Otitis externa is the most common ear disease in dogs, with a reported prevalence of 10% to 20%.1,2 Fifty percent to 89% of dogs with chronic otitis externa may have concurrent otitis media.3,4 The most common bacterial pathogens isolated from ears of dogs with otitis externa are Staphylococcus intermedius, Pseudomonas aeruginosa, Proteus spp, and β-hemolytic streptococcus.4–10 These same bacterial organisms have also been isolated from middle ears of dogs with otitis media.4,11
Cell surfaces of gram-negative bacteria are damaged when exposed to EDTA.12 Tromethamine is a synthetic buffer that enhances the chelating effect of EDTA.13 Gram-negative bacteria exposed to tromethamine-EDTA have increased permeability to extracellular solutes and leakage of intracellular solutes14; are sensitized to lysozyme, bactericides, and antimicrobials15–17; and release lipopolysaccharide, protein, phospholipids, and divalent cations from their cell walls.18 Cell walls of gram-positive bacteria are more resistant to the effects of tromethamine-EDTA than gram-negative bacteria.19 The in vitro activity of tromethamine-EDTA, with and without the addition of antimicrobial agents, against gram-positive and gramnegative bacterial organisms has been studied.17,19–23 However, few numbers of isolates were evaluated in each study, and no study evaluated the in vitro activity of a product containing tromethamine-EDTA against multiple clinical isolates of the 4 most common bacterial otic pathogens.
One commercially available ERa contains benzyl alcohol in addition to tromethamine-EDTA. Benzyl alcohol has traditionally been used as an antiseptic, but presently is used as a preservative.24 Benzyl alcohol has been found to have a greater inhibitory effect against gram-negative bacteria such as P aeruginosa and Escherichia coli than gram-positive bacteria.25 However, the in vitro effect of benzyl alcohol in a solution of tromethamine-EDTA on gram-positive and gram-negative otic pathogens has not been evaluated.
The purpose of the study reported here was to evaluate the in vitro activity of an ERa containing tromethamine, EDTA, and benzyl alcohol on bacterial pathogens from dogs with otitis. We hypothesized that this ER would have antibacterial activity against gramnegative and gram-positive otic pathogens and that the addition of benzyl alcohol would further enhance its activity against gram-negative pathogens.
Materials and Methods
Bacteria—Staphylococcus spp (n = 11; S intermedius [7] and Staphylococcus spp [4]), P aeruginosa (5), Proteus spp (5), βhemolytic streptococcus (11), and 1 ATCC control strain of each organism (S intermedius ATCC 29663, P aeruginosa ATCC 9027, Proteus vulgaris ATCC 6380, and Streptococcus pyogenes ATCC 51339) were used. The sample size for each organism was generated from results of a preliminary study based on the expected difference in bacterial count of the organisms in solution (ER) between time 0 and 15 minutes; the level of significance was set at α = 0.05 and the power of 80%.
Sample collection of clinical isolates—Samples for bacterial culture were obtained from client-owned dogs evaluated by The Ohio State University Veterinary Teaching Hospital dermatology or soft tissue surgery service for bacterial otitis externa or otitis media. Approval by the institutional review committee on the care and use of client-owned animals at The Ohio State University was obtained prior to obtaining samples for the study. Client consent was obtained for sample collection. To obtain samples of otic exudate from dogs with otitis, a sterile cotton-tipped applicator was used. In dogs with otitis externa, a sterile cotton-tipped applicator was inserted into the external ear canal, and a sample was obtained from the junction of the vertical and horizontal ear canal (Table 1). In dogs with otitis media, samples were obtained from the middle ear cavity via myringotomy or directly from the tympanic bulla during bulla osteotomy by use of a sterile cotton-tipped applicator. Thirtytwo bacterial organisms were obtained from ears of 17 dogs. Ear swab specimens were placed into sterile tubes and transported within 1 hour to the veterinary teaching hospital microbiology laboratory, plated on sheep blood agar and MacConkey's agar, and incubated at 35°C for 18 to 24 hours. Organisms were identified morphologically and by routine biochemical testing. Staphylococcal organisms were identified by use of the coagulase test and a commercial identification system.b
Number of bacterial isolates obtained from the external ear canal or the middle ear cavity from 17 dogs with otitis.
