Exploration of antibiofilm and antibacterial activity of eugenol and cinnamic acid on Pseudomonas aeruginosa isolates from dogs

Introduction

In canine clinical practice, Pseudomonas aeruginosa is an important opportunistic pathogen associated with persistent infections of the ear, skin and wounds. It has been associated with several chronic and difficult-to-manage infections, including deep pyoderma, otitis externa, wound infections and urinary tract infections (Hillier et al., 2006; Hariharan et al., 2006; Mekic et al., 2011) [13,15,22] . The therapeutic management of infections caused by this organism is complicated by its ability to withstand antimicrobial treatment through intrinsic, acquired and mutational mechanisms of resistance (Livermore, 2002; Linder et al., 2005) [19,20] . The increasing occurrence of Multidrug-Resistant (MDR) P. aeruginosa highlights the need to investigate alternatives or complementary approaches to conventional antimicrobial therapy. This concern becomes particularly relevant in biofilm-associated infections, where the presence of a biofilm can contribute to persistence and reduced susceptibility to antimicrobial treatment. Consequently, compounds derived from plants have attracted considerable attention as potential sources of antimicrobial agents. Plants contain numerous biologically active secondary metabolites, including flavonoids, alkaloids, terpenoids and tannins, several of which have demonstrated antimicrobial activity in vitro (Cowan et al., 1999; Lewis et al., 2006) [6,18] . The antibacterial potential of plant-derived compounds has been investigated for many years, following the identification of naturally occurring compounds with therapeutic properties (Schmitz, 1985) [33] . Such phytochemicals may therefore provide useful candidates for investigation against resistant bacterial pathogens.

In our earlier investigations, the same canine clinical isolate collection was characterized with respect to antimicrobial resistance and ESBL production (Swathi et al., 2024) [36,37] . The present study extends this work by examining the biofilm-forming characteristics of these isolates and evaluating the in-vitro antibacterial and antibiofilm activities of eugenol and cinnamic acid.

Materials & Method Sample collection

A total of 97 clinical samples were obtained from dogs presenting with different clinical conditions. Ear swabs were collected from cases of otitis externa, while skin swabs were obtained from dogs with superficial pyoderma or chronic wounds. Sterile cotton-tipped swabs were used for sample collection. The clinical samples used in the present investigation were derived from the same canine sample collection previously described (Swathi et al.2024a, b) [36,37]

Isolation and characterization of Pseudomonas aeruginosa

The collected ear, skin and wound swabs were directly inoculated into Brain Heart Infusion (BHI) broth and incubated at 37 °C for 24 h. Following incubation, cultures showing turbidity were subsequently streaked onto Pseudomonas Isolation Agar (PIA) and maintained at 37 °C for 24-48 h. Colonies exhibiting the characteristic translucent appearance with green pigmentation were provisionally considered Pseudomonas spp. based on their colonial morphology. These colonies were further examined by Gram staining. Species-level confirmation of the P. aeruginosa isolates was subsequently performed by PCR using oligonucleotide primers targeting species-specific genes (Spiker et al.,). The isolation and preliminary characterization of the canine P. aeruginosa isolates have been described previously (Swathi et al., 2024a, b) [36,37]

Antibiotic sensitivity test

The antimicrobial susceptibility profile of the confirmed P. aeruginosa isolates was assessed by the Kirby-Bauer disc diffusion technique (Bauer et al., 1966) [1] . Ten antibiotics commonly used in veterinary practice were included in the test panel. The diameter of the inhibition zones produced around the antibiotic discs was measured and used for interpretation of the susceptibility or resistance pattern of individual isolates.

Multiple antibiotic resistance index

The multiple antibiotic resistance (MAR) index was calculated as the proportion of antibiotics to which an isolate was resistant relative to the total number of antibiotics tested (Osundiya et al., 2013) [24] .

MAR index = a/b

Where a represents the number of antibiotics against which the isolate exhibited resistance and b represents the total number of antibiotics to which the isolate was exposed.

