In vitro Antimicrobial Activity Assessment of Zymox Otic Solution Against a Broad Range of

In vitro Antimicrobial Activity Assessment of Zymox Otic Solution Against a Broad Range of Microbial Organisms

Rajvinder Atwal, PhD*

*Laclede Inc., Rancho Dominguez, California

This research was performed in the Laboratory Services Division, TherImmune Research Corporation, Gaithersburg, Maryland.

KEY WORDS: Zymox Otic solution, antimicrobial activity, bacteria, microorganisms

Abstract

The goal of this study was to assess the in vitro antimicrobial activity of Zymox Otic solution against a broad panel of microbial organisms. Serial twofold dilutions of Zymox Otic were used in microbroth dilution assays against 3 Gram-negative bacteria (Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa), 4 Gram-positive bacteria (Staphylococcus aureus, Streptococcus intermedius, Streptococcus pyogenes, and Corynebacterium diphtheriae), and 2 fungi (Candida albicans and Malassezia pachydermatis). The results of these assays showed that Zymox Otic inhibited the growth of every organism tested at various test compound dilutions. Zymox Otic had the greatest amount of antimicrobial activity against S. pyogenes exhibiting a minimal inhibitory concentration (MIC) at a 1:32 dilution of the test compound. A 1:16 dilution of Zymox Otic effectively inhibited the growth of S. aureus, E. coli, S. intermedius, C. albicans, P. aeruginosa, C. diphtheriae, and M. pachydermatis; and a 1:8 dilution of Zymox Otic effectively inhibited the growth of P. mirabilis.

Materials and Methods

The materials and reagents were used in the study are shown in Table 1.

The minimal inhibitory concentration (MIC) of Zymox Otic against each microorganism was determined using microbroth dilution analysis. Bacterial and fungal strains were obtained from American Type Culture Collection (ATCC) and cultured according to the manufacturer's specifications. For each organism tested, 10 mL of sterile liquid broth media was inoculated with a freshly plated, single-colony isolate and incubated overnight to establish logarithmic growth. The specific media and growth condition used for the individual organisms is detailed in Table 2. After overnight incubation, the bacteria or fungus was pelleted by centrifugation (3100 x g, 5 min), the supernatant discarded, and the pellet suspended in 0.9% sterile saline solution (9 g/L sodium chloride) to an optical density at 625 nm equal to 0.1. This density was assumed to be equivalent to 1 x 108 colony-forming units (CFU)/mL for dosing purposes. The suspended inoculum was diluted 1:100 in sterile culture media to a concentration of 1 x 106 CFU/mL. One hundred microliters of the diluted inoculum was added to triplicate wells of a 96-well plate containing 100 mL of freshly prepared Zymox Otic solution serially diluted twofold in the respective sterile culture media. One hundred microliters of the diluted inoculum was also added to triplicate wells containing 100 mL of serial twofold dilutions of a positive control antibiotic and to triplicate wells containing 100 mL of media only. This inoculation scheme yielded final concentrations of each microbial organism estimated to be 5 x 105 CFU/mL and Zymox Otic solution ranging from a high-test dilution of 1:2 (inoculum mixed with 100 mL of undiluted Zymox Otic) to a low test of 1:2048. The plates were incubated 16 to 48 hours at 370C, and the inhibition of bacterial growth was determined by measuring the optical density at 625 nm for of each well using a Spectramax 384 plate reader. The MIC for each compound was defined as the lowest compound dilution that completely inhibited growth. The results are presented as the percentage of growth relative to the no-compound control and were calculated using the formula:

100% x OD₆₂₅X

OD₆₂₅NC

where OD₆₂₅X is the average OD₆₂₅of the triplicate test samples and OD₆₂₅NC is the average OD₆₂₅of the triplicate no compound control samples.