| Organism | External ear canal | Middle ear–myringotomy | Middle ear–bulla osteotomy |
|---|---|---|---|
| Staphylococcus spp | 6 | 3 | 2 |
| Pseudomonas aeruginosa | 3 | 1 | 1 |
| Proteus spp | 3 | 0 | 2 |
| β-Hemolytic streptococcus | 10 | 0 | 1 |
In dogs with otitis externa, samples were obtained from the junction of the vertical and horizontal ear canal. In dogs with otitis media, samples were obtained from the middle ear cavity via myringotomy or directly from the tympanic bulla during bulla osteotomy.
Testing of solutions and bacterial organisms was performed at an off-site good manufacturing practice microbiology laboratory, designated as the TML. To store organisms for shipping to the TML, bacterial growth of each organism from the pure culture was lifted off the plate via a sterile cottontipped applicator, transferred to a tube containing BHI broth, and mixed until a turbid suspension of the organism was formed. Each organism was frozen in the tube containing BHI broth at −80°C prior to shipment to the TML. Once all organisms had been collected and frozen, organisms were prepared for shipment to the TML as follows: individual BHI broth tubes containing organisms were thawed in an incubator, mixed to resuspend organisms, streaked for isolation individually on blood agar plates, and placed back into an incubator for 18 to 24 hours at 35°C. Plates were removed from the incubator, wrapped with laboratory film,c and shipped with ice packs via 2-day delivery to the TML. After arrival at the TML, bacterial strains were prepared on soybean-casein digest agard plates. The prepared microbial cultures were harvested by use of sterile 0.9% NaCl solution and adjusted to approximately 108 organisms/mL.
Test solutions and antimicrobial testing—Solutions evaluated in the study included the ERa containing EDTA, tromethamine, and benzyl alcohol; EDTA and tromethamine (ER – BA); and purified water (water base of ER; control [H2O]). For each test solution, 10-mL aliquots were transferred into 36 sterile, capped tubes. Each tube was inoculated with 0.1 mL of one of the test organisms to give a final microbial concentration of 105 to 106 microorganisms/mL. Samples were kept at 22.5 ± 2.5°C during the study.
Sampling—Samples were retrieved from each tube at 0, 15, 30, 45, and 60 minutes after inoculation. Samples were diluted with product diluent (soybean-casein digest broth with supplemental 4% polysorbate-20 and 1% lecithin) such that 1 mL was expected to yield between 30 and 300 colonies. The number of viable microorganisms was determined by use of the standard plate count technique.26,27
Standard plate count technique—One milliliter of appropriately diluted product was transferred onto each of 2 sterile Petri dishes. Fifteen to 20 mL of soybean-casein digest agar that had previously melted and cooled to approximately 45°C was added. Petri dishes were covered, mixed by tilting or rotating dishes, and allowed to solidify at 22.5 ± 2.5°C. Petri dishes were inverted and incubated for 48 to 72 hours at 32.5 ± 2.5°C. After incubation, plates were examined for growth, colonies were counted, and the mean of the 2 plates was expressed as the number of CFU per milliliter.
Statistical analysis—The mean number of CFU per milliliter from 2 plates for each organism in each solution obtained at 0, 15, 30, 45, and 60 minutes was calculated. Because the initial (time 0) inoculum varied from isolate to isolate, the outcome in analysis was the proportion of bacterial growth in CFU per milliliter at each time relative to time 0. Making everything proportional to time 0 adjusted for the variation at the start. The proportion was modeled by use of a repeated-measures analysis in a commercially available statistical software package.e Time and solution were factors, and an interaction term between time and solution was also included in the model. The analysis accounted for nonindependence by use of an autoregressive correlation structure for measurements obtained on the same solution with time. Separate models were constructed for each of the 4 organisms. Within an organism model, all pairwise comparisons of solution-time combinations were evaluated by use of the Tukey-Kramer adjustment for multiple comparisons. Values of P < 0.05 were considered significant.
