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Article

Antibacterial In Vitro Properties of Silver Sulfadiazine in Combination with Tris-EDTA and N-Acetylcysteine Against Pseudomonas aeruginosa Isolates from Dogs with Suppurative Otitis

by
Ioanna Papadogiannaki
1,
Rosario Cerundolo
1,*,
Jennifer Plant
2,
Elizabeth Villiers
2,
Jenny Littler
3,
Anika Wisniewska
4 and
Panagiotis Sgardelis
5
1
Dermatology Section, Dick White Referrals, Veterinary Specialist Centre, Station Farm, London Road, Six Mile Bottom, Suffolk CB8 0UH, UK
2
Microbiology Section, Antech Laboratory, Dick White Referrals, Veterinary Specialist Centre, Station Farm, London Road, Six Mile Bottom, Suffolk CB8 0UH, UK
3
Warwick Antimicrobial Screening Facility, University of Warwick, Interdisciplinary Biomedical Research Building, Gibbet Hill, Coventry CV4 7AL, UK
4
Microbiology and Molecular Biology, Antech Diagnostics Limited, Unit 1, Titan Business Centre, Tachbrook Park, Warwick CV34 6RR, UK
5
Norfolk & Norwich University Hospital, Colney Ln, Norwich NR4 7UY, UK
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2025, 16(7), 138; https://doi.org/10.3390/microbiolres16070138
Submission received: 3 April 2025 / Revised: 18 June 2025 / Accepted: 20 June 2025 / Published: 1 July 2025
(This article belongs to the Special Issue Veterinary Microbiology and Diagnostics)
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Versions Notes

Abstract

Pseudomonas aeruginosa otitis is common in dogs and antibiotic-resistant strains are often isolated. We are unaware of reports evaluating the combination of silver sulfadiazine (SSD) with a biofilm disruptor solution containing Tris-EDTA (tromethamine-ethylenediaminetetraacetic acid) and N-acetylcysteine (Tris-NAC). Forty-eight P. aeruginosa strains from dogs with suppurative otitis were analysed using the agar well diffusion method. A volume of 70 μL of Tris-NAC, a water solution of 10% SSD and their combination in equal amount was pipetted into the designated wells. After incubation, the diameter of the inhibition zone was measured. A synergy experiment using the checkerboard assay was performed to look at any potential synergistic effects of SSD and Tris-NAC against only 10 randomly selected isolates of P. aeruginosa. The samples tested with Tris-NAC + 10% SSD solution, compared with the samples tested with 10% SSD alone, demonstrated significantly higher inhibition zones against P. aeruginosa, p < 0.00001. The checkerboard assay results showed an additive effect between the two compounds. The use of 10% SSD could be evaluated as a therapeutic tool against strains of P. aeruginosa if combined with Tris-NAC.

1. Introduction

Otitis externa (OE) is a relatively common disease in dogs. A retrospective study of dogs examined in primary veterinary practices in the United Kingdom (UK) reported an incidence of 7.3% [1]. Canine recurrent or chronic OE can be a frustrating condition to manage, affecting the quality of life of dogs and their owners, and is often an expensive condition to treat in addition to the cost of addressing the underlying cause and its treatment. In recent years it has become common to isolate resistant bacteria from the external ear canals of dogs with otitis as a result of the repeated use of topical and/or systemic antibiotics [2,3,4].
Pseudomonas aeruginosa (P. aeruginosa), which is a ubiquitous, Gram-negative (GN), rod-shaped aerobic bacterium, is listed as one of the ‘critical priority pathogens’ in the World Health Organisation’s (WHO) list of bacteria for which new antibiotics are urgently needed [5]. P. aeruginosa poses a particular threat due to its widespread environmental distribution, intrinsic resistance to many classes of antimicrobials and commonly acquired resistance to antimicrobials often considered the treatment of choice, such as fluoroquinolones or aminoglycosides [6,7,8]. P. aeruginosa is the most common GN isolate in canine suppurative otitis [9,10]. Thus, there is a need for alternative treatment options to the commercially available otic products containing different combinations of antibiotic/antifungal/steroid medications.
SSD is a metal–organic compound that is obtained from the reaction of silver nitrate (30.2%) with sulfadiazine (69.8%) and has extensive antimicrobial activity [11]. In human medicine, SSD alone has proven to be an effective topical agent against P. aeruginosa strains isolated from burn wounds [12]. Similarly, in veterinary medicine, a study reported SSD’s effectiveness against 80% of all the P. aeruginosa isolated from canine otitis [13]. For many years, SSD cream or powder has been formulated as an otic solution in veterinary practices to treat P. aeruginosa otitis in dogs, although whether biofilm was produced by the isolates was unknown in the past years.
Recent studies have shown that the biofilm, a complex biological system composing of exopolysaccharides, proteins, extracellular DNA and biomolecules, is the most important factor in limiting the efficacy of topical antibiotic treatments against P. aeruginosa [14,15]. N-Acetylcysteine (NAC) has often been used as a biofilm disruptor and has recently become available on the veterinary market as the topical solution Tris-NAC (Nextmune, Italy), containing a combination of Tris–EDTA and NAC. Studies have reported that Tris-EDTA significantly reduces the minimum bactericidal concentrations (MBCs) and the minimum inhibitory concentration (MIC)s of marbofloxacin and gentamicin for multidrug resistant (MDR) strains of P. aeruginosa in vitro [16].
To the best of the authors’ knowledge, to date, there have been no in vitro studies to show whether the combination of Tris-EDTA and NAC with SSD might have better growth inhibition than SSD alone against resistant strains of P. aeruginosa. The aim of this study was to test and compare in vitro whether the addition of Tris-EDTA and NAC demonstrated an additive effect when compared with 10% SSD alone against P. aeruginosa strains isolated from dogs suffering with suppurative otitis.