Detection of ESBL production

Phenotypic screening for extended-spectrum β-lactamase (ESBL) production was initially carried out using the Phenotypic Screening Test (PST). Isolates identified as suspected ESBL producers were subsequently evaluated by the Double Disk Synergy Test (DDST), following the CLSI (2020) guidelines. The confirmed P. aeruginosa isolates were further examined by multiplex PCR (m-PCR) for the ESBL-associated genes blaTEM, blaSHV and blaOXA (Spiker et al.,). The phenotypic and molecular ESBL detection procedures were based on the methodology previously described by Swathi et al. (2024b) [37] Detection of biofilm formation Biofilm-forming ability was assessed using three phenotypic approaches: the tube method (Christensen et al., 1985) [2] , Congo Red Agar (CRA) method (Freeman et al., 1989) [9] and Microtiter Plate (MTP) assay, with modifications based on Robinson et al. (2019) [32] . The presence of the biofilmassociated genes ppyR and pslA was investigated according to the procedure described by Pournajaf et al. (2018) [27] .

Microtiter plate assay

For quantitative assessment of biofilm formation, 180 µL of Tryptic Soy Broth (TSB) was dispensed into the wells of the first row of a microtiter plate, followed by addition of 20 µL of bacterial suspension adjusted to 0.5 McFarland turbidity. Three wells containing sterile TSB without bacterial inoculum were included as negative controls. The plates were incubated at 37 °C for 24 h. After incubation, the culture contents were removed and each well was washed three times with 250 µL phosphatebuffered saline (PBS; pH 7.2). The plates were then inverted and heat-fixed at 60 °C for 60 min. Subsequently, 200 µL methanol was added to each well and allowed to remain for 15 min at room temperature to fix the adherent cells. The wells were then stained with 150 µL of 0.1% crystal violet for 15 min at room temperature. Following staining, excess dye was discarded and the wells were rinsed with sterile distilled water. The plates were inverted and allowed to dry at room temperature for 15-30 min. Biofilm-associated crystal violet was subsequently eluted by adding 150 µL of 95% ethanol to each well, followed by incubation for 30 min at room temperature. The Optical Density (OD) was measured at 570 nm using a Multiskan GO ELISA plate reader (Thermo Scientific).

Interpretation of the MTP assay

The cut-off Optical Density (ODc) was calculated as three standard deviations above the mean OD of the negativecontrol wells. Based on the measured OD values, isolates were categorized as follows  Non-biofilm producers: OD ≤ ODc  Weak biofilm producers: ODc < OD ≤ 2 × ODc  Moderate biofilm producers: 2 × ODc < OD ≤ 4 × ODc  Strong biofilm producers: OD > 4 × ODc

Biofilm inhibition study

Biofilms were first established in microtiter plates using the procedure described above. For evaluation of biofilm inhibition, the established biofilms were exposed separately to cinnamic acid and eugenol by adding 200 µL of the respective phytochemical preparation to the wells and incubating for 1 h (Coelho et al., 2013) [3] . Untreated wells containing 180 µL TSB and 20 µL bacterial suspension were maintained for the same duration as positive controls. Following treatment, the liquid from each well was removed and the biofilms were washed twice with sterile water. The extent of inhibition was determined by comparing the OD obtained from untreated control wells with that of phytochemical-treated wells. Absorbance was recorded at 630 nm using a Multiskan GO microtiter plate reader (Thermo Scientific). The following controls were included  Extract control: TSB containing the respective phytochemical extract.  Positive control: TSB containing the bacterial suspension.  Negative control: TSB containing sterile distilled water.  Media control: TSB alone.

The percentage inhibition of biofilm formation was calculated using

Determination of minimum inhibitory concentration of phytochemicals

The Minimum Inhibitory Concentrations (MICs) of eugenol and cinnamic acid were determined using the resazurinbased turbidometric assay described by Teh et al. (2017) [38] , with minor modifications. Resazurin was used as an oxidation-reduction indicator for assessing bacterial growth.

Preparation of resazurin solution

Resazurin was prepared in sterile distilled water at a concentration of 6.75 mg/mL. The dye is initially blue and, following reduction by oxidoreductase activity in viable cells, undergoes a colour transition through pink to colourless resorufin-related products.

Preparation of bacterial inoculum

Pure cultures of the P. aeruginosa isolates were inoculated into 5 mL Mueller-Hinton Broth (MHB) and incubated for 24 h at 37 °C. The resulting suspensions were adjusted to the turbidity of a 0.5 McFarland standard using sterile MHB. Subsequently, 10 µL of the adjusted suspension was dispensed into each well of U-bottom 96-well microtiter plates.