Results and Discussion

The results of our analysis indicated that Zymox Otic had antimicrobial activity against all organisms tested (Table 3 and Figs. 1 through 9). Zymox Otic had the greatest amount of antimicrobial activity against Streptococcus pyogenes, exhibiting an MIC at a 1:32 dilution of the original test compound. A 1:16 dilution of Zymox Otic effectively inhibited the growth of Staphylococcus aureus, Escherichia coli, Streptococcus intermedius, Candida albicans, Pseudomonas aeruginosa, Corynebacterium diphtheriae, and Malassezia pachydermatis; and a 1:8 dilution of Zymox Otic effectively inhibited the growth of Proteus mirabilis. All of the control antimicrobial compounds had effective MICs within published ranges for the organisms tested. The Zymox Otic compound formed a gelatinous precipitate at the highest concentrations (1:2 and 1:4 dilution, Figs. 1 through 9 panel C) leading to an increase in the measured optical density at the high-test concentrations relative to the intermediate concentrations. For Malassezia pachydermatis (Fig. 9, panels A and C), one well for each of the test concentration triplicates had a significantly higher OD₆₂₅ than the other 2 replicates. Examination of these wells indicated that the fungus failed to evenly disperse during culturing and formed a mat on the top of the culture media. The presence of this mat inflated the optical density measured for these wells. Because this phenomenon was not observed in the no-compound control wells, the graph of the M. pachydermatis results that includes these data indicates an artificial enhancement of growth by the Zymox Otic compound at the higher concentrations (Fig. 9 panel C).

Table 1. Materials and Reagents Used in the Study

Staphylococcus aureus ATCC 29213

Escherichia coli ATCC 25922

Streptococcus pyogenes ATCC 10389

Proteus mirabilis ATCC 14153

Streptococcus intermedius ATCC 27335

Candida albicans ATCC 10231

Pseudomonas aeruginosa ATCC 27853

Corynebacterium diphtheriae ATCC 14779

Malassezia pachydermatis ATCC 42756

96-well U-bottom plates Falcon 353227

TSA II 5% sheep blood agar plates Remel 01202

Disposable bacterial spreaders VWR WLS23286

Trypticase soy agar BBL 211043

YM broth Difco 271120

YM agar Difco 271210

Sabouraud dextrose broth Difco 238220

Nutrient broth Difco 233000

Brain heart infusion media Remel 452471

Sabouraud dextrose agar emmons Remel 454471

Trypticase soy broth BD 211768

Saline 0.9% LC234601 Fisher

Mueller Hinton II broth BD 297310
(Cation adjusted)

Ampicillin Fisher BP1760-25

Gentamycin Sigma G1522

Amphotericin B Fisher BP264520

Bifinazole Fisher ICN15485601

Zymox Otic solution Laclede

Table 2. Growth Media and Culture Conditions Used to Culture and Assess the Antimicrobial Activity of Zymox Otic for Each Respective Organism

Staphylococcus aureus Trypticase soy broth 37˚C/ambient air/16 hr

Escherichia coli Trypticase soy broth 37˚C/ambient air/16 hr

Streptococcus pyogenes Mueller Hinton II broth (Cation adjusted) 37˚C/ambient air/16 hr

Proteus mirabilis Mueller Hinton II broth (Cation adjusted) 37˚C/ambient air/16 hr

Streptococcus intermedius Brain heart infusion media 37˚C/5% CO₂/16 hr

Candida albicans YM broth 37˚C/ambient air/24 hr

Pseudomonas aeruginosa Mueller Hinton II broth (Cation adjusted) 37˚C/ambient air/16 hr

Corynebacterium diphtheriae Trypticase soy broth 37˚C/ambient air/24 hr

Malassezia pachydermatis Sabouraud dextrose broth 37˚C/ambient air/48 hr

Table 3. Minimal Inhibitory Concentration (MIC) of Zymox Otic and Control Compounds

