A Quantitative Determination of the Antibacterial Efficiency of Fibrous Air Filters Based on the Disc Diffusion Method

In this study, we fabricated air filters coated with Ag nanoparticles and evaluated their antibacterial efficiencies using colony counting method (CCM) and disc diffusion method (DDM) as a function of Ag nanoparticle content. Two kinds of bacterium Escherichia coli (Gram-negative bacterium), and Staphylococcus epidermidis (Gram-positive bacterium) were tested. As the amount of antibacterial agent increased, the inhibition zone width (IZW), which is the result of DDM, gradually increased and reached the maximum value, while the number of colony forming unit (CFU), which is the result of CCM, decreased and became zero. We derived a correlation equation using the CCM and DDM results, which was used to define the quantitative antibacterial efficiency of the filters according to DDM results.


INTRODUCTION
Heating, ventilating, and air conditioning (HVAC) systems, which are designed and operated to provide improved air quality in indoor environments, are required to not only ensure acceptable temperature, humidity, and air movement, but also to control various particle pollutants or biological aerosols in order to maintain human health (Barhate and Ramakrishna, 2007;Yu et al., 2009).In order to prevent bioaerosol contamination of air filters, antimicrobial treatments are used.It has been shown that treatment of fibrous air filters with antimicrobial agents can inhibit the growth of microorganisms, and that the use of such agents causes no significant difference in the filtration efficiency for either bioaerosols or inert test aerosols (Foarde and Hanley, 1999).
There are two primary kinds of antibacterial effect assessment methods: the colony count method (CCM) and the disc diffusion method (DDM).The CCM, which has been standardized by various standards institutes such as the American Association of Textile Chemists and Colorists (AATCC), the Korean Agency for Technology and Standards (KS), and International Organization for Standardization (ISO), is based on the concept of colony forming unit (CFU) numbers (AATCC, 2004;KS, 2008, ISO 2006).This test method provides a quantitative procedure for the evaluation of the degree of antibacterial activity and is widely used in microbiology and immunology.Perelshtein et al. (2009) tested the antibacterial activities of a CuO-cotton fabric composite against Escherichia coli (Gram negative) and Staphylococcus aureus (Gram positive) cultures by CFU counting, and demonstrated a significant bactericidal effect, even in 1% coated fabrics.Likewise, Niu et al. (2009) synthesized cross-linked chitosan (CCTS) silver-loaded nano-SiO 2 composites (CCTS-SLS) and evaluated their antibacterial activity, while Wei et al. (2009) studied the antibacterial effects of chitosan-based silver nanoparticles.Both of these studies employed the CCM to demonstrate the high antibacterial activities of CCTS-SLS and chitosanbased silver nanoparticles against both Gram-positive and Gram-negative bacteria.
The CCM is widely used to evaluate the antibacterial performances of air filters treated with antibacterial agents.Miaśkiewicz-Peska and Łebkowska (2011) evaluated the antibacterial ability of silver-nitrate-treated air filters against five bacterial strains, Micrococcus luteus, Maoricolpus roseus, Bacillus subtilis, Pseudomonas luteola, and Pseudomonas putida, and demonstrated a decrease in the amount of bacteria on the filter surfaces.Likewise, Yoon et al. (2008) studied the antibacterial activity of silver-treated activated carbon fiber (ACF) filters against two kinds of bacteria, E. coli, and B. subtilis, and showed that the colony ratio decreased with increasing amounts of ACF coating.Ratnesar-Shumate et al. (2008) studied about the physical capture efficiency (PRE) and the viable bacterial removal efficiency (vBRE) of the novel biocidal (Iodine-treated) filter medium and reported that the vBRE is 2-log higher than the PRE against the two bacterial strains of M. roseus and E. coli.
The DDM (also known as the Kirby-Bauer method), which is standardized by the Clinical and Laboratory Standards Institute (CLSI), is based on the appearance of a belt-like inhibition zone around an antimicrobial agent in a Petri dish containing nutrient agar and bacteria after incubation for approximately 24 hours (CLSI, 2009).The inhibition zone diameter is correlated with the minimal inhibitory concentrations (MICs) of strains known to be susceptible or resistant to various antimicrobial agents.It has been reported that a qualitative procedure that clearly demonstrates antibacterial activity as compared with a lack of such activity in an untreated specimen may be acceptable if bacteriostatic activity is the only parameter being evaluated (CLSI, 2009).In the DDM, an inhibition zone appears around a specimen coated with an antibacterial agent when the antibacterial agent diffuses from the specimen into the agar.Thus, the solubility of the agent and its molecular size can affect the size of the zone, with larger inhibition zone diameters indicating stronger antibacterial ability.Many researchers have used the DDM to evaluate the antibacterial ability of various antimicrobial agents.McGill et al. (2009) compared the antibiotic resistance profiles of 75 Campylobacter isolates of food and human clinical origin using the DDM.Similarly, Palazzo et al. (2007) studied the susceptibility of 58 coagulase-negative Staphylococci strains and 58 S. aureus strains to oxacillin by the quantitative DDM.
However, the DDM has limited usefulness for testing the performance of antibacterial air filters, since it cannot simulate what will transpire on a filter when contaminated with a bacterial species in an aerosol form.However, the antimicrobial tendency or relative antibacterial ability of a filter sample can be obtained by the DDM.We previously reported the use of the DDM to determine the performance of antibacterial air filters employing either copper or silver as an antibacterial agent.Specifically, a metallic agent was coated onto ACF filters by an electroless deposition method, and the DDM results indicated that the inhibition zone diameter increased with increasing amounts of antibacterial agent (Byeon et al., 2007;Yoon et al., 2008).
Although measurements of inhibition zone sizes and CFU numbers are tedious and prone to transcription errors, the results are nevertheless reliable, cost-effective, highly reproducible, and easy to experimentally confirm (Lestari et al., 2008;Wei et al., 2009).While results obtained using the DDM or the CCM can accurately represent antibacterial efficiency, both methods are only applicable to culturable bacteria that proliferate at a rate sufficient to form colonies, which can result in underestimation of the total number of cells due to VBNC (viable but non-culturable) cells that proliferate only under certain conditions.
Quantitative antibacterial ability cannot be evaluated by the results of the DDM by itself, since the DDM can only indicate the qualitative antibacterial ability as the diameter of the inhibition zone.However, if it were possible to quantitatively evaluate antibacterial ability by the DDM, such an approach would be both convenient and economical compared with CCM since it would not require serial dilution of bacterial cultures to obtain a suitable number of colonies, nor would it require counting to analyze antibacterial characteristics.In this study, we fabricated Agnanoparticle-coated air filters and evaluated the antibacterial abilities of the filter using both the CCM and the DDM as a function of the amount of Ag nanoparticles.We then derived a correlation equation between the CCM and DDM results to define the quantitative antibacterial efficiency of filters using the DDM.