Results
In all 4 analyses, including the control strains, the time-solution interaction was significant, which indicated that various solutions had different effects with time. For Staphylococcus spp, a significant decrease in bacterial growth was detected for ER at 60 minutes (P = 0.001), compared with time 0. Neither ER – BA nor H2O had any effect on growth of Staphylococcus spp (Figure 1).
Bacterial growth of Staphylococcus spp in 3 solutions (ER containing EDTA, tromethamine, and benzyl alcohol, diamond; ER – BA, square; and purified water [water base of ER, control], triangle) with time. The means of the number of CFU per milliliter for each Staphylococcus spp organism (n = 11; Staphylococcus intermedius [7] and Staphylococcus spp [4]) in each solution at each time were calculated. Because the initial (time 0) inoculum of each Staphylococcus spp organism varied from isolate to isolate, the outcome in the analysis was the proportion of bacterial growth in CFU per milliliter (proportional CFU per milliliter) at each time relative to time 0. Values are reported as mean ± SD proportional CFU per milliliter. *Within a solution, value differs significantly (P < 0.05) from the value at time 0.
Citation: American Journal of Veterinary Research 67, 6; 10.2460/ajvr.67.6.1040
For β-hemolytic streptococcus, there was a significant decrease in bacterial growth for ER at all times (15, 30, 45, and 60 minutes; P < 0.001); for ER – BA at 60 minutes (P < 0.005); and for H2O at 30 (P = 0.002), 45 (P = 0.002), and 60 minutes (P < 0.001), compared with time 0. Ear rinse was significantly more effective in decreasing bacterial growth (P < 0.001) at all times (15, 30, 45, and 60 minutes), compared with both ER – BA and H2O (Figure 2).
Bacterial growth of β-hemolytic streptococcus in 3 solutions with time. The means of the number of CFU per mL for each β-hemolytic streptococcal organism (n = 11) in each solution at each time were calculated. Because the initial (time 0) inoculum of each β-hemolytic streptococcal organism varied from isolate to isolate, the outcome in the analysis was the proportion of bacterial growth in CFU per milliliter (proportional CFU per milliliter) at each time relative to time 0. Values are reported as mean ± SD proportional CFU per milliliter. †Within a time, ER value differs significantly (P < 0.05) from ER – BA value. ‡Within a time, ER value differs significantly (P < 0.05) from H2O value. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 67, 6; 10.2460/ajvr.67.6.1040
For P aeruginosa, there was a significant decrease in bacterial growth for ER at all times (15, 30, 45, and 60 minutes; P < 0.001) and for ER – BA at 30, 45, and 60 minutes (P < 0.001), compared with time 0. Purified water (H2O) had no effect on growth of P aeruginosa. Ear rinse significantly decreased bacterial growth at all times (15, 30, 45, and 60 minutes), compared with H2O (P < 0.001; Figure 3).
Bacterial growth of Pseudomonas aeurginosa in 3 solutions with time. The means of the number of CFU per milliliter for each P aeruginosa organism (n = 5) in each solution at each time were calculated. Because the initial (time 0) inoculum of each P aeruginosa organism varied from isolate to isolate, the outcome in the analysis was the proportion of bacterial growth in CFU per milliliter (proportional CFU per milliliter) at each time relative to time 0. Values are reported as mean ± SD proportional CFU per milliliter. See Figure 1 and 2 for remainder of key.
Citation: American Journal of Veterinary Research 67, 6; 10.2460/ajvr.67.6.1040
For Proteus spp, there was a significant decrease in bacterial growth for ER at 30 (P = 0.003), 45 (P < 0.001), and 60 (P < 0.001) minutes, compared with time 0. Neither ER – BA nor H2O had any effect on growth of Proteus spp. Ear rinse significantly decreased bacterial growth at 30, 45, and 60 minutes, compared with ER – BA (P < 0.001) and H2O (P = 0.007; Figure 4).
Bacterial growth of Proteus spp organisms in 3 solutions with time. The means of the number of CFU per milliliter for each Proteus spp organism (n = 5) in each solution at each time were calculated. Because the initial (time 0) inoculum of each Proteus spp organism varied from isolate to isolate, the outcome in the analysis was the proportion of bacterial growth in CFU per (proportional CFU per milliliter) at each time relative to time 0. Values are reported as mean ± SD proportional CFU per milliliter. See Figures 1 and 2 for remainder of key.
