Special Issue:

Emerging and Re-emerging Animal Health Challenges in Low and Middle-Income Countries

Effect of Acidocin Purified from Lacotobacillus acidophilus and Biofilm Extract on the Cell Membrane of Staphylococcus aureus

H.A. Abdul-Ratha1*, Sahar Mahdi Hayyawi2, Essam F. AL-Jumaily3

1Al-Farahidi university, College of Science, Iraq