MATERIALS AND METHODS

Study area: The study was carried out at Microbiology laboratory, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Malaysia from November, 2018-June, 2019.

Fabrication of CS-CaCO₃NP and OTC-CS-CaCO₃NP: Fabrication and characterization of CS-CaCO₃NP and Oxytetracycline loaded calcium carbonate aragonite nanoparticle (OTC-CS-CaCO₃NP) as well as in vitro release of OTC from CS-CaCO₃NPwere done as described in our previous study²⁶.

Bacterial culture and staining characteristics: Corynebacterium pseudotuberculosis used in this work was obtained from the Bacteriology Laboratory of Faculty of Veterinary Medicine, Universiti Putra Malaysia, Malaysia. Blood agar was used to culture the bacteria at 37°C for 48 hrs. Gram staining was done to confirm the staining characteristic of the organism.

Antibacterial mode of action: High-Resolution Transmission Electron Microscopy (HR-TEM) was performed to study the effect of cockle shell nanoparticles on bacterial cell integrity and to further explain the mechanism of action of the drug. Control and C. pseudotuberculosis treated with OTC-CS-CaCO₃ NP, CS-CaCO₃NP and OTC for 24 hrs were fixed and prepared for viewing by HR-TEM. Briefly, bacteria were fixed in fixation solution (2% glutaraldehyde+2% paraformaldehyde dissolved in 0.12 M Sorensen’s buffer, pH 7.4) and then stained by (2%) Osmium Tetroxide solution for 1 hr at 4°C. Embedding sections were cut on an MT6000 ultramicrotome (Sorvall, New Castle, DE). Thin sections (0.1-0.05 mm) of the embedded bacterial cell ultrastructural changes induced in C. pseudotuberculosis treated with OTC-CS-CaCO₃ NP, CS-CaCO₃NP and OTC were examined under the electron microscope (Hitachi H-7100, Hitachi Ltd., Tokyo, Japan).

Corynebacterium pseudotuberculosis biofilm formation: Bacterial colonies were collected from the blood agar plate and dissolved in Tryptic Soy Broth (TSB) with 2% FBS. The density of the bacterial suspension was standardized by comparison with 1.0 McFarland standard (3.0×10⁸ CFU mL^–1). This was incubated at 37°C overnight. Biofilm growth, staining and quantification were done as described by. Overnight C. pseudotuberculosis culture was diluted (1:30) into fresh TSB to give an approximate bacterial count of 1.0×10⁷ CFU mL^–1. Total 100 μL of the diluted culture was added into each well of the 96 well non-tissue culture treated plates. This was then incubated at 37°C on a rocker at 10 rpm for 6, 12 and 24 hrs at 10% CO₂ to evaluate biofilm formation. Following incubation, the unattached C. pseudotuberculosis cells were discarded by turning the plate over and shaking out the liquid. Then the plate was gently submerged in a small tub of water and the water was gently shaken out to remove the remaining media and unattached planktonic cells. About 125 μL of a 0.1% solution of crystal violet was added to each well of the 96 well plates for 10-15 min and incubated at room temperature. After incubation, the plate was submerged in a clean plate of water to completely wash out the crystal violet. This was then blotted dry on a paper tissue and the plates turned upside down to dry overnight. To quantify the number of biofilms formed, 125 μL of 30% acetic acid in water to each well of the microtiter plate to solubilize the crystal violet. The 96 well plates were then incubated at room temperature for 10-15 min after which 125 μL of the solubilized biofilm stained with crystal violet was then transferred to a new microtiter plate. The biofilms were quantified by reading the absorbance in a microplate reader (TEKAN Infinite M200PRO) (Tekan, Seestrasse, Mannedorf Switzerland) at 600 nm using 30% acetic acid in water as the blank. The experiment was done 3 times on different days and the average Optical Density (OD) at 600 nm was determined. The results were interpreted as follows: Positive (>0.24), weak (>0.12 and <0.24), or negative: (<0.12)²⁷.

