Antimicrobial and Antibiofilm Activity of Cinnamon Oil on Staphylococcus aureus Isolated from Cattle Milk with Mastitis

Bovine mastitis is one of the leading disease of animals, negatively affecting economy and welfare of dairy industry. Treatment failures are increasing antibiotic use, as some cases do not respond to standard therapies (Pedersen et al., 2021). Staphylococcus aureus due to its pathogenicity and ability to form biofilm is the most imperative and widespread environmental pathogen which spread through other contaminants, apart from contaminated milk of mastitis cows (Vargová et al., 2023). Biofilm are the communities of bacteria attached to a surface with a slime layer. It protects bacteria against treatments and help in persistence of bacterial infections. (Pizauro et al., 2021). Plant essential oils (EOs) with their antibacterial, antiviral, antifungal and insecticidal agents could be a better alternative to antibiotic therapy against multidrug-resistant bacteria (Razni et al.,2019; Caneschi et al., 2023; Shrivastava et al., 2020). Cinnamaldehyde present in Cinnamon oil increase membrane permeability and compromise membrane integrity, leading to the removal of preformed biofilms and significantly reducing biofilm formation (74.7 and 69.6%, respectively). (Budri et al., 2015; Ganic et al., 2022; Didehdar et al., 2022; Mohini and Alexander, 2024). Present study focuses on the use of essential oils with test drug cefoperazone, against biofilm formation.

A total of 164 bovines (cow = 113 and Buffalo = 51) from dairy farms were screened in and around Rewa (M.P., India) for mastitis by California Mastitis test (CMT) and clinical mastitis (CM) as abnormal milk, abnormal udder and /or abnormal cow (Constable et al., 2017; Rust et al., 2023).  Collected milk samples (300 µl) was inoculated in 30 ml of Mueller-Hinton broth supplemented with 6.5% NaCl, incubated at 35°C for 16-20 hours. Work was conducted after prior permission of IAEC (No71/IAEC/Vety/Rewa/6.4. 2022).

One loopful of pre-enriched culture was streaked into Mannitol Salt agar plates (35°C for 16-20 h) and thereafter in Baird-Parker agar plate supplemented with egg yolk-tellurite (at 35°C for 18-24 h). Colony was streaked onto a Tryptone soy agar plate (HI Media) incubated at 35 °C for 18-24 hour to confirm Staphylococcus aureus. The colonies were checked for purity by Gram’s staining for gram’s reaction, cellular morphology. Later Staphylococcus aureus was phenotypically characterized by standard biochemical tests, Novobiocin and Polymyxin B susceptibility (EFSA  2012). Biofilm producing ability of Staphylococcus aureus was phenotypically characterized by following methods.

Congo red agar method

The Congo red Agar was prepared  as per the earlier method with slight modifications (Kaiser et al., 2013; Raksha et al., 2020).

Light microscopy method

2 ml of overnight bacterial culture in tryptone soy broth (TSB) supplemented with 0.25% glucose (TSB-glucose). diluted 1:40 in TSB-glucose, was used per petri plate (30 mm) containing a cover slip. Incubated at 37°C for 18 h, gentle washing thrice with 1-2 ml of sterile phosphate-buffered saline (PBS), air dried and stained with 0.1% crystal violet for 10 minutes. Cover slips were put on a clean oil free glass slide and was observed under 40´of light microscope (S LPA U and SJG, 2020).

Microtiter plate assay method

Overnight bacterial culture was diluted 1:40 in tryptone soy broth (TSB) supplemented with 0.25% glucose (TSB-glucose), 200 µl of this cell suspension was inoculated in sterile, 96-well microtiter plates. After incubation at 37°C for 18 h, wells were gently washed thrice with 200 µl of sterile phosphate-buffered saline (PBS), air dried and stained with 0.1% crystal violet for 10 minutes. The biofilms were washed with distilled water, dried at 37°C for 15 min and destained with 200 μl of the destaining solution [50% (v/v) ethanol, 50% (v/v) glacial acetic acid], The amount of released stain was quantified by measuring the absorbance at 490 nm (A490) with a micro plate reader (Campo-Pérez et al.,2023). For scanning electron microscope (SEM) analysis, Glass slides were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 2-12 hours at 4°C followed by gradual dehydration (by 70, 85, 90 and 100% ethanol). The samples were air-dried, sputter-coated with colloidal gold and observed under an EVO18 scanning electron microscope at an operating voltage of 15 kV. For confocal microscope analysis, Glass slides having biofilm were fixed by absolute methanol for 2 minutes, air-dried and further subjected to staining with 0.1% acridine orange stain (dissolved in PBS) for 5 minutes. Finally, stained slides were examined with a confocal laser scanning microscope (CLSM) (Fig 1, 3).