Preparation of phytochemical solutions

For cinnamic acid, a stock solution of 20 mg/mL was prepared by dissolving 20 mg of cinnamic acid in 1 mL Dimethyl Sulfoxide (DMSO). The eugenol stock preparation was obtained by diluting 1 mL of 98% (v/v) eugenol with a small quantity of Tween 80, maintaining the final Tween 80 concentration below 0.5%. The resulting preparation had a concentration of 16,420 µg/mL, based on a molecular weight of 164.2 g/mol, and the final volume was adjusted to 10 mL using sterile distilled water.

Preparation of antibiotic solution

Amoxicillin was used as the reference antibiotic. Its stock solution was prepared by dissolving 10 mg of the antibiotic in 1 mL sterile distilled water.

MIC assay

The assay was performed in U-bottom 96-well microtiter plates (Tarsons, Kolkata). Each well in the first row initially received 100 µL MHB. An equal volume (100 µL) of either cinnamic acid or eugenol preparation was then introduced into the first well. Two-fold serial dilution was performed sequentially by transferring 100 µL from one well to the next, continuing through the twelfth well. After completion of the dilution series, 100 µL was discarded from the final well. A 10 µL aliquot of the 0.5 McFarland-adjusted bacterial suspension was added to each well and mixed thoroughly. Each isolate was tested in duplicate. The plates were incubated overnight at 37 °C. Following incubation, 10 µL of resazurin solution (6.75 mg/mL) was added to every well, and the plates were incubated for a further 4 h at 37 °C. The wells were examined visually for a colour change from purple through pink to colourless. The lowest concentration of phytochemical associated with the observed colour change was recorded as the MIC.

Controls

The following controls were included in the assay:  Extract control: MHB containing the respective phytochemical extract.  Antibiotic control: MHB containing amoxicillin.  Growth control: MHB containing the bacterial suspension.  Sterility control: MHB containing sterile distilled water.  Media control: MHB alone.

Results & Discussion

Sampling, isolation and characterization of P. aeruginosa: In this study, a total of 97 samples were collected, of which 45 samples were from otitis cases and 52 samples were from pyoderma and chronic wounds of dogs. Out of 97 samples enriched using BHI broth, 35 samples predominantly produced characteristic green pigmentation on PIA and also have shown pink colour rods on Gram's staining. All these 35 samples were subjected to species specific confirmation by PCR using 16S rRNA and yielded a specific PCR product at 956 bp. This present study corelates with the published results on the prevalence of P. aeruginosa which reported to be ranging from 6.5 % to 43 % (Foster and DeBoer 1998; Colombini et al., 2000; Quinn et al., 2011; Falodun and Musa 2020) [4,8,20,28] .

Antibiotic sensitivity profile of P. aeruginosa

The antimicrobial susceptibility profile of the 35 confirmed P. aeruginosa isolates, previously characterized by our group (Swathi et al., 2024a) [36] , was considered as the baseline antimicrobial-resistance profile for the present investigation. Antimicrobial susceptibility testing was carried out for all 35 PCR-confirmed P. aeruginosa isolates using the Kirby-Bauer disc diffusion method with ten antibiotics. The greatest proportion of resistant isolates was recorded for tetracycline (97.14%), followed by ampicillin (94.28%). Resistance to aztreonam, streptomycin and enrofloxacin was 91.42% for each antibiotic, while resistance to co-trimoxazole, amoxiclav and gentamicin was 88.57%, 80% and 74.28%, respectively. Ceftriaxone showed resistance in 65.71% of isolates, whereas imipenem exhibited the lowest resistance rate (11.42%). Importantly, all 35 isolates (100%) fulfilled the criteria for Multidrug Resistance (MDR), while 30 isolates (85.71%) were classified as Extensively Drug-Resistant (XDR). All isolates also showed MAR index values ≥0.2. These baseline resistance characteristics were previously reported for this isolate collection (Swathi et al., 2024a) [36] . The low resistance observed against imipenem (11.42%) was comparable with the 14% reported by Plokarz et al. (2023) [25] . In contrast, the resistance rates for enrofloxacin and gentamicin in the present investigation were considerably higher than those reported by Ramanarayana (2022) [30,31] , who recorded resistance of 9.61% and 21.15%, respectively. Such differences between studies may reflect variation in antimicrobial-use practices and treatment protocols across geographical regions. The proportion of MDR isolates observed in the present study was also comparable with the 97.56% MDR reported by Costa et al. (2022) [5] . Furthermore, the MAR findings were consistent with Ramanarayana (2022) [30,31] , where all 55 P. aeruginosa isolates had MAR indices above 0.2.