Citation: American Journal of Veterinary Research 67, 6; 10.2460/ajvr.67.6.1040
Discussion
In the study reported here, ER significantly decreased in vitro bacterial growth of P aeruginosa and β-hemolytic streptococcal organisms within 15 minutes, Proteus spp within 30 minutes, and Staphylococcus spp within 60 minutes. In an in vitro study by Wooley and Jones,17 by use of tromethamine-EDTA without benzyl alcohol, P aeruginosa was rapidly lysed; β-hemolytic streptococcus and P vulgaris were also lysed, although not as rapidly as P aeruginosa; and there was no decrease in Staphylococcus aureus numbers.
Other studies28,29 have evaluated the in vitro effect of tromethamine-EDTA and lysozyme as well as tromethamine-EDTA and SDS, both of which were added to enhance the antimicrobial effects of tromethamine-EDTA. The addition of lysozyme to tromethamine-EDTA resulted in a rapid decrease in bacterial numbers of P aeruginosa; however, there was no effect on S aureus or Streptococcus equi. The combination of tromethamine-EDTA and SDS had synergistic antimicrobial activity in vitro against P aeruginosa as well as P vulgaris and additive antimicrobial activity against S aureus. In our study, addition of benzyl alcohol significantly increased the antimicrobial activity of tromethamine-EDTA on β-hemolytic streptococcus and Proteus spp organisms.
The resistance of gram-positive bacteria to tromethamine-EDTA without benzyl alcohol is expected, since tromethamine-EDTA damages bacteria by releasing divalent cations from cell walls. Gram-positive bacteria have teichoic acid, which functions as a binding agent for divalent cations, in their cell walls.30 In our study, even with the addition of benzyl alcohol, a significant decrease in bacterial growth of Staphylococcus spp was detected only after 60 minutes of contact time with ER. On the other hand, ER significantly decreased growth of β-hemolytic streptococcus as early as 15 minutes. The difference in reduction of bacterial growth in vitro between β-hemolytic streptococcus and Staphylococcus spp (both gram-positive organisms) in ER may be attributable to a difference in the type of teichoic acid in the cell walls of these organisms.31
Tromethamine-EDTA in combination with antimicrobial agents in vitro has been evaluated for enhanced antimicrobial activity against gram-negative and gram-positive bacteria.17,19–23 A decrease in the minimum inhibitory concentration of S aureus was detected when tromethamine-EDTA was added to specific antimicrobial agents in vitro.23 However, this decrease was not as marked as that reported against gram-negative organisms.23 Synergistic activity in vitro was reported for combinations of tromethamine-EDTA and aminoglycoside antimicrobials against S intermedius, Proteus mirabilis, and P aeruginosa.19 The in vivo efficacy of tromethamine-EDTA in combination with a topically administered antimicrobial agent used in 8 dogs with bacterial otitis externa resulted in resolution of clinical signs of otitis and the bacterial infection within 7 to 15 days.32 Therefore, combining an antimicrobial with tromethamine-EDTA may improve its antibacterial efficacy, which is especially important for gram-positive bacterial ear infections caused by Staphylococcus spp.
In our study, sampling times of test solutions with the bacterial inoculum were at 15, 30, 45, and 60 minutes. For both β-hemolytic streptococcus and P aeruginosa, significant decreases in vitro of bacterial organisms in ER were detected at all times, beginning at 15 minutes. In a clinical situation, it is unlikely that the entire amount of any topically applied solution would remain in the ear for 15 minutes. However, samples were not obtained before 15 minutes, and the decrease in bacterial organisms in vitro may have occurred earlier than 15 minutes. In addition, although an ear canal will not remain completely full of a topically applied solution for extended periods, it is likely that some of the solution will remain in the canal in contact with bacterial organisms.