Preparation of Silver-Deposited Air Filters
Antibacterial-treated filter samples were fabricated by coating HEPA filters with silver nanoparticles.Silver nanoparticles were generated by the spark discharge method with a silver metal electrode and a 2 kV-1 mA power source.The electrical circuit consisted of a resistance of 0.5 MΩ and a capacitance of 1 nF.Clean air at 1.5 L/min was used as the carrier gas.When a high voltage was applied to the silver electrodes, the air temperature inside the spark channel was increased beyond a critical value, which was sufficient to sublimate part of the electrode.The resulting silver vapor cooled rapidly downstream of the spark, where it nucleated and condensed (Byeon et al., 2008;Tabrizi et al., 2009).Finally, the generated particles were transported by the carrier gas to coat the HEPA filter (Fig. 1).A scanning mobility particle sizer (SMPS, 3936N22 Custom, TSI Inc., USA) was used to evaluate the size distribution and the amount of silver particles.The SMPS system consisted of a classifier controller (3080, TSI Inc., USA), a differential mobility analyzer (DMA, 3081, TSI Inc., USA), a condensation particle counter (CPC, 3022A, TSI Inc., USA), and an aerosol charge neutralizer (Soft X-ray charger 4530, HCT Co., Ltd., Korea) with a sampling air flow rate of 0.3 L/min.
The size distribution of particles is shown in Fig. 2. The mode diameter of the generated silver nanoparticles was approximately 19 nm, the total number and mass concentrations were 4.31 × 10 7 particles/cc, and 252 µg/m 3 , respectively.The geometric standard derivation of the generated particles was 1.39.The total number and mass concentrations downstream from the filter were 5.5 × 10 3 particles/cc, and 0.57 µg/m 3 , respectively.The penetrations (P = C down /C up ) of the silver particles were 1.3 × 10 -4 in number base, and 2.3 × 10 -3 in mass base, respectively.The coating areal density (ρ areal ) in number of Ag particles/cm 2 was calculated using Eq.(1) for each coating time t : where Q  is the carrier gas flow rate, A is the effective cross-sectional area of the filter sample, and C is the total mass concentration of silver aerosol nanoparticles.The superscripts "up" and "down" refer to the upstream and downstream locations of the filter sample, respectively.Table 1 summarizes the various coating areal densities for different coating times.The coating areal density increased linearly with the coating time.