The antimicrobial and anti-biofilm effect of cinnamon oil on biofilm producing Staphylococcus aureus

Commercially available (100% pure) Cinnamon oil were used after dilution in the DMSO by drop method for the study in the concentration range of 1%, 2% and 3%.  Phytochemical analysis of cinnamon was done for saponins, flavonoid, steroids/terpenes, Tannins, alkaloids, Resins, Phenols and glycosides (Effiong et al., 2020).

Quantitative analysis of essential oil by gas chromatography

Phytochemical analysis was performed through GCMS-QP2010 Plus equipment (outsourced from JNU New Delhi). Experimental Conditions were Ion Source Temp: 220.00°C; Interface Temp: 270.00°C;Solvent Cut Time: 2.50 min;Detector Gain Mode: Relative; Detector Gain: 0.00 kV; Threshold: 100; Column Oven Temp 50.0°C; Injection Temp -260.00°C; Injection Mode-Split Flow; Control Mode-Linear; Velocity Pressure-69.0 kPa Total Flow-137.3 mL/min; Column Flow-1.21 mL/min; Linear Velocity 39.9 cm/sec; Purge Flow-3.0 mL/min; Split Ratio-110.0Sample Inlet Unit :GC (Effiong et al., 2020).

Antibiofilm effect of cinnamon oil by Microtiter plate assay method

Fresh overnight bacterial culture (adjusted to 0.5 McFarland standards) (20µl) was inoculated in each well with, cinnamon oil (1%, 2% and 3%) Cefoperazone (5 µgm/ml) 200 µl per well and MHB, DMSO used as positive and negative controls, respectively plate incubated at 37°C for 18 h without any shaking. Stained with 1% crystal violet, washed, destained and absorbance recorded at an optical density of 490 nm. The percentage inhibition was calculated as (Sarah et al., 2023).
                 
                           

Determination of Minimum inhibitory concentration of Cefoperazone by Resazurin-based 96-well plate micro dilution method

The inoculums were prepared in accordance with the CLSI recommendation (as per 0.5 McFarland standard). diluted by 1:100 in MHB broth. Dilutions of the antibiotic prepared with the CLSI recommendation in the range of 256µgm/ml to 0.25 µg/ml (Peng et al.,2022). 100 µl, added in each column leaving column 11 and 12 for positive and negative control. 100 µl of the adjusted (0.5 McFarland standard) bacterial suspension (diluted by 1:100 in MHB) was added to each well. In each column 17 different biofilm isolates were added in triplicates In column 11 only MH broth and in column 12 MH broth with bacterial inoculums was added as negative and positive control respectively. After incubation for 24 h at 37°C, resazurin (0.015 %, 30 µl per well) was added), colour was observed after incubation of 30 minutes. Columns with no colour change (blue colour) were scored as above the MIC value (Elsheikh et al., 2016).

MIC of cinnamon oil by Resazurin-based 96-well plate micro dilution method

After dilution of cinnamon oil in the concentration range of 6%,5%,4%,3%,2%,1%,0.5%,0.4%. in the MH broth with DMSO as emulsifier MIC was performed like antibiotic. Color change was observed with resazurin (0.015%,), columns with no colour change were scored as above the MIC value.

Determination of sub-minimum inhibitory concentrations (sub-MICs) of antibiotics and its effect on biofilm formation

The MIC and Sub-MIC for the tested cefoperazone were determined, as per the standards of CLSI. Culture diluted to 1:100. Cefoperazone diluted in the range from 1/2,1/4 and 1/8 μg/mL of the MIC values of cefoperazone determined previously. Staphylococcus aureus was sub-cultured on Mueller Hinton, all wells in the first antibiotic-broth plate were dispensed with 50 μL of culture, while the wells of the second antibiotic-broth plate were inoculated with 50 μL of free culture. The same procedure was repeated to the antibiotic-free broth plates as control. After incubation for 72 hrs 30 μL of 0.1% crystal violet was added to each well and incubated at room temperature for 30 min. The bound bacteria were quantified by addition of ethanol 70% and measurement of the dissolved crystal violet at optical density (OD) of 630 nm using 96-flat wells microtiter plate The effect of sub -MIC level of antibiotic on the biofilm was estimated.