2. Material and Methods

2.1. Ethics

Ethical approval was not required since the present study was carried out on diagnostic samples collected during routine veterinary activities and submitted to our internal microbiology laboratory for diagnostic purposes.

2.2. Samples, Study Period and Bacteriological Investigation

Between June and December 2023, 48 P. aeruginosa isolates were isolated from dogs suffering with otitis who were presented to our hospital dermatology service (n = 24) or from swabs submitted from other veterinary practices to our microbiology laboratory for bacterial culture and susceptibility testing. The specimens were non-duplicate and were presumed to be epidemiologically unrelated since they came from different animals belonging to different families and presented on different days.

2.3. Isolation of Pure Cultures

Samples were plated from charcoal swabs or E-swabs onto Columbia horse blood agar (Oxoid Blood Agar Base) that was incubated in 5% CO2 at 35–37 °C for 15–18 h; colistin-nalidixic acid agar (CNA, which is selective for Gram-positive organisms) and Sabouraud dextrose (which promotes growth of yeast and fungi) agar (Oxoid, Basingstoke, UK) were both incubated in aerobic conditions for 24–48 h at 35 to 37 °C. For samples with mixed growth, subcultures were performed to obtain a pure growth of each isolate. To make sure there were no additional growth of other organisms in the Pseudomonas colony, before loading it onto the Vitek, subcultures of P. aeruginosa samples were obtained via incubation on cystine–lactose–electrolyte-deficient (CLED) medium at 36–37 °C for 18 h (P. aeruginosa grows as green colonies with a matt surface and a rough periphery on CLED medium). Identification was performed using the Vitek 2 [17].