Preparation of Bacterial Solution
Two kinds of bacteria were selected for the antibacterial tests: E. coli (ATCC 11775) as a Gram-negative bacterium, and Staphylococcus epidermidis (ATCC 14990) as a Grampositive bacterium.Both bacteria were prepared by liquid culture, in which the desired bacteria were suspended in BD ® Difco TM Nutrient Broth.This liquid broth consists of approximately 3 g beef extract and 5 g peptone per liter.After inoculation, the liquid broth with bacteria was grown overnight in a shaking incubator (DSS 6001, Dasol MI-Tech, Korea) at a constant temperature of 37°C.The bacterial media was then diluted with nutrient broth to an optical density (OD) of 0.002 as measured using a photospectrometer (Libra S12, Biochrom Ltd., UK) at a wavelength of 600 nm.

Bacterial Test
The filter samples for bacterial test were circular in shape (1 cm in diameter).The CCM consisted of four steps (Fig. 3(a)).First, 1 mL of inoculated bacterial solution was added to 19 mL of nutrient broth.Eight circular filter samples were then placed into the solution and incubated for 30 minutes with shaking at 37°C.Filters lacking an antibacterial agent were used as a control.Next, the incubated solution was serially diluted with de-ionized water (DI water) to obtain a countable number of colonies, and 10 µL of each diluted solution was spread onto the surface of 87 × 15 mm petri dishes containing 15 mL of nutrient agar.The plates were then cultivated in an incubator at 37°C for approximately 24 hours, after which the number of CFUs per sample were determined by counting.
The DDM also consisted of four steps (Fig. 3(b)).First, 100 µL of bacterial solution was spread onto the surface of 50 × 15 mm Petri dishes containing 5 ml of nutrient agar.Next, one piece of a circular filter sample was pasted onto each culture dish.Plates were then cultivated in an incubator at a temperature of 37°C for approximately 24 hours, after which the inhibition zone width (IZW) of the filter sample was measured with Absolute Digimatic calipers (CD-20CP, 500 series, Mitutoyo Corp., Japan) to an accuracy of 0.01 mm.Four different locations (3, 6, 9, and 12 o'clock) around each filter sample were independently measured and used to calculate an average value.A filter with no antibacterial agent was used as a control.For the DDM, the amount of bacteria per filter sample was set such that it was similar to the number of bacteria for the CCM.The results of each experimental method were averaged from a dozen replications.Antibacterial efficiency as based on the CCM (η CCM ) was calculated by the following equation: where the subscript '0' indicates the control case (bacterial concentration when exposed to a filter sample without silver nanoparticles).