Phenotypic screening of staphylococcus aureus for biofilm production

Out of 164 bovines (cow = 113 and Buffalo = 51) screened for mastitis by California Mastitis test (CMT), 24 samples were found to be mastitis positive and 17 characterized as Staphylococcus aureus. Among the 17 Staphylococcus aureus samples 70% (n=12) were positive for biofilm formation on Congo red agar, where as 52% samples (n=8) were positive for biofilm on light microscopy (Fig 2). All the 17 samples showed biofilm production through micro titer plate method with almost 90% sensitivity. Subclinical mastitis is of concern in veterinary hospitals because contagious mastitis pathogens might be unknowingly transmitted to susceptible cows and then back to their farm of origin. Crystal violet staining binds to negatively charged molecules of bacteria and exopolysaccharides and is therefore used to quantify the total biomass of these systems (Fig 4).



Multiple studies observed that majorities of Staphylococcus aureus isolate from bovine mastitis cases can be studied in vitro by this assay (Bonsaglia et al., 2020). Congo red agar method giving very little correlation with the other methods with specificity (92%) and very low accuracy (41%), some reports do not recommend the Congo red agar method for biofilm detection in their study (Othmany et al., 2021). In a study undertaken previously (Sandasi et al., 2010) isolates showing biofilm formation by micro titer plate or tissue culture plate was 70 (64.7%), non or weak biofilm producers were 40 (36.3%) (Table 1).



Regional data from India also showed that out of 152 isolates tested, the number of biofilm producers identified by Micro titer plate method was 53.9 % and non-biofilm producers were 46%.

GC-MS analysis of cinnamon oil

Phytochemical screening of the Cinnamon oil was undertaken through GC-MS analysis led to the identification of a number of compounds. The various components present in the cinnamon oil Pinene alpha, BICYCLO [2.2.1] HEPTANE, 2, 2-DIMETHYL-3-METHYLENE- BENZALDEHYDE, Pinene beta-Phellandrene alpha-Terpinene alpha-Cymene para-, Phellandrene beta , CYCLOHEXENE, 1-METHYL-4-(1-ETHYLETHYLIDENE)- Linalool, ACETIC ACID, PHENYLMETHYL ESTER ,Terpinen-4-ol, OCTADIEN-3-OL, DIMETHYL-, PROPANOATE, 3-Phenylacrylaldehyde ,Cinnamaldehyde, 2-PROPENAL, 2-METHYL-3-PHENYL- Hexa-2,4-dienylbenzene, Eugenol ,Copaene alpha-Benzene, 1,3-hexadienyl- Caryophyllene, 2-Propen-1-ol, 3-phenyl-, acetate, (E)- Humulene alpha-, PHENOL, 2-METHOXY-4-(2-PROPENYL)-, ACETATE, Caryophyllene oxide Tetramethyl-12-oxabicyclo[9.1.0]dodeca-3,7-diene BENZOIC ACID, PHENYLMETHYL ESTER, .alpha.-Phellandrene, dimer Myrac aldehyde (Fig 5).Phytochemical screening of the cinnamon oil showed presence of Saponins Alkaloids Steroids/ Terpenes Tannins Alkaloids Resins in cinnamon oil phenols and Flavonoids were absent in cinnamon oil. Twenty-nine compounds were identified (Table 2, Fig 5).




Cinnamaldehyde was identified as the main compound with the highest peak area percentage of 70.61%, Eugenol was the second major compound detected with area % of 17.54%. (1.21%). Ainane et al., (2019) showed that the main component of the essential oil of cinchona bark was cinnamaldehyde (89.31%) and (69.15%) respectively which were similar to our results. Tsukatani et al. (2020) also suggested inhibitory activities in 498 different plant foodstuffs on biofilm formation of Streptococcus mutans by micro titer plate assay and concluded five of the extracts with potent inhibitory activities which further correlates with our finding. The antibacterial activity of Cinnamomum cassia (cinnamon) essential oil (EO) alone and in combination with some classical antibiotics against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa as reported earlier further correlates with our finding of antibiofilm and antimicrobial activity of essential oil (Atki et al., 2021). The bioactive components of essential oils supposed to be affecting the integrity of cell membranes by altering membrane permeability, resulting in leakage of electrolytes and loss of important intracellular contents (Kegang et al., 2023).