2.4. Antibiotic Susceptibility Testing

Antibiotic susceptibility testing was performed on all isolates using the Thermo Fisher™ Sensititre ARIS HiQ AST System. Isolated colonies of Pseudomonas aeruginosa that had been incubated overnight on Columbia Blood Agar (Oxoid™, PB0123A, Basingstoke, UK) were added to 5 mL of demineralized water (Thermo Fisher, T3339, Waltham, MA, USA) to prepare suspensions equivalent to 0.5 MacFarland using the Sensititre Nephelometer instrument. 10 µL of inocula were aseptically transferred to 10 mL of Sensititre cation-adjusted Mueller–Hinton broth (Thermo Fisher, T3462) using 10 µL calibrated loops to give a final concentration of 5 × 104–5 × 105 cfu/mL, according to the manufacturer’s manual. 50 µL of bacterial suspensions were dispensed into custom made 96-well Gram-negative AST Sensititre plates (Thermo Fisher, ADGB1GNF) using the Sensititre AIM™ Automated Inoculation Delivery System. Gram-negative AST Sensititre plates were designed to test for P. aeruginosa antimicrobial sensitivity against Amikacin, Gentamicin, Enrofloxacin, Marbofloxacin, Pradofloxacin, Polymyxin B, Imipenem, Ceftazidime and Cefoxitin at increasing concentrations, according to CLSI (Clinical and Laboratory Standards Institute) and EUCAST (European Committee on Antimicrobial Susceptibility Testing) guidelines [18,19]. The MIC breakpoints for the tested fluoroquinolones, Polymyxin B and aminoglycosides are shown in Table 1. Adhesive seals were applied, and AST plates were placed in the Aris HiQ instrument that had been designed to incubate and auto-read plate utilising fluorescence detection.
P. aeruginosa is intrinsically resistant to amoxicillin clavulanate, cephalexin, cefovecin, trimethoprim sulfamethoxazole, florfenicol, tetracyclines, fusidic acid and neomycin, and so these antibiotics were not tested.
Any isolate phenotypically identified as P. aeruginosa species was included in the study. Isolates were then collected and stored frozen in ‘Protect beads’ (Scientific Labs, Nottingham, UK). Firstly, a single ‘Protect’ bead from each of the isolates was placed into a meat broth bottle (Oxoid, Cooked Meat Medium, Basingstoke, UK). Following incubation for 24 to 48 h, isolates were plated onto Columbia horse blood agar and incubated at 35 to 37 °C in 5% CO2 for 15–18 h. Colonies were then collected and subsequently plated onto Mueller–Hinton (M–H) agar (Oxoid, Basingstoke, UK). In a biosafety cabinet, colonies of Pseudomonas were taken using a sterile cotton swab and suspended in saline solution (Thermo Fisher, Waltham, MA, USA) to a 0.5 MacFarland. Turbidity of the isolates was then measured using a nephelometer (Thermo Fisher) to ensure the correct concentration was spread onto the M–H plate.

2.5. Antibacterial Studies by Agar Diffusion Method

Wells of 6 mm diameter were then cut into the agar using a sterile 6 mm biopsy punch. Three wells were cut into each plate. A volume of 70 μL of each solution, Tris-NAC, a water solution of 10% SSD (Sigma-Aldrich 98%, St. Louis, MO, USA) and their combination in equal amount, was pipetted into the designated well of the plate. The authors checked that the pipette volumes were contained exclusively within the agar plates prior to incubation. Plates were incubated for 15 to 18 h at 36 °C. Following incubation, the diameter of the inhibition zone of the wells was measured. Two separate measurements were taken diagonally across the well, from one leading zone edge to the other. The average zone diameter was then calculated for each of the preparations [7].

2.6. Synergy Experiment—Checkboard Assay

A synergy experiment using the checkerboard assay was performed to look at any potential synergistic effects of SSD and Tris-NAC against 10 randomly selected P. aeruginosa clinical isolates [20,21,22].
Firstly, MICs were conducted following the broth microdilution method. In a 96-well plate (CytoOne), a 2-fold series dilution of Tris-NAC and SSD was briefly performed in cation-adjusted Mueller–Hinton broth (CAMHB). A bacterial inoculation of 10 P. aeruginosa clinical isolates was prepared using the MacFarland 0.5 standard in PBS, which was diluted to 1 in 100 in CAMHB. Then 100 µL of this was added to each well and incubated for 18–20 h at 37 °C. The lowest concentration of compound to inhibit the growth of the organism was classed as the MIC.
A SSD solution of 0.3% was made using sterilised distilled water. This was based on MIC results obtained via the broth microdilution method. Tris-NAC was made following instructions from the manufacturer (by mixing the powder in the cap with the liquid in the bottle). In a 96-well plate (CytoOne), a 2-fold series dilution was made of both compounds. The assay is composed of a 2-fold series dilution in CAMHB of Tris-NAC and SSD only and then a combination of both compounds at various concentrations. An increasing concentration of SSD and Tris-NAC was added to the columns and rows, respectively. The isolates were made up to the MacFarland 0.5 standard in PBS, from which a 1 in 100 dilution in CAMHB was performed. A 100 μL aliquot of inoculum was pipetted into each well, with the exception of the negative controls. Plates were incubated at 37 °C for 24 h. The experiment was performed in triplicate for each isolate.
The fractional inhibitory concentration (FIC) and fractional inhibitory concentration index (FICI) were calculated in the following way [22]:
F I C = M I C   o f   a n t i b i o t i c   i n   c o m b i n a t i o n M I C   o f   a n t i b i o t i c   a l o n e
FICI = ΣFIC of antibiotics
The type of synergy response is then produced according to the FICI result; a score of <0.5 = synergy, 0.5–4 = additive and >4 = antagonistic was obtained [22].