In our study, the ER formulation with benzyl alcohol had enhanced antiseptic activity against β-hemolytic streptococcus and Proteus spp, compared with a solution of tromethamine and EDTA alone. These results also support the role of benzyl alcohol as a preservative to protect the solution from contamination. Virtually all veterinary otic medications are packaged in multiple-dose containers in which contact of the applicator tip with the infected ear canal has the potential to contaminate the product. Thus, an effective preservative is important to minimize the possibility of spreading or perpetuating infection through the repeated use of the medication.
Ear rinse significantly decreased bacterial growth in vitro of P aeruginosa and β-hemolytic streptococcus within 15 minutes and Proteus spp within 30 minutes. The ER formulation with benzyl alcohol had significantly increased efficacy in reducing bacterial growth of β-hemolytic streptococcus and Proteus spp in vitro. On the basis of our results, ER may be useful in the treatment of infectious otitis. Future studies evaluating the in vivo efficacy of ER as a sole treatment for otic infections caused by β-hemolytic streptococcus, P aeruginosa, and Proteus spp and of ER in combination with an antimicrobial agent for bacterial otic infections caused by Staphylococcus spp are warranted.
ABBREVIATIONS
| ER | Ear rinse |
| ATCC | American Type Culture Collection |
| TML | Testing microbiology laboratory |
| BHI | Brain heart infusion |
| ER – BA | ER without benzyl alcohol |
T8 Solution, IVX Animal Health Inc, St Joseph, Mo.
ID 32 Staph, bioMerieux Inc, Durham, NC.
ParafilmM, American National Can, Menasha, Wis.
Soybean-casein digest agar, BD Diagnostic Systems, Sparks, Md.
PROC MIXED in SAS, version 9.1, SAS Institute Inc, Cary, NC.
References
- 1
Rosser EJ. Causes of otitis externa. Vet Clin North Am Small Anim Pract 2004;34: 459–468.
- 2
Griffin CE. Otitis externa and otitis media. In:Griffin CE, Kwochka KW, MacDonald JM, eds.Current veterinary dermatology. St Louis: Mosby Year Book Inc, 1993;244–262.
- 3
Spreull JSA. Treatment of otitis media in the dog. J Small Anim Pract 1964;5: 107–152.
- 4
Cole LK, Kwochka KW & Podell M, et al. Evaluation of radiography, otoscopy, pneumotoscopy, impedance audiometry and endoscopy for the diagnosis of otitis media. In:Thoday KL, Foil KL, Bond R. eds.Advances in veterinary dermatology. Vol 4. Oxford, England: Blackwell Publishing Ltd, 2002;49–55.
- 5
Blue JL, Wooley RE. Antibacterial sensitivity patterns of bacteria isolated from dogs with otitis externa. J Am Vet Med Assoc 1977;171: 362–363.
- 6
Kowalski JJ. The microbial environment of the ear canal in health and disease. Vet Clin North Am Small Anim Pract 1988;18: 743–754.
- 7
Marshall MJ, Harris AM, Horne JE. The bacteriological and clinical assessment of a new preparation for the treatment of otitis externa in dogs and cats. J Small Anim Pract 1983;24: 413–421.
- 8
McCarthy G, Kelly WR. Microbial species associated with the canine ear and their antibacterial sensitivity patterns. Ir Vet J 1982;36: 53–56.
- 9
Nesbitt GH, Schnitz JA. Chronic bacterial dermatitis and otitis: a review of 195 cases. J Am Anim Hosp Assoc 1977;13: 442–450.
- 10
Sharma VD, Rhoades HE. The occurrence and microbiology of otitis externa in the dog. J Small Anim Pract 1975;16: 241–247.
- 11
Colombini S, Merchant SR, Hosgood G. Microbial flora and antimicrobial susceptibility patterns from dogs with otitis media. Vet Dermatol 2000;11: 235–239.
- 12↑
Roberts NA, Gray GW, Wilkinson SC. The bactericidal action of ethylenediaminetetra-acetic acid on Pseudomonas aeruginosa. Microbios 1970;7–8:189–208.
- 13↑
Goldschmidt MC, Wyss O. The role of Tris in EDTA toxicity and lysozyme lysis. J Gen Microbiol 1967;47: 421–431.