Staphylococcus aureus Ampicillin (mg/mL) 1

Zymox (dilution) 1:16

Escherichia coli Ampicillin (mg/mL) 16

Zymox (dilution) 1:16

Streptococcus pyogenes Ampicillin (mg/mL) 0.04

Zymox (dilution) 1:32

Proteus mirabilis Ampicillin (mg/mL) 2

Zymox (dilution) 1:8

Streptococcus intermedius Ampicillin (mg/mL) 25

Zymox (dilution) 1:16

Candida albicans Amphotericin B (mg/mL) 1.56

Zymox (dilution) 1:16

Pseudomonas aeruginosa Gentamicin (mg/mL) 0.78

Zymox (dilution) 1:16

Corynebacterium diphtheriae Ampicillin (mg/mL) 0.39

Zymox (dilution) 1:16

Malassezia pachydermatis Bifonazole (mg/mL) 0.1

Zymox (dilution) 1:16

The listed MIC for each of the control compounds is given in µg/mL and the MIC for Zymox is listed as a dilution of the original starting material in the respective growth media

Figure 1. Determination of minimal inhibitory concentration of ampicillin (B) and Zymox Otic (C) against Staphylococcus aureus.

Figure 1A

Ampicillin (µg/mL) 256 128 64 32 16 8 4 2 1 0.5 0.25 0

Sample 1 0.043 0.042 0.045 0.049 0.042 0.199 0.583 1.053 1.206 1.235 1.234 1.279

Sample 2 0.043 0.042 0.045 0.046 0.047 0.199 0.648 1.042 1.166 1.196 1.203 1.236

Sample 3 0.042 0.044 0.052 0.067 0.05 0.147 0.606 1.028 1.175 1.193 1.204 1.229

Average 0.043 0.043 0.047 0.054 0.046 0.182 0.612 1.041 1.182 1.208 1.214 1.248

% Control 3.4 3.4 3.8 4.3 3.7 14.6 49 83 95 97 100 100

Zymox (dilution) 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 1:2048 0

Sample 1 0.271 0.054 0.066 0.057 1.119 1.09 0.985 0.95 0.926 0.939 1.024 1.223

Sample 2 0.108 0.256 0.117 0.062 1.054 1.092 0.995 0.95 0.952 0.942 1.042 1.217

Sample 3 0.177 0.066 0.062 0.061 1.074 1.096 0.996 0.979 0.967 0.983 1.058 1.272

Average 0.185 0.125 0.082 0.060 1.082 1.093 0.992 0.960 0.948 0.955 1.041 1.237

% Control 15.0 10.0 6.6 5.8 87.0 88.0 80.0 78.0 77.0 77.0 84 100

Figure 1C

Figure 1B

Figure 2A.

Ampicillin (µg/mL) 256 128 64 32 16 8 4 2 1 0.5 0.25 0

Sample 1 0.043 0.042 0.045 0.049 0.042 0.199 0.583 1.053 1.206 1.235 1.234 1.279

Sample 2 0.043 0.042 0.045 0.046 0.047 0.199 0.648 1.042 1.166 1.196 1.203 1.236

Sample 3 0.042 0.044 0.052 0.067 0.05 0.147 0.606 1.028 1.175 1.193 1.204 1.229

Average 0.043 0.043 0.047 0.054 0.046 0.182 0.612 1.041 1.182 1.208 1.214 1.248

% Control 3.4 3.4 3.8 4.3 3.7 14.6 49.1 83.4 94.7 96.8 97.2 100

Zymox (dilution) 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 1:2048 0

Sample 1 0.271 0.054 0.066 0.057 1.119 1.09 0.985 0.95 0.926 0.939 1.024 1.223

Sample 2 0.108 0.256 0.117 0.062 1.054 1.092 0.995 0.95 0.952 0.942 1.042 1.217

Sample 3 0.177 0.066 0.062 0.061 1.074 1.096 0.996 0.979 0.967 0.983 1.058 1.272

Average 0.185 0.125 0.082 0.060 1.082 1.093 0.992 0.960 0.948 0.955 1.041 1.237

% Control 15.0 10.1 6.6 4.8 87.5 88.3 80.2 77.6 76.6 77.2 84.2 100

Figure 2. Determination of minimal inhibitory concentration of ampicillin (B) and Zymox Otic (C) against Escherichia coli.