2.7. Statistical Methods

Data Analysis

The data was analysed for their mean and standard deviation. The Shapiro–Wilk test was used to assess the normality of the distribution, which revealed differences in I variance between the two groups. Consequently, Welch’s t-test was applied to compare group means. A p-value of <0.05 was considered statistically significant. Data analysis was performed using SPSS v26.

3. Results

3.1. Antibiotic Susceptibility Testing

Clinical isolates of P. aeruginosa were often resistant to at least one of the antipseudomonal antibiotic categories but did not meet the criteria to be called multidrug resistant (MDR) [23]. Some strains of P. aeruginosa isolated were susceptible to the fluoroquinolone group (18/48 susceptible to enrofloxacin; 34/48 susceptible to marbofloxacin) or the aminoglycoside group (28/48 susceptible to gentamicin; 37/48 susceptible to amikacin and 48/48 susceptible to tobramycin). Also 47/48 were susceptible to polymyxin B (Table 2).

3.2. Antibacterial Studies by Agar Diffusion Method

The solutions used in this study have shown a mean inhibition zone of 0 mm for Tris-NAC (no inhibition at all for any tested strain), 15.9 mm (minimum value 9 mm; maximum value 23 mm) for the 10% SSD and 27.7 mm for the combination of Tris-NAC and 10% SSD (minimum value 19 mm; maximum value 32 mm) (Figure 1 and Table 3 and Table 4).

3.3. Synergy Experiment—Checkboard Assay

Individual compound therapy showed that SSD was more active, with a lower concentration needed to cause the inhibition of P. aeruginosa strains compared with Tris-NAC, as can be seen in Table 5. The antibacterial activity of the combined therapy was tested against 10 randomly selected P. aeruginosa isolates using the checkerboard assay to assess synergy, as seen in Table 5. The combination of Tris-NAC and SSD did lower the MIC values compared to individual therapy; however, this effect was shown to be additive rather than synergistic or antagonistic, with a FICI score of between 0.68 and 1.5 when tested against each isolate.