- 14↑
Leive L. A nonspecific increase in permeability in Escherichia coli produced by EDTA. Proc Natl Acad Sci U S A 1965;53: 745–750.
- 15
Brown MRW, Richards RME. Effect of ethylenediamine tetraacetate on the resistance of Pseudomonas aeruginosa to antibacterial agents. Nature 1965;207: 1391–1393.
- 16
Russel AD. Effect of magnesium ions and ethylenediamine tetra-acetic acid on the activity of vancomycin against Escherichia coli and Staphylococcus aureus. J Appl Bacteriol 1967;30: 395–401.
- 17↑
Wooley RE, Jones MS. Action of EDTA-Tris and antimicrobial agent combinations on selected pathogenic bacteria. Vet Microbiol 1983;8: 271–280.
- 18↑
Leive L, Shovlin VK, Mergenhagen SE. Physical, chemical, and immunological properties of lipopolysaccharide released from Escherichia coli by ethylenediaminetetraacetate. J Biol Chem 1968;243: 6384–6391.
- 19↑
Sparks TA, Kemp DT & Wooley RE, et al. Antimicrobial effect of combinations of EDTA-Tris and amikacin or neomycin on the microorganisms associated with otitis externa in dogs. Vet Res Commun 1994;18: 241–249.
- 20
Wooley RE, Jones MS & Gilbert JP, et al. In vitro action of combinations of antimicrobial agents and EDTA-tromethamine on Pseudomonas aeruginosa. Am J Vet Res 1983;44: 1521–1524.
- 21
Wooley RE, Jones MS & Gilbert JP, et al. In vitro effect of combinations of antimicrobial agents and EDTA-tromethamine on certain gram-positive bacteria. Am J Vet Res 1983;44: 2167–2169.
- 22
Farca AM, Nebbia P, Re G. Potentiation of the in vitro activity of some antimicrobial agents against selected gram-negative bacteria by EDTA-tromethamine. Vet Res Commun 1993;17: 77–84.
- 23↑
Farca AM, Nebbia P, Re G. Potentiation of antibiotic activity by EDTA-tromethamine against three clinically isolated gram-positive resistant bacteria. An in vitro investigation. Vet Res Commun 1994;18: 1–6.
- 24↑
Harvey SC. Antiseptics and disinfectants; fungicides; ectoparasiticides. In:Gilman AG, Goodman LS, Rall TW, et al, eds.The pharmacological basis of therapeutics. 7th ed.New York: MacMillan Publishing Co, 1985;959–979.
- 25↑
Karabit MS, Juneskans OT, Lundgren P. Studies on the evaluation of preservative efficacy-II. The determination of antimicrobial characteristics of benzyl alcohol. J Clin Hosp Pharm 1986;11: 281–289.
- 26
Antimicrobial effectiveness testing. In:The United States pharmacopeia. 28th ed.Rockville, Md: The United States Pharmacopeia Inc, 2005;2242–2243.
- 27
Microbial limits test. In:The United States pharmacopeia. 28th ed.Rockville, Md: The United States Pharmacopeia Inc, 2005;2246–2251.
- 28
Wooley RE, Blue JL. In vitro effect of EDTA-tris-lysozyme solutions on selected pathogenic bacteria. J Med Microbiol 1975;8: 189–194.
- 29
Wooley RE, Dickerson HW, Engen WR. Treating canine otitis externa with an EDTA-TRIS-SDS combination. Vet Med 1988;83: 1088–1091.
- 30↑
Cutinelli C, Galdiero F. Ion-binding properties of the cell wall of Staphylococcus aureus. J Bacteriol 1967;93: 2022–2023.
- 31↑
Cutinelli C, Wooley RE. Gram-positive pyogenic bacteria. In:Ofek I, Doyle RJ, eds.Bacterial adhesion to cells and tissues. New York: Chapman & Hall, 1994;136–170.
- 32↑
Farca AM, Piromalli G & Maffei F, et al. Potentiating effect of EDTA-Tris on the activity of antibiotics against resistant bacteria associated with otitis, dermatitis, and cystitis. 1997;38:243–245.