Antibiofilm activity by micro titer plate assay method

Antibiofilm activity of essential oils by micro titer plate assay method was measured using the dye crystal violet (CV) at 490 nm wavelength. (In triplicates) (Mean±S.E.). Maximum inhibition of cinnamon oil was observed at 3% concentration with O.D values ranging from 0.021 ± 0.005 to 0.084±0.007 with per cent inhibition of 96% to 86% respectively. In 2% and 1% concentration per cent inhibition was 16 to 20%. (Table 3,4, Fig 6).





In situ visualization of S. aureus biofilm

At the 5,10,15 KX magnification, SEM and confocal images of the S. aureus control group and the cinnamon oil treatment group at MIC were shown (Fig 1, 3).





The treated group had reduced biofilm, slime production and dismantling of cell layers on one another. As the microorganisms grew in the media supplemented with glucose, the biofilm production and growth rate were higher at MBC compared to the growth when MIC was determined. This reduction in the biofilm thickness and dismantling of the cell indicates the cinnamon oil inhibitory effect.

Determination of MIC of cefoperazone and essential oil against biofilm positive isolate

MIC of cefoperazone was in the range of 32 µgm/ml to 8 µgm/ml Minimal inhibitory concentration (MIC) states in vitro planes of susceptibility or resistance of explicit bacterial strains to applied antibiotic (Table 5) Cinnamon oil showed MIC of 0.5% in almost all the samples.



Dependable assessment of MIC has a significant effect on the optimal of a therapeutic strategy, which marks efficiency of an infection therapy. In view of our observations, Sader et al. (2020) reported MIC breakpoints of Cefoperazone sulbactam combination in the commercial product to be 16 µg/ml for susceptible organisms and 64 µg/ml for resistant organisms. Earlier studies showed MIC in the range of 32 µgm/ml to 2 µgm/ml which correlate with our findings. Comparison of MICs in different antibiotics is based on how far the MIC is as of the breakpoint, the site of the infection and other contemplations, such as the age, species and health of the animal (Millezi et al., 2019).

A positive correlation between cinnamon oil and cefoperazone MICs, was observed may be associated with antimicrobial resistance in some cases. In particular, increased resistance to multiple antibiotics was reflected in increased susceptibility to cinnamon oil. Although highly hypothetical but this suggests that acquired resistance may decrease the probability of the bacterium to counteract the activity of this essential oil (Jadhav et al., 2018; Van et al., 2022). The sub- MIC effect bacterial virulence factor expression and rational use of antimicrobials in clinical practice. No effect of sub-MIC of cefoperazone was observed by us on the biofilm production differing with the earlier findings where sub-MICs of most antibiotics inhibit the development of S. aureus biofilm, oxacillin, cefazoline, mupirocin and rifampicin even promoted staphylococcal biofilm formation. Bernardi et al (2021); Chen et al (2021) reported the increase in the ability to form biofilms over a concentration range from 1/2 to 1/64 of the MIC concentration. Efficacy of bacteriophage was also evaluated against Staphylococcus in mastitis (Srujana et al., 2022).

On the basis of our findings, we can conclude that presence of biofilm producing organism is very detrimental for proper treatment of Mastitis. Sensitivity of Congo red agar method, Light microscopy method is lower than Microtiter plate method for detection of biofilm production. Cinnamon oil showed antimicrobial and antibiofilm producing ability in all the concentration used (1%, 2%, 3%) Cefoperazone could be used in the biofilm producing Staphylococcus aureus causing mastitis in bovines but reduced concentration of cefoperazone has no effect on the biofilm formation as reported earlier since, cefoperazone showed no antibiofilm effect at sub-MIC concentration.

Authors are highly thankful to Dean, College of veterinary science and A.H. Rewa for providing necessary facilities. We are also thankful to Director, Advance Instrumentation Research facility, Jawaharlal Nehru University and All India Institute of Medical Sciences New Delhi for necessary help in our research work.

Disclaimers

The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

Informed consent

All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee (certificate attached).

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.