4. Discussion

The CLSI states that “P. aeruginosa may develop resistance during prolonged therapy with all antimicrobial agents; therefore, isolates that are initially susceptible might become resistant within 3–4 days of initiating therapy and repeat culture might be needed” [18]. All cases of chronic otitis referred to the Dermatology Service had been previously treated on multiple occasions with the most commonly used otic medications licensed for dogs. Therefore, the daily challenge has been to develop a protocol to deal with and successfully treat these infections while minimising the use of antibiotics. This prompted an in vitro study to test the combination of Tris-EDTA and NAC (Tris-NAC) with 10% SSD against a large number of P. aeruginosa strains. This combination (Tris-NAC with 10% SSD) has shown a significant inhibition of growth for all the tested strains. On the contrary, 10% SSD alone only caused a partial inhibition of bacterial growth, while Tris-NAC alone had no efficacy.
The methodology used in this study has been adapted and modified from previous studies [7,24]. An agar well diffusion technique has been used rather than impregnated discs (Kirby–Bauer). Wells were made and filled with an equal amount of the selected solutions. The bacterial susceptibility observed in this study was the result of the antibacterial action of the solution diffusing through the agar. The pH of the solutions tested was not evaluated as a previous study has shown poor correlation between the pH value and in vitro anti-Pseudomonas activity [7].
It is well known that EDTA damages the outer cell wall membrane of GN bacteria by chelating divalent cations, destroying the biofilm of P. aeruginosa in vitro [25,26]. This causes the release of lipopolysaccharides and renders the bacteria more permeable to other agents [27]. Tris is an alkaline buffer that potentiates the chelating action of EDTA [28]. It has been demonstrated that Tris-EDTA has a synergistic effect with various antibiotics and chlorhexidine [29,30,31]. Another study has reported that adding just Tris-EDTA (without the NAC) to SSD has shown no effect against P. aeruginosa [32].
The production of biofilm by P. aeruginosa strains isolated from dogs with otitis is common, with variability in the degree of the in vitro biofilm production between isolates [33]. The ability to produce biofilm could be an important virulence factor by facilitating the establishment of resistant infections in canine otitis. P. aeruginosa has been reported to produce extrapolymeric substance wherever conditions are favourable for bacterial colonisation [34]. It also produces proteases and other enzymes, which can lead to extensive ulceration and inflammation in the ear canal [33].
NAC has an inhibitory effect on various steps of biofilm formation by reducing the adhesion and formation of P. aeruginosa biofilm. It has been suggested that 20 mg/mL and 60 mg/mL of NAC would be the optimal concentrations for eardrops in human practice to prevent biofilm formation and to eradicate preformed biofilms, respectively [35]. It has also been reported to have an antibacterial effect by inhibiting clinically relevant and antibiotic-resistant bacteria in vitro against bacteria isolated from dogs with otitis externa. A further study has shown a poor interaction between NAC and enrofloxacin or gentamicin at the concentration tested in vitro against various bacteria isolated from dogs with otitis [24,36]. It is unknown why in this study the Tris-NAC alone had no efficacy against the tested strains, considering that both NAC and Tris-EDTA have been reported to have antibacterial activity [14,36]. It is unknown if this was due to, e.g., its concentration or stability issues. It is possible that only when Tris-NAC is used in combination with SSD that NAC and/or the Tris-EDTA component has an additive effect or the SSD may diffuse easily over the plate, thus facilitating the in vitro antibacterial activity, although the true mechanisms remain unknown. As bacteria embedded within a biofilm are more resistant to antimicrobials than the planktonic form, understanding the biofilm-forming ability of clinical isolates and their susceptibility, in comparison with their counterpart planktonic form, is important for further understanding the interaction effect between Tris-NAC and SSD.
The advantage of using SSD instead of other antibiotics is that it has soothing properties, is not an irritant and may enhance epidermal barrier repair by reducing inflammation [37]. Ulcerated and very inflamed ear canals are very common findings in dogs with P. aeruginosa otitis. Silver exerts its antimicrobial effects by binding to multiple cellular components and, as such, bacterial resistance to it is relatively low and, if it does occur, it does not confer resistance to other antibiotic classes [38].
In this study the checkerboard assay showed that MIC results for Tris-NAC were consistent at 12.5% across all the isolates. SSD had lower average MICs ranging from 0.0006% to 0.0064%. The combination of both compounds showed an additive effect rather than synergistic or antagonistic. It had been suggested to the authors that testing 10 isolates would be sufficient. However, it would have been ideal to test a larger number of isolates to confirm our results. The additive effect might be relevant in a clinical setting dealing in dogs with Pseudomonas spp. otitis, but further in vivo studies are needed to confirm our laboratory findings.
The in vitro study has shown that the SSD concentrations required to inhibit P. aeruginosa growth could be easily achieved clinically; although the results cannot be converted directly to the clinical setting, it is possible that the topical use of similar SSD concentrations could kill bacterial isolates commonly reported as resistant to the commercially available antibiotics. In fact, another in vitro study performed by Von-Silva Tarouca et al. reported that all the antimicrobial agents tested, including SSD, should achieve higher concentrations in the ear canal [13]. In Von-Silva Tarouca’s study, the MIC for SSD was actually slightly higher than the one reported in a previous study where MICs for an enrofloxacin–silver sulfadiazine combination were determined for resistant P. aeruginosa isolates [39]. Nevertheless, a conclusion cannot be made about whether the efficacy on those studies was due to the enrofloxacin or the SSD itself; however, Von-Silva Tarouca et al. suggested a potential synergy between SSD and enrofloxacin against P. aeruginosa.