Figure 2C

Figure 2B

Figure 3. Determination of minimal inhibitory concentration of ampicillin (B) and Zymox Otic (C) against Streptococcus pyogenes.

Figure 3A.

Ampicillin (µg/mL) 5 2.5 1.25 0.625 0.31 0.156 0.078 0.039 0.019 0.009 0.005 0

Sample 1 0.309 0.307 0.306 0.308 0.304 0.307 0.303 0.306 0.699 0.942 1.008 1.054

Sample 2 0.311 0.304 0.306 0.306 0.31 0.307 0.303 0.304 0.819 0.996 1.059 1.097

Sample 3 0.309 0.308 0.306 0.306 0.307 0.305 0.305 0.306 0.81 0.998 1.071 1.119

Average 0.310 0.306 0.306 0.307 0.307 0.306 0.304 0.305 0.776 0.979 1.046 1.090

% Control 28.4 28.1 28.1 28.1 28.2 28.1 27.9 28.0 71.2 89.8 96.0 100.0

Zymox (dilution) 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 1:2048 0

Sample 1 1.15 1.282 0.387 0.329 0.327 0.99 1.267 1.262 1.233 1.206 1.12 1.124

Sample 2 0.934 1.297 0.404 0.339 0.328 0.869 1.085 1.085 1.112 1.099 1.073 1.098

Sample 3 1.034 0.576 0.365 0.342 0.342 0.916 1.051 1.112 1.077 1.035 1.05 1.053

Average 1.039 1.052 0.385 0.337 0.332 0.925 1.134 1.153 1.141 1.113 1.081 1.092

%Control 95.2 96.3 35.3 30.8 30.4 84.7 103.9 105.6 104.5 102.0 99.0 100.0

Figure 3C

Figure 3B

Figure 4. Determination of minimal inhibitory concentration of ampicillin (B) and Zymox Otic (C) against Proteus mirabilis.

Figure 4A.

Ampicillin (µg/mL) 100 50 25 12.5 6.25 3.125 1.56 0.78 0.39 0.195 0.09 0

Sample 1 0.057 0.046 0.045 0.046 0.049 0.054 0.1 0.958 1.104 1.063 1.041 0.966

Sample 2 0.045 0.043 0.048 0.046 0.048 0.05 0.091 0.935 1.154 1.115 1.075 0.881

Sample 3 0.046 0.043 0.048 0.045 0.045 0.05 0.095 0.931 1.136 1.067 1.052 0.837

Average 0.049 0.044 0.047 0.046 0.047 0.051 0.095 0.941 1.131 1.082 1.056 0.895

%Control 5.5 4.9 5.3 5.1 5.3 5.7 10.7 105.2 126.5 120.9 118.0 100

Zymox (dilution) 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 1:2048 0

Sample 1 0.265 0.276 0.071 0.331 0.877 1.006 1.026 0.988 0.957 0.931 0.906 0.875

Sample 2 0.081 0.381 0.078 0.279 0.862 0.941 0.964 0.916 0.878 0.845 0.849 0.82

Sample 3 0.102 0.306 0.075 0.44 0.855 1.058 1.005 0.983 0.883 0.91 0.922 0.947

Average 0.149 0.321 0.075 0.350 0.865 1.002 0.998 0.962 0.906 0.895 0.892 0.881

%Control 17.0 36.4 8.5 39.7 98.2 113.7 113.4 109.3 102.9 101.7 101.3 100.0

Figure 4C

Figure 4B

Figure 5. Determination of minimal inhibitory concentration of ampicillin (B) and Zymox Otic (C) against Streptococcus intermedius.

Figure 5A.

