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 OD625 of the triplicate test samples and OD625NC is the average OD625 of 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