Currently, there is only one veterinary otic preparation containing a combination of 1% SSD and 0.5% enrofloxacin [40]. The routine use of this product in countries where it is available might have led to the selection of the strains of P. aeruginosa now resistant to enrofloxacin or SSD. The concentration of SSD used in this study is higher than that present in the commercially available combination of SSD and enrofloxacin [40], and is much higher than the concentration shown to be effective in killing P. aeruginosa in one of the above in vitro studies [13].
SSD is considered to be safe in the middle ear; therefore, it should be considered as a valid treatment option for dogs suffering from P. aeruginosa otitis, even if the integrity of the tympanic membrane cannot be demonstrated. In contrast, polymyxin B and gentamicin are considered potentially ototoxic [41]. As this is an in vitro study, the authors cannot predict if the 10% concentration of SSD might cause in vivo local irritation or issues with systemic absorption, especially if the tympanic membrane is ruptured. Therefore, in vivo toxicity testing would be required in future studies.
This study has shown there is an additive effect between Tris-NAC and SSD, caused by the ability of the Tris-NAC to disrupt the biofilm, by disrupting the bacterial cell wall and enhancing the activity of the SSD or by both mechanisms. Unfortunately, the P. aeruginosa isolates were not tested to confirm if they were biofilm producer, although a slimy exudate was frequently present when samples were collected from patients. Future studies should quantify the biofilm biomass and test efficacy in both planktonic and sessile forms.
It is unknown if in vitro susceptibility testing can approximate an in vivo biofilm production testing. There is disagreement within the literature regarding the appropriate test to determine antibiotic MIC values for bacterial biofilms [42]. Unfortunately, none of the tests used in other published studies can be considered the gold standard as none can mimic the complexity of the microenvironment of an inflamed ear and the real biofilm activity production of the P. aeruginosa in dogs with otitis.
The culture and susceptibility of the ear canal is normally recommended in dogs with otitis not responding to empirical topical antimicrobial therapy. It is important to remember that bacterial culture and susceptibility (C&S) reports are based on the amount of the antibacterial drug in serum concentrations that is required to kill the organism [43]. This makes the interpretation of ear C&S reports challenging; breakpoints for topical administration do not exist. However, when used topically, these drugs can achieve much greater concentrations than those tested and may still be effective, especially when their use is combined with a product like Tris-EDTA [41].
Another limitation of our study is that susceptibility to some compounds such as SSD or drug combinations (additive/synergistic effects) are rarely reported. While susceptibility testing has limitations, results provide guidance to veterinarians regarding which antimicrobials may be more likely to be effective if systemic administration is considered. In our study the inhibition zone caused by the Tris-NAC and 10% SSD combination is numerically much larger than the one caused by the 10% SSD alone.
Although a 1% SSD solution has been reported to be effective against P. aeruginosa based on the MIC testing performed in the Von Silva-Tarouca study [13], we found that both 1% SSD and 2% SSD were still not enough to have a large inhibition zone on the plated P. aeruginosa strains selected for this study (unpublished data). The high concentration of SSD combined with the use of the biofilm disruptor (NAC) used in this study likely promotes SSD permeability to the deep layer of the biofilm, thus avoiding the selection of resistant mutants of bacteria that leads to resistant strains. If this combination could be applicable to clinical settings in dogs with Pseudomonas otitis, it might have the additional benefit of stimulating tissue regeneration and increasing the rate of re-epithelialization, as dogs with Pseudomonas otitis often have ulcerated ear canals.
Before this combination is applicable in a clinical setting to treat Pseudomonas otitis, it is important to remember that if multiple drugs are mixed in-house or by a compounding pharmacy, clinicians should be aware that potency, stability over time, purity, efficacy, sterility and safety can all be affected in the final product, so further studies are needed. The use of ‘home-made’ topical compounding solutions containing an ear cleanser and an injectable antibiotic (aminoglycosides and fluoroquinolones) is common in veterinary practice. However, little information is available on the most appropriate combination as well as their chemical and physical stability when applied topically into dogs’ ears. It has been often suggested that topical application of aminoglycosides and fluoroquinolones may overcome their in vitro resistance shown against some P. aeruginosa isolated from dogs with otitis [13].
The P. aeruginosa isolates from this study had a high frequency of susceptibility to polymyxin B and aminoglycosides. These antibiotics could still be highly effective for canine otitis; however, according to the CLSI M100 [44], there may be some ‘intrinsic’ resistance to aminoglycosides, not to mention that systemic regimens could be potentially toxic at MICs appropriate for Pseudomonas with aminoglycosides. Furthermore, the antibiotics’ efficacy can be reduced by organic debris in the ear canal due to the binding and inactivation of drugs, resulting in active concentrations much lower than applied concentrations. In the authors’ clinical experience, the use of these commercially available otic products containing aminoglycosides and fluoroquinolones has been previously unsuccessful in completely eradicating Pseudomonas otitis in our referred patients; therefore, there is a need for a different diagnostic–therapeutic approach.