Ampicillin (µg/mL) 100 50 25 12.5 6.25 3.125 1.56 0.78 0.39 0.195 0.098 0

Sample 1 0.059 0.068 0.13 0.208 1.497 1.62 1.654 1.611 1.554 1.629 1.612 1.633

Sample 2 0.065 0.068 0.071 0.152 1.347 1.595 1.525 1.512 1.508 1.519 1.525 1.531

Sample 3 0.063 0.065 0.069 0.166 1.271 1.508 1.556 1.53 1.521 1.524 1.527 1.615

Average 0.062 0.067 0.090 0.175 1.372 1.574 1.578 1.551 1.528 1.557 1.555 1.593

% Control 3.9 4.2 5.6 11.0 86.1 98.8 99.1 97.4 95.9 97.8 97.6 100

Zymox (dilution) 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 1:2048 0

Sample 1 0.157 0.197 0.192 0.21 1.52 1.339 1.318 1.324 1.329 1.35 1.434 1.633

Sample 2 0.094 0.249 0.21 0.105 1.48 1.372 1.348 1.358 1.374 1.362 1.426 1.531

Sample 3 0.124 0.157 0.179 0.127 1.559 1.406 1.392 1.411 1.404 1.42 1.515 1.615

Average 0.125 0.201 0.194 0.147 1.520 1.372 1.353 1.364 1.369 1.377 1.458 1.593

% Control 7.8 12.6 12.2 9.2 95.4 86.1 84.9 85.6 85.9 86.5 91.5 100.0

Figure 5C

Figure 5B

Figure 6. Determination of minimal inhibitory concentration of amphotericin B (B) and Zymox Otic (C) against Candida albicans.

Figure 6A.

Amphotericin B (µg/mL) 100 50 25 12.5 6.25 3.125 1.56 0.78 0.39 0.195 0.098 0

Sample 1 0.05 0.046 0.044 0.044 0.045 0.047 0.045 0.713 0.925 0.974 1.024 1.107

Sample 2 0.056 0.061 0.047 0.059 0.052 0.051 0.046 0.855 0.947 0.92 1.013 1.153

Sample 3 0.062 0.051 0.048 0.046 0.043 0.047 0.044 1.121 0.818 1.201 1.145 1.111

Average 0.056 0.053 0.046 0.050 0.047 0.048 0.045 0.896 0.897 1.032 1.061 1.124

%Control 5.0 4.7 4.1 4.4 4.2 4.3 4.0 79.8 79.8 91.8 94.4 100

Zymox (dilution) 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 1:2048 0

Sample 1 0.107 0.087 0.071 0.055 1.146 1.33 1.317 1.189 1.142 1.086 1.202 1.107

Sample 2 0.103 0.087 0.065 0.057 0.942 1.177 1.139 1.141 1.125 1.049 1.003 1.153

Sample 3 0.091 0.084 0.057 0.056 1.02 1.241 1.138 1.241 1.091 0.971 1.039 1.111

Average 0.100 0.086 0.064 0.056 1.036 1.249 1.198 1.190 1.119 1.035 1.081 1.124

%Control 8.9 7.7 5.7 5.0 92.2 111.2 106.6 105.9 99.6 92.1 96.2 100.0

Figure 6C

Figure 6B

Figure 7. Determination of minimal inhibitory concentration of gentamicin (B) and Zymox Otic (C) against Pseudomonas aeruginosa.

Figure 7A.

