indicate that its residual antimicrobial activity survives far more water washings than either LSH or GSH. This is because the latter are water-soluble, while the quaternary ammonium compounds in ABC have limited water solubility. Within the limitations of such laboratory studies, the RAPA method looks promising for evaluating the antimicrobial properties of hand sanitizer activity against various microorganisms. Future studies should be done using oral microorganisms. a. b. c. d. e. f.

Fisher Scientific, Pittsburgh, PA, USA. Colgate-Palmolive Co., Piscataway, NJ, USA. GOJO Industries, Akron, OH, USA. FiteBac, Kimmerling Holdings Group, Atlanta, GA, USA. Invitrogen, Carlsbad, CA, USA. Becton Dickinson, San Jose, CA, USA.

Acknowledgement: To Mrs. Michelle Barnes for her outstanding secretarial support. Disclosure statement: Drs. Baban, Tay and Pashley and Mrs. Liu had no commercial interest in any of the products used in this study. This study was not supported by any manufacturer. Dr. Baban is an Assistant Professor of Oral Biology, Mr. Liu is a Research Assistant in Oral Biology, Dr. Tay is a Professor of Endodontics and Dr. Pashley is an Emeritus Regents Professor of Oral Biology, College of Dental Medicine, Georgia Health Sciences University, Augusta, Georgia, USA.

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