5. Conclusions

Overall, the two compounds are additives when combined together, meaning that the MICs are generally lower or the same when combined but not enough to be classed as synergistic. The use of SSD, at a high concentration, could be evaluated as an important therapeutic tool against the highly resistant strains of P. aeruginosa. Furthermore, this combination could be a safe, effective and cost-effective way to control Pseudomonas otitis in dogs. The findings of this in vitro study should be confirmed by an in vivo clinical trial as it will allow for one to spare the use of antibiotics like aminoglycosides and fluoroquinolones, which are sometimes the only antibiotics still showing to be effective on the tested isolates [10].

Author Contributions

Conceptualization, R.C. and I.P.; methodology, R.C. and I.P.; formal analysis, P.S.; investigation, I.P., R.C., J.P., J.L. and A.W.; writing—original draft preparation, R.C. and I.P.; writing—review and editing, R.C., I.P., E.V., J.L. and A.W.; supervision, R.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article.

Acknowledgments

The authors are grateful to Kerry Brundell, VN for helping to collect swabs from affected dogs and Thomas King for helping with the laboratory work.

Conflicts of Interest

R.C. has performed consultancy work for Nextmune, Italy. I.P., J.P., E.V., J.L., A.W. and P.S. have no conflicts of interest.

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Microbiolres 16 00138 g001
Figure 1. The inhibition zone for three solutions is shown for one of the P. aeruginosa isolates tested, as an example.
Figure 1. The inhibition zone for three solutions is shown for one of the P. aeruginosa isolates tested, as an example.
Microbiolres 16 00138 g001
Table 1. MIC Breakpoints (µg/mL) for the tested antibiotics [18,19].
Table 1. MIC Breakpoints (µg/mL) for the tested antibiotics [18,19].
MIC Breakpoints (µg/mL)
AntibioticSensitiveIntermediateResistant
Marbofloxacin/Orbifloxacin≤12≥4
Enrofloxacin≤0.5From 1 to 2≥4
Gentamicin≤24≥8
Polymyxin B≤4/8
Ceftazidime≤816≥32
Amikacin≤48≥16
Table 2. Isolates tested and their susceptibility pattern for fluoroquinolones, aminoglycosides, ceftazidime and polymyxin B.
Table 2. Isolates tested and their susceptibility pattern for fluoroquinolones, aminoglycosides, ceftazidime and polymyxin B.
Samples Lab No.MarbofloxacinEnrofloxacinGentamicinTobramycinAmikacinCeftazidimePolymyxin B
421785RRSSSSS
421746SISSSSS
421810SSSSSSS
421843IRSNDSSS
421834RRSSSSS
421769SISSSSS
421931RRSSSSS
421904SSSSSSS
421987RRSSSSS
422166SISSSSS
422047SISSSSS
417558SSSSSSS
416724SSSSSSS
422227SSSSSSS
422705RRSNDSSS
422706RRSNDSSS
422726RRSSSSS
422849SISSSSS
422850SISSSSS
423069SSSNDSSS
423118IRSSSSS
418698RRSSSSS
418699SSSSSSS
423865SSSSSSS
423764SSSSSSS
423888RRSSSSS
425028SSSSSSS
425696SSSSSSS
425608SSSSSSS
425607SSSSSSS
425604RRSSSSS
425609SSSSSSS
425610SSSSSSS
425201SSSSSSS
424715SSSSSIS
424710SISSSSS
425205SSSSSSS
425571RRSSSSS
425570RRSSSSS
425941SSSSSSS
425951SSSSSSS
425955IRSSSSS
425898SSSSSSS
426085SSSSSSR
426170IRSSSSS
426160RRSSSSS
425968RRSSSSS
426210SSSSSSS
S: sensitive; I: intermediate; R: resistant; ND: not done.
Table 3. Minimum and maximum values of zone diameter for the inhibition of growth of P. aeruginosa cultures for the otic preparations used in the study.
Table 3. Minimum and maximum values of zone diameter for the inhibition of growth of P. aeruginosa cultures for the otic preparations used in the study.
Growth Inhibition Zone of P. aeruginosa Cultures
Otic SolutionMinimum Value (mm)Maximum Value (mm)
10% SSD923
Tris-NAC + 10% SSD1932
Table 4. Values of the mean and standard deviation of the zone of inhibition for Tris-NAC + 10% SSD and 10% SSD.
Table 4. Values of the mean and standard deviation of the zone of inhibition for Tris-NAC + 10% SSD and 10% SSD.
Values of the Mean, Standard Deviation and Variance of the Zone of Inhibition for the Tris-NAC + 10% SSD and 10%SSD
Otic SolutionTris-Nac + 10% SSD10% SSD
N=4848
Mean27.715.9
Standard deviation2.63.3
Welch’s t-test analysis p < 0.00001
The 48 samples that were tested with Tris-NAC + 10% SSD solution (mean = 27.7 mm; SD = 2.6), compared with the same samples that were tested with 10% SSD alone (mean = 15.9 mm; SD = 3.3), demonstrated significantly higher inhibition zones against P. aeruginosa, p < 0.00001.
Table 5. MICs of Tris-NAC and SSD against P. aeruginosa clinical isolates. These were performed in CAMHB, and MICs are shown as a percentage. MIC and fractional inhibitory concentration (FICI of Tris-NAC and SSD against 10 P. aeruginosa clinical isolates. Each MIC is presented as a percentage (%). The FICI synergy result is also presented. A FICI score of <0.5 = synergy, 0.5–4 = additive and >4 = antagonistic was used.
Table 5. MICs of Tris-NAC and SSD against P. aeruginosa clinical isolates. These were performed in CAMHB, and MICs are shown as a percentage. MIC and fractional inhibitory concentration (FICI of Tris-NAC and SSD against 10 P. aeruginosa clinical isolates. Each MIC is presented as a percentage (%). The FICI synergy result is also presented. A FICI score of <0.5 = synergy, 0.5–4 = additive and >4 = antagonistic was used.
P. aeruginosa IsolateAverage Individual MICAverage MIC CombinedFractional Inhibitory Concentration (FICI) ΣFICSynergy Result
Tris-NAC (%)Silver Sulfadiazine (%)Tris-NAC (%)Silver Sulfadiazine (%)
42596812.50.00142.6040.001261.14Additive
42608512.50.00642.8640.005060.94Additive
42595112.50.00383.3850.002040.79Additive
42616012.50.00076.2500.000711.49Additive
42695512.50.00060.3900.000511.21Additive
42594110.40.00125.2080.000550.92Additive
42617012.50.00083.2550.000510.93Additive
42589812.50.00122.2130.000610.68Additive
49621012.50.00094.1670.000511.00Additive
41819512.50.00066.2500.000611.50Additive
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Papadogiannaki, I.; Cerundolo, R.; Plant, J.; Villiers, E.; Littler, J.; Wisniewska, A.; Sgardelis, P. Antibacterial In Vitro Properties of Silver Sulfadiazine in Combination with Tris-EDTA and N-Acetylcysteine Against Pseudomonas aeruginosa Isolates from Dogs with Suppurative Otitis. Microbiol. Res. 2025, 16, 138. https://doi.org/10.3390/microbiolres16070138