RESULTS AND DISCUSSION
The experimental results for E. coli and S. epidermidis from both the DDM and the CCM analyses are shown in Fig. 4. To generalize the results of the CCM and the DDM, we defined parameters for dimensionless bacterial concentration (CFU * ) and dimensionless inhibition zone width (IZW * ) using the following equations, respectively.* 0 where IZW max is the maximum inhibition zone width.As the coating areal density (ρ areal ) increased, IZW * increased while CFU * decreased.The decrease of CFU * and the increase of IZW * can be expressed by the following exponential equations: where α and β are fitting coefficients (dimensionless).
The value of CFU * for a filter without antibacterial activity should be equal to unity, while the value of IZW * approaches zero under the same conditions.On the other hand, CFU * and IZW * should tend towards to zero and unity, respectively, as the coating areal density ρ areal increases.Table 2 summarizes the various coefficients for the two microorganisms used in this study.
A correlation equation can be obtained by eliminating ρ areal from Eq. ( 5) and Eq. ( 6): Thus, the decrease in CFU * depends on the ratio of the coefficients α and β, which were 1.61 and 0.76 for E. coli and S. epidermidis, respectively.From Eq. (2) and Eq. ( 7) the quantitative antibacterial efficiency by the DDM can be expressed as: Fig. 5 shows η DDM for various coating areal densities.The derived equation shows that antibacterial efficiencies tend to decrease with increasing coating areal density.
Therefore, the quantitative antibacterial efficiency of any antibacterial agent against a given bacterium can be easily determined once the parameters, α, β, and IZW max for that bacterium are known.The inhibition zone width (IZW) gradually increases with an increasing amount of antibacterial agent; however, IZW can become saturated when the amount of the antibacterial agent exceeds a given amount.In order to determine the value of parameter β, IZW max should be known.In Fig. 4 it is shown that IZW * is nearly the unity when CFU * is zero.Therefore, the value of IZW max can be defined as the value of IZW at the coating areal density when CFU * is zero (ρ areal = ρ CFU*=0 ).Even though various IZW values can exist when ρ areal = ρ CFU*=0   (Fig. 4(a)), the differences between these values are negligible, and the differences lead to negligible changes in the quantitative antibacterial efficiency.In this study, IZW max was chosen to be 2 mm, which was the result obtained for a coating areal density of 4.25 µg/cm 2 , accordingly.Once IZW max is determined, the coefficient β can be obtained from Eq. ( 6) for an arbitrary value of ρ areal that is smaller than ρ areal,max .Likewise, the coefficient α can be obtained from Eq. ( 5) using the value of ρ areal obtained by CCM.

CONCLUSIONS
In this study, we proposed a methodology by which a quantitative antibacterial efficiency can be obtained using the DDM.Even though our results were used to investigate the antibacterial performance of silver nanoparticles against E. coli and S. epidermidis, the quantitative antibacterial efficiency of any antibacterial agent against a given bacterium can be easily determined once the parameters α, β, and IZW max have been obtained for that bacterium.
Our proposed methodology can be used to reduce the time and costs inherent to repetitive antibacterial tests.However, our methodology is applicable only to culturable bacteria, which proliferate at a sufficient rate to form colonies, which may result in an underestimation of cells due to VBNC cells that proliferate only under certain conditions.In order to expand the methodology to non-culturable bacterium, more studies will be necessary to obtain a correlation between the DDM and direct counting methods such as fluorescence microscopy analysis.In this study, the incubation time of test bacteria was 24 hours.If different incubation time is needed, the parameters of α, β, and IZW max will have different values, however, the overall correlation trend between CFU * and IZW * will be unchanged.

Fig. 1 .
Fig. 1.Experimental schematic for coating of air filters with silver aerosol nanoparticles.

Fig. 3 .
Fig. 3. Schematic of the process used to match (a) the CCM and (b) the DDM results.

Fig. 4 .
Fig. 4. Experimental results for (a) E. coli and (b) S. epidermidis based on both the CCM and the DDM.

Table 1 .
Coating areal densities of Ag-coated air filter samples.

Table 2 .
Coefficients for E. coli and S. epidermidis.