MIC and MBEC of CS-CaCO₃NP, OTC-CS-CaCO₃NP and OTC Against C. pseudotuberculosis: The MIC of CS-CaCO₃NP, OTC-CS-CaCO₃ NP and OTC was determined using the microtiter plate assay method. Total 100 μL of sterile Mueller Hinton Broth (MHB) was added to 96 well microtiter plates then 100 μL of CS-CaCO₃NP, OTC-CS-CaCO₃ NP and OTC prepared in MHB were added to the first well and 2-fold serial dilutions were done (2000-15.6 μg mL^–1). Treatment was not added to the last row to serve as the control. A 5 μL of diluted overnight cultures (1:100) of C. pseudotuberculosis (1.0×10⁸ CFU mL^–1) in TSB supplemented with 2% FBS was added to the treatment rows except for the control. This was incubated overnight at 37°C for 24 hrs. MIC was determined, visually assessing the turbidity in the CS-CaCO₃NP, OTC-CS-CaCO₃ NP and OTC 96 well plates and further confirmed using a micro ELISA auto reader (TEKAN Infinite M200PRO) (Tekan, Seestrasse, Mannedorf, Switzerland) at a wavelength of 600 nm. The lowest concentration that inhibited bacterial growth (showed no visible growth after 24hrs incubation) was assigned as the MIC.

The MBEC was done as described by Mulla et al.²⁸ with modifications. Overnight C. pseudotuberculosis culture (From TSB with 2% FBS) was diluted (1:30) into fresh MHB (with 2% FBS) broth for biofilm assay. This is to give an approximate bacterial count of 1.0×10⁷ CFU mL^–1 in sterile glass tubes. A 150 μL of the diluted culture was added into each well of the 96 well microtiter plate and incubated at 37°C on a rocker at 10 rpm for 24 hrs at 10% CO₂.

Biofilm formation was confirmed by staining the 96 well plates with 0.1% crystal violet for 10-15 min. The plates were then submerged in a tube with water, shaken out to remove the excess dye and blotted dry on a stack of paper towels to remove the excess cells and dye.

Mechanical disruption of biofilms was done by ultrasonic disruption of the biofilms in the 96 well microtiter plates after adding 150 μL of MHB with 2% FBS for 5 min and then incubated at 37°C for 24 hrs with OTC-CS-CaCO₃ NP, CS-CaCO₃NP and OTC (2000-15.6 μg mL^–1). MBEC was determined by inspecting the plates visually for turbidity and confirmed by reading the absorbance at 600 nm in a 96-well plate reader (TEKAN Infinite M200PRO) (Tekan, Seestrasse, Mannedorf Switzerland) as described by Harrison et al.²⁹.

Biofilm inhibition assay: Inhibition of C. pseudotuberculosis biofilm formation by OTC-CS-CaCO₃NP and OTC was studied spectrophotometrically using crystal violet assay as described previously³⁰. Overnight C. pseudotuberculosis culture was diluted (1:30) into fresh TSB to give an approximate bacterial count of 1.0×10⁷ CFU mL^–1. A 100 μL of the diluted culture was added into each well of the 96 well non-tissue culture treated plates containing different concentrations of OTC-CS-CaCO₃NP NP and OTC ranging from 2500-78.13 μg mL^–1 obtained by 2-fold serial dilution in MHB and incubated at 37°C for 24 hrs on a rocker. The wells were then washed with sterile PBS to ensure the removal of unattached planktonic C. pseudotuberculosis. A 125 μL of a 0.1% solution of crystal violet solution was added to each well for 15 min. After incubation, the crystal violet solution in the wells was removed and excess crystal violet was washed out with sterile PBS. Total 125 μL of 30% acetic acid in water was added to each well of the microtiter plate to solubilize the crystal violet stain for 10-15 min, then the solubilized biofilm was transferred to a new microtiter plate and absorbance was measured spectrophotometrically at 600 nm. The percentage of biofilm inhibition was calculated as follows:

Morphological changes of C. pseudotuberculosis biofilms exposed to OTC-CS-CaCO₃NP
Scanning electron microscopy: Total 96 well plates with the bacterial biofilm were incubated for 24 hrs at 37°C with MBEC of CS-CaCO₃NP, OTC-CS-CaCO₃ NP and OTC. The supernatant was removed and rinsed with PBS, the biofilm was then disrupted by sonication then collected with pipettes into centrifuge tubes. The control and treated biofilms were centrifuged at 3000 rpm for 5 min after which the supernatant was removed and was fixed with glutaraldehyde for 6 hrs and further prepared for analysis using SEM. Briefly, the bacterial biofilm was fixed in fixation solution (2% glutaraldehyde+2% paraformaldehyde dissolved in 0.12 M Sorensen’s buffer, pH 7.4), dehydrated with graded levels of acetone 35, 50, 75, 90 and 100% sequentially. Then stained by (2%) Osmium Tetroxide solution for 2 hrs at 4°C and was examined and photographed using a scanning electron microscope (JSM-6400 Scanning Microscope) (Jeol, USA).

Fluorescent microscopy: Plates with control biofilm and biofilm treated with MBEC of CS-CaCO₃NP, OTC-CS-CaCO₃NP and OTC were incubated for 24 hrs at 37°C. The plates were rinsed with PBS then treated with 6 μL of Acridine orange/Ethidium bromide (1 and 3 mg mL^–1, respectively) was added to each well, then incubated in the dark for 20 min. Excess dye was removed from each well and the wells were viewed under a fluorescent microscope before the dyes leak out of the biofilm. The wells were photographed by a fluorescence microscope (Motic Asia, Hong Kong) with a FITC filter (green) for live cells a TRITC filter (red) for dead biofilms as described by Skogman et al.³¹.

RESULTS

Cultural and staining characteristics of C. pseudotuberculosis: The result of Fig. 1a revealed the colonial morphology of C. pseudotuberculosis on 5% sheep blood agar. It presented as small (about 1 mm in diameter) with whitish and translucent colonies which did not produce hemolysis following 48 hrs of incubation at 37°C. Gram staining the smear made from the cultured C. pseudotuberculosis showed a pleomorphic Chinese Letter-like presentation which stained Gram-positive (Fig. 1b).

Fig. 1(a-b):

Cultural and staining characteristics of C. pseudotuberculosis (a) C. pseudotuberculosis after 48 hrs of incubation at 37°C grew as small (1 mm diameter) white, opaque and dry colonies with no haemolysis and (b) C. pseudotuberculosis stained Gram-positive displaying the characteristic Chinese-letter presentation (magnification ×100)

MIC and MBEC of CS-CaCO₃NP, OTC-CS-CaCO₃NP and OTC against C. pseudotuberculosis: The MIC values of OTC-CS-CaCO₃NP and OTC that inhibited the growth of C. pseudotuberculosis in Mueller Hinton broth was 125 and 500 μg mL^–1, respectively. Whereas the MBEC was 250 and >2000 μg mL^–1 (Table 1).

Antibacterial mode of action of OTC-CS-CaCO₃NP: TEM studies were done to observe the antibacterial effect of OTC-CS-CaCO₃NP on C. pseudotuberculosis cell walls. The images obtained indicated OTC-CS-CaCO₃NP that disrupted the integrity of the cell wall as shown in Fig. 2a-d.

Fig. 2(a-d):

TEM images of C. pseudotuberculosis (a) Control, (b) CS-CaCO3NP treated bacterial showing the normal characteristic electro dense (red arrow) and a plasma membrane layer (yellow arrow) of Corynebacterium spp., (c) OTC treated bacteria showing the mild dissolution of membrane layers (blue arrow) and (d) OTC-CS-CaCO3NP) showing dissolution of cells (green arrow) (scale bar 200 μm)

The control C. pseudotuberculosis cell (Fig. 2a) appeared with a complete cell wall and plasma membrane showing the normal structure of C. pseudotuberculosis having a close association between the thick electron-dense layer (yellow arrow) and the plasma membrane (blue arrow). All the layers were also present in the CS-CaCO₃NP treated group (Fig. 2b) implying that CS-CaCO₃NP had no antibacterial effect on C. pseudotuberculosis.

Treatment with OTC caused the bacteria cells to have slightly reduced thickness of the electron-dense layer with the majority of the cells remaining undamaged (Fig. 2c).