ESBL profile of P. aeruginosa isolates

The ESBL characteristics of the 35 P. aeruginosa isolates used in the present study have been reported previously by our group (Swathi et al., 2024b) [37] . Briefly, phenotypic confirmation by DDST identified 21 (60%) isolates as ESBL producers. All 35 confirmed P. aeruginosa isolates were initially evaluated by phenotypic screening for ESBL production. Resistance to at least one of the tested cephalosporins resulted in their classification as suspected ESBL producers. The DDST was subsequently performed using cefotaxime, ceftazidime, ceftriaxone and aztreonam positioned around a central amoxiclav disc. A positive synergy or "ghost" phenomenon was observed in 21 isolates, corresponding to 60% of the tested population. The 35 PCR-confirmed isolates were further examined for the presence of blaSHV, blaOXA and blaTEM. Amplification products of 564 bp, 713 bp and 800 bp were obtained for blaSHV, blaOXA and blaTEM, respectively. At least one of the investigated ESBL genes was detected in 15 isolates (42.85%). Among the individual genes, blaOXA was detected most frequently, occurring in 11 isolates (31.42%), followed by blaSHV in nine isolates (25.71%) and blaTEM in seven isolates (20%).

The phenotypic ESBL frequency observed here was comparable with the 70.15% reported by Laudy et al. (2017) [16] . The frequencies of blaSHV and blaTEM were also considered in relation to earlier reports by Shacheraghi et al. (2010) and Ramanarayana et al. (2022) [30,31] . Differences in the distribution of individual ESBL genes among studies may be associated with geographical variation, as suggested in the original manuscript. The phenotypic and genotypic ESBL findings were consistent with our earlier report based on this isolate collection (Swathi et al., 2024b) [37] .

Biofilm formation by P. aeruginosa isolates

The three phenotypic assays used for biofilm detection tube assay, Congo Red Agar (CRA) method and microtiter plate (MTP) assay showed differences in the distribution of biofilm-producing isolates. Using the tube method, 3 isolates (8.57%) were classified as non-adherent, 8 (22.85%) as weak producers, 14 (40%) as moderate producers and 10 (28.57%) as strong producers. These findings differed from those of Raheem et al. (2023) [29] , who reported 5% non-adherent, 5% weak and 90% strong biofilm-forming isolates. Differences in categorization may partly arise from the subjective interpretation associated with visually assessing the intensity of biofilm formation. The CRA assay classified all 35 isolates (100%) as biofilm producers. Within this group, 17.14% were weak, 45.71% moderate and 37.14% strong producers. The observation that all isolates produced biofilm was comparable with the findings of Locatelli et al. (2004) [21] and Freitas and Barth (2002) [10] , who also reported biofilm production among all P. aeruginosa strains examined. Quantitative assessment using the MTP assay similarly identified all 35 isolates as biofilm producers. Of these, 8.57% were weak, 62.85% moderate and 28.57% strong producers. The manuscript notes that 1% glucose was incorporated into TSB to promote biofilm development, consistent with the approach reported by Hassan et al. (2011) [14] . The 100% biofilmpositive finding obtained by CRA and MTP was also comparable with the report of Ramanarayana (2022) [30,31] . [15,23,26,27] .