AMA Style

Papadogiannaki I, Cerundolo R, Plant J, Villiers E, Littler J, Wisniewska A, Sgardelis P. Antibacterial In Vitro Properties of Silver Sulfadiazine in Combination with Tris-EDTA and N-Acetylcysteine Against Pseudomonas aeruginosa Isolates from Dogs with Suppurative Otitis. Microbiology Research. 2025; 16(7):138. https://doi.org/10.3390/microbiolres16070138

Chicago/Turabian Style

Papadogiannaki, Ioanna, Rosario Cerundolo, Jennifer Plant, Elizabeth Villiers, Jenny Littler, Anika Wisniewska, and Panagiotis Sgardelis. 2025. "Antibacterial In Vitro Properties of Silver Sulfadiazine in Combination with Tris-EDTA and N-Acetylcysteine Against Pseudomonas aeruginosa Isolates from Dogs with Suppurative Otitis" Microbiology Research 16, no. 7: 138. https://doi.org/10.3390/microbiolres16070138

APA Style

Papadogiannaki, I., Cerundolo, R., Plant, J., Villiers, E., Littler, J., Wisniewska, A., & Sgardelis, P. (2025). Antibacterial In Vitro Properties of Silver Sulfadiazine in Combination with Tris-EDTA and N-Acetylcysteine Against Pseudomonas aeruginosa Isolates from Dogs with Suppurative Otitis. Microbiology Research, 16(7), 138. https://doi.org/10.3390/microbiolres16070138

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