Furthermore, treatment of C. pseudotuberculosis with OTC-CS-CaCO₃NP resulted in changes in the morphology of the bacterial surface. After 24 hrs, OTC-CS-CaCO₃NP caused the outer layers of the cells to be flattened out with reduced thickness, membrane dissolution, broken cell membrane and presence of vacuoles within the bacterial cells (Fig. 2d). TEM analysis further revealed that OTC treated C. pseudotuberculosis showed intact bacterial cells (Fig. 3a) whereas treatment with OTC-CS-CaCO₃NP resulted in leakage of the cytoplasmic components to the outer environment through the disruption of the cell wall (Fig. 3b).

Corynebacterium pseudotuberculosis biofilms formation: C. pseudotuberculosis biofilms significantly increased from 0.32±0.05 at 6 hrs to 2.17±0.72 and 2.94±0.25 at 12 and 24 hrs respectively, at 600 nm optical density (Fig. 4).

Biofilm inhibition assay: OTC-CS-CaCO₃NP and OTC at concentrations from >MBEC to the sub-MBEC (2500-78.13 μg mL^–1) were utilized to study the antibiofilm activity against C. pseudotuberculosis using crystal violet biofilm assay as shown in Fig. 2b-c. Biofilm formations by C. pseudotuberculosis were significantly reduced by OTC-CS-CaCO₃NP at concentrations of 156.3 and 78.13 μg mL^–1 (p<0.05) (Fig. 5a).

Fig. 3(a-b):

TEM images of C. pseudotuberculosis (a) After treatment with OTC showing intact bacterial cells and (b) OTC-CS-CaCO3NP showing the dissolution of cells and presence of vacuoles (red arrow), broken cell membrane (blue arrow) and detachment of cytoplasmic membrane from the bacterial cell wall (green arrow) (scale bar 1 μm)

Fig. 4:

Biofilm formation (Optical density600) of C. pseudotuberculosis by crystal violet assay

Whereas the OTC treated biofilms were significantly reduced starting from 625-78.13 μg mL^–1 (p<0.05) (Fig. 5b).

Morphological changes assessment
Scanning electron microscopy: Visualization of C. pseudotuberculosis biofilms with scanning electron microscopy and fluorescence microscopy showed morphological differences in the OTC-CS-CaCO₃NP and OTC treated biofilm architectures. The control and CS-CaCO₃NP treated C. pseudotuberculosis displayed the characteristic 3-dimensional morphology showing rod-shaped, smooth-surfaced bacteria cells closely packed together. Also, the cell membranes of the bacterial cells were intact (Fig. 6a-b) whereas C. pseudotuberculosis treated with OTC revealed bacterial cells in the biofilm less compactly held together, with blebbing and roughness on the outer surfaces of the cells (Fig. 6c). OTC-CS-CaCO₃NP treated biofilms showed roughness on the outer surface of the bacteria seen in the biofilm. Most of the bacterial cells in the biofilm were digested and having fewer bacterial cells with swollen, rough, irregular and non-uniform shapes (Fig. 6d). This result shows that OTC-CS-CaCO₃NP destroyed the cell membrane, with a roughness of the outer surface of the bacteria seen in the biofilm. Most of the bacterial cells in the biofilm were digested and having fewer bacterial cells with swollen, rough, irregular and non-uniform shapes.

Fluorescence microscopy: Visual examination of fluorescence images of untreated biofilms and CS-CaCO₃NP, OTC-CS-CaCO₃NP and OTC treated biofilms showed colours depicting the different stages of cell death (Fig. 7a-d). Control and biofilms treated with CS-CaCO₃NP showed live bacterial cells (bright green) (Fig. 7a-b). Whereas OTC-CS-CaCO₃NP treated biofilms had dead bacterial cells (Red), early apoptotic bacterial cells (greenish-yellow), late apoptotic bacterial cells (Orange-red) and dying cells (faint green) (Fig. 7c).