Gentamicin (µg/mL) 100 50 25 12.5 6.25 3.125 1.56 0.78 0.39 0.195 0.098 0

Sample 1 0.042 0.041 0.041 0.041 0.041 0.042 0.042 0.045 0.446 1.268 1.289 1.412

Sample 2 0.044 0.042 0.043 0.041 0.042 0.042 0.042 0.046 0.478 1.182 1.241 1.343

Sample 3 0.048 0.042 0.053 0.042 0.04 0.039 0.039 0.055 0.399 1.136 1.225 1.394

Average 0.045 0.042 0.046 0.041 0.041 0.041 0.041 0.049 0.441 1.195 1.252 1.383

% Control 3.2 3.0 3.3 3.0 3.0 3.0 3.0 3.5 31.9 86.4 90.5 100

Zymox (dilution) 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 1:2048 0

Sample 1 0.197 0.251 0.068 0.077 1.303 1.146 1.278 1.328 1.279 1.26 1.278 1.412

Sample 2 0.093 0.278 0.081 0.074 1.194 1.121 1.233 1.259 1.25 1.284 1.264 1.343

Sample 3 0.094 0.215 0.072 0.071 1.275 1.163 1.299 1.337 1.304 1.282 1.3 1.394

Average 0.128 0.248 0.074 0.074 1.257 1.143 1.270 1.308 1.278 1.275 1.281 1.383

% Control 9.3 17.9 5.3 5.4 90.9 82.7 91.8 94.6 92.4 92.2 92.6 100.0

Figure 7C

Figure 7B

Figure 8. Determination of minimal inhibitory concentration of ampicillin (B) and Zymox Otic (c) against Corynebacterium diphtheriae.

Figure 8A.

Ampicillin (µg/mL) 100 50 25 12.5 6.25 3.125 1.56 0.78 0.39 0.195 0.098 0

Sample 1 0.05 0.049 0.049 0.048 0.048 0.049 0.049 0.049 0.05 0.157 0.215 0.22

Sample 2 0.05 0.049 0.049 0.055 0.05 0.048 0.063 0.059 0.049 0.177 0.208 0.226

Sample 3 0.048 0.047 0.049 0.049 0.049 0.048 0.048 0.048 0.048 0.171 0.208 0.236

Average 0.049 0.048 0.049 0.051 0.049 0.048 0.053 0.052 0.049 0.168 0.210 0.227

% Control 21.7 21.3 21.6 22.3 21.6 21.3 23.5 22.9 21.6 74.0 92.5 100

Zymox (dilution) 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 1:2048 0

Sample 1 0.191 0.206 0.141 0.058 0.154 0.167 0.176 0.188 0.197 0.219 0.236 0.22

Sample 2 0.101 0.268 0.196 0.061 0.172 0.204 0.216 0.225 0.227 0.232 0.246 0.226

Sample 3 0.12 0.126 0.18 0.064 0.175 0.231 0.225 0.233 0.239 0.25 0.256 0.236

Average 0.137 0.200 0.172 0.061 0.167 0.201 0.206 0.215 0.221 0.234 0.246 0.227

%Control 60.4 88.0 75.8 26.8 73.5 88.3 90.5 94.7 97.2 102.8 108.2 100.0

Figure 8C

Figure 8B

Figure 9. Determination of minimal inhibitory concentration of Bifonazole (B) and Zymox Otic (C) against Malassezia pachydermatis.

Figure 9A.

Bifonazole (mg/mL) 25 12.5 6.25 3.125 1.56 0.781 0.39 0.195 0.0975 0.049 0.024 0

Sample 1 0.056 0.062 0.06 0.074 0.064 0.058 0.059 0.07 0.065 0.061 0.504 0.573

Sample 2 0.058 0.06 0.061 0.083 0.075 0.077 0.076 0.075 0.064 0.233 0.545 0.631

Sample 3 0.051 0.053 0.057 0.061 0.072 0.058 0.078 0.0650.078 0.075 0.064 0.162 0.544

Average 0.055 0.058 0.059 0.073 0.070 0.064 0.071 0.073 0.068 0.119 0.404 0.583

% Control 9.4 10.0 10.2 12.5 12.1 11.0 12.2 12.4 11.7 20.5 69.3 100

Zymox (dilution) 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 1:2048 0

Sample 1 0.282 0.2 0.159 0.07 0.18 1.743 1.747 0.428 1.954 1.894 1.954 0.573

Sample 2 0.509 0.231 0.149 0.068 0.061 0.584 0.5 0.377 0.435 0.583 0.462 0.631

Sample 3 0.497 0.193 0.177 0.065 0.16 0.416 0.496 1.894 0.5 0.527 0.536 0.544

Average 0.429 0.208 0.162 0.068 0.134 0.914 0.914 0.900 0.963 1.001 0.984 0.583

%Control 73.7 35.7 27.7 11.6 22.9 156.9 156.9 154.4 165.3 171.9 168.9 100.0

Figure 9C

Figure 9B