Biofilm inhibition by eugenol and cinnamic acid

All 35 P. aeruginosa isolates identified as biofilm producers by the phenotypic assays were subsequently evaluated for inhibition of established biofilms using the microtiter plate assay. Both eugenol and cinnamic acid demonstrated inhibitory activity, although the magnitude of inhibition varied with the compound and concentration used. Eugenol produced mean inhibition rates of 43.144±2.638% at 0.4 mg/mL and 20.983±2.010% at 0.2 mg/mL. Cinnamic acid produced corresponding mean inhibition rates of 55.873±2.050% at 2.5 mg/mL and 47.625±2.146% at 1.25 mg/mL. Thus, increasing the concentration of either phytochemical resulted in greater mean biofilm inhibition under the conditions evaluated. Although cinnamic acid produced a higher absolute inhibition at the concentrations tested, eugenol achieved a comparable level of inhibition at substantially lower concentrations. This observation suggests that eugenol may have greater potency on a concentration basis in the present experimental system. However, this interpretation should be presented as a comparative observation from the tested concentrations, rather than as a definitive claim of superior antibiofilm activity. The present findings are comparable with those of Zhou et al. (2013) [40] , who reported approximately 43% inhibition of P. aeruginosa biofilm formation with eugenol at a sub-inhibitory concentration. In the present study, a similar inhibition percentage was obtained, although at a higher concentration. The difference may reflect variation in the characteristics of the bacterial isolates and experimental conditions. [39] also investigated the antibiofilm activity of Cinnamomum verum essential oil and reported a 50% reduction in an established 24-h bacterial biofilm at 1 mg/mL. The findings from the present study show a comparable level of inhibition with the tested phytochemicals, although direct comparison should be made cautiously because the experimental preparations and conditions were not identical. Letsididi et al. (2018) [17] reported ≥50% inhibition of biofilm formation using transcinnamic-acid nanoemulsion at 0.39 mg/mL. In comparison, the present study required 2.5 mg/mL cinnamic acid to achieve a mean inhibition exceeding 50%. The greater activity reported for the nanoemulsion preparation may be related to the formulation and physicochemical characteristics of the delivery system; however, this should be regarded as a possible explanation rather than a demonstrated mechanism in the present study.

Minimum inhibitory concentration of the phytochemicals

The MIC values demonstrated variation among the P. aeruginosa isolates. Eugenol showed MIC values ranging from 6.41 to 51.31 µg/mL, whereas the corresponding values for cinnamic acid ranged from 31.25 to 500 µg/mL based on the individual isolate results presented in Table 2.

The reference antibiotic, amoxicillin, showed MIC values ranging from 15.625 to 1000 µg/mL in the tabulated results. The lower MIC values observed with eugenol compared with cinnamic acid indicate greater inhibitory activity of eugenol against the tested isolates under the conditions of this assay. This observation is particularly noteworthy because all isolates evaluated in the present study were characterized as MDR. The finding is consistent with the report of Costa et al. (2022) [5] , who demonstrated antimicrobial activity of eugenol against MDR P. aeruginosa isolates. [11] investigated the action of isoeugenol against P. aeruginosa and reported significant antibacterial activity with MIC of 64 µg/mL which is in correlation with the upper range value of MIC for eugenol obtained in the current study. Wijesinghe et al. (2021) [39] reported the MIC value of Cinnamon essential oil on P. aeruginosa was 0.5 mg/ mL (i.e., 500 µg/ mL) which was similar to the present study.

Due to the increased prevalence of MDR and XDR P. aeruginosa isolates, the need of novel agents for the treatment and management of biofilm associated infections is high. As one of the reasons for the increased MDR and XDR strains is due to the biofilm forming ability of the isolates, targeting them with the agents that have the potential antibacterial as well as the anti-biofilm activity helps in reduction of the persistent infections. Addition of eugenol and cinnamic acid to destabilize and inhibit biofilm formation is a promising approach to treat chronic infections. However, further studies are required for the clinical use of these agents.

Conclusion

The present study identified a high frequency of antimicrobial resistance and biofilm-forming ability among P. aeruginosa isolates obtained from canine clinical infections. All confirmed isolates exhibited multidrug resistance, while a substantial proportion showed an extensively drug-resistant phenotype and phenotypic ESBL production. The detection of biofilm-associated genes further supports the ability of these isolates to persist in clinical environments.

Both eugenol and cinnamic acid demonstrated antibacterial and antibiofilm activity against the tested isolates. Cinnamic acid produced greater mean biofilm inhibition at the concentrations evaluated, whereas eugenol showed lower MIC values in the present assay. These findings indicate that both phytochemicals warrant further investigation as potential adjunct or alternative approaches for controlling resistant and biofilm-associated P. aeruginosa infections in dogs. Further studies using larger isolate collections and appropriate in-vivo models are required before their therapeutic application can be established.

Funding source of research

The authors are very much grateful to Sri Venkateswara Veterinary University, Tirupati, Andhra Pradesh, India, for providing the facilities to carry out the work.

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