Fig. 5(a-b):

Antibiofilm formation activity by crystal violet assay (a) OTC-CS-CaCO3NP antibiofilm activity on C. pseudotuberculosis and (b) OTC antibiofilm activity on C. pseudotuberculosis

Fig. 6(a-d):

Scanning electron microscope image of C. pseudotuberculosis biofilm (a) intact cell membrane and smooth cell surface, (b) Treatment with CS-CaCO3NP showing intact cell membrane and smooth cell surface, (c) Treatment with OTC showing bacterial cells with pleomorphic shapes, roughness and blebbing on the cell surfaces and (d) Treatment with OTC-CS-CaCO3NP showing digested bacterial cells with irregular shapes, rough cell surface and membrane

However, treatment of biofilms with OTC resulted in viable bacterial cells (bright green), early apoptotic cells (greenish-yellow) and a few late apoptotic cells (orange-red) (Fig. 7d).

Fig. 7(a-d):

Fluorescence microscope image of C. pseudotuberculosis biofilm (a) Viable cells (The bright green coloured biofilms indicate live biofilms), (b) Treatment with CS-CaCO3NP and stained by acridine orange/ethidium bromide (The bright green coloured biofilms indicate live biofilms, (c) Treatment with OTC (The green colour depicts viable biofilms, the greenish-yellow = early apoptotic biofilms and orange-red = late apoptotic biofilms) and (d) Treatment with OTC-CS-CaCO3NP Showing dead biofilms (red), early apoptotic biofilms (greenish-yellow) and late apoptotic biofilms (orange-red)

DISCUSSION

In the present study, the planktonic form of C. pseudotuberculosis was highly susceptible to OTC compared to the biofilm form which agrees with the results of Olson et al.⁵. The lower MIC of OTC-CS-CaCO₃NP compared to that of OTC in the present study means that encapsulation of OTC in CS-CaCO₃NP nanoparticles enhanced the antibacterial sensitivity of OTC against C. pseudotuberculosis. The antibacterial effect of OTC-CS-CaCO₃NP may be due to the large surface area of the CS-CaCO₃NP conjugated with a considerable number of OTC molecules that bind to the outer surface of C. pseudotuberculosis which enables easy entry into the bacterium. Furthermore, the lower MIC of OTC-CS-CaCO₃NP as against that of OTC may have been caused by a synergistic mechanism of action of both OTC and CS-CaCO₃NP in the OTC-CS-CaCO₃NP. Nanoparticles exert synergistic antibacterial effect by disruption of the cell wall integrity because of adsorption of the negatively charged CS-CaCO₃NP on the surface of C. pseudotuberculosis, combined with the regular mechanism of action of the antibiotic-loaded nanoparticle¹⁹ and in this case OTC which is via protein synthesis inhibition. MBEC values in the present study for both OTC-CS-CaCO₃NP and OTC suggest that the formation of biofilms by C. pseudotuberculosis is the major reason why CLA in animals is unresponsive to antibiotics because biofilms can withstand very high concentrations of antibiotics that would readily destroy the planktonic form of bacteria³².

The close association of the thick electron-dense layer and the plasma membrane seen in the C. pseudotuberculosis cells on HR-TEM is characteristic of Corynebacterium species³³. An earlier study by Burkovski,³³ revealed that the electro dense layer is made up of peptidoglycans while the electro light area is composed of mycolic acid is which plays a major role in the pathogenicity and permeability of Corynebacterium species. All the layers were also present in the CS-CaCO₃NP treated group implying that CS-CaCO₃NP had no antibacterial effect on C. pseudotuberculosis. Furthermore, in the OTC-CS-CaCO₃NP group, there was no clear demarcation of these layers as there is a spread out of the outer envelope of the bacteria. Therefore, we suggest that the antibacterial mechanism of action of OTC-CS-CaCO₃NP could be via 2 pathways: (i) Damage to the outer envelope of C. pseudotuberculosis disrupting the gradient of the electrochemical potential of ions across the membrane (Fig. 3b). (ii) OTC-CS-CaCO₃NP may have increased the rate of Reactive Oxygen Species (ROS) within the organism as evidenced by the high amounts of vacuoles seen in C. pseudotuberculosis. This agrees with Mohamed et al.¹⁴ who reported that the antibacterial mechanism of action of gold nanoparticles against C. pseudotuberculosis was due to induction of oxidative stress and cell membrane damage. Mukherjee et al.¹² reported that loading tetracyclines into calcium phosphate nanoparticles block the tetracycline efflux pump in Shigella flexneri 2a which resulted in high amounts of tetracycline in the cell and hence improved antibacterial activity. OTC-CS-CaCO₃NP may also block the tetracycline efflux pump in the bacteria cells which results in increased concentration of the OTC within the bacteria cell as explained by Mukherjee et al.¹². However, in the OTC treated C. pseudotuberculosis although some cells were damaged, the cell envelopes were intact with no obvious disorganization. This is similar to what was observed by Pan et al.¹⁹ on HR-TEM after treating Bacillus subtillis with free gentamicin and gentamicin-loaded CaCO₃ nanoparticles. The similarities may be because CS-CaCO₃NP enhanced the penetration of loaded into it, therefore, increasing damage to the bacteria envelope.

The increasing biofilm formation between 6-24 hrs is expected because C. pseudotuberculosis readily forms biofilms following culture in TSB with 2% FBS at 10% CO₂ as reported by Yaacob et al.⁵. High in vitro biofilm formation capacity is an indication that C. pseudotuberculosis readily forms biofilms following culture in TSB with 2% FBS at 10% CO₂ as reported by researchers⁵, Since TSB with 2% FBS at 10% CO₂ mimics the environment of the organism in host tissue⁵,then it can be said that similar conditions are necessary for the growth of C. pseudotuberculosis biofilms in vitro and in vivo. Moreover, the absorbance at 600 nm is proportional to the number of biofilms formed. The C. pseudotuberculosis biofilm inhibition assay revealed that greater than 50% of the C. pseudotuberculosis was viable after 24 hrs following OTC treatment. The results suggest that OTC-CS-CaCO₃NP maintained high biofilm inhibition up to 156.3 μg mL^–1 whereas biofilm inhibition was concentration-dependent for OTC.

The C. pseudotuberculosis in the control and CS-CaCO₃NP treated groups were predominantly green implying that the bacterial cells within the biofilms were viable as the stain (acridine orange) used enters the cells of intact viable cells and stain the nucleus green^34,35. For the OTC-CS-CaCO₃NP treated biofilms, more red colouration was present and this may have been due to destruction to the cytoplasmic membrane of C. pseudotuberculosis done by OTC-CS-CaCO₃NP which caused the influx of the ethidium bromide dye into the cytoplasm to stain the nucleus red. Furthermore, in the OTC-CS-CaCO₃NP treated group, the early and late apoptotic cells (greenish-yellow and orange-red, respectively) show that OTC-CS-CaCO₃NP exerted its effect on C. pseudotuberculosis biofilms since most of the cells were in the different stages of cell death. On the other hand, OTC treated biofilms exerted a mild antibiofilm effect as there were predominantly viable bacterial cells within the biofilm with few early apoptotic cells. Thus, from this study, it is clear that OTC was successfully released from OTC-CS-CaCO₃NP to the bacterial cells after penetrating the biofilm which prevented the proliferation of new biofilms as well as the destruction of existing biofilm. Similar reports were noted by Mu et al.³⁵ Consequently, the SEM and fluorescent microscopy results from this work results agree with the MIC, MBEC and TEM results.

CONCLUSION

This study reveals that OTC-CS-CaCO₃NP exerts a better antibacterial and antibiofilm effect against C. pseudotuberculosis compared to free OTC based on the MIC, MBEC and biofilm inhibition studies. The improved antibiofilm effect is attributed to the damage to the outer envelope of C. pseudotuberculosis caused by OTC-CS-CaCO₃NP as seen on HR-TEM. Thus, OTC-CS-CaCO₃NP may provide a promising new strategy for the treatment of C. pseudotuberculosis infection. Therefore, further studies investigating the potential of OTC-CS-CaCO₃NP for the effective treatment of C. pseudotuberculosis in vivo is necessary.

SIGNIFICANCE STATEMENT

This study discovered that the antibiofilm effect of OTC-CS-CaCO₃NP is more pronounced than that of free OTC which can be beneficial in C. pseudotuberculosis treatment. Our findings coupled with the increasing use of calcium-based nanoparticles opens uncover promising perspectives for OTC-CS-CaCO₃NP in the treatment of caseous lymphadenitis.

ACKNOWLEDGMENTS

This project was financed by Geran Putra, Universiti Putra Malaysia (Grant Code: GP/2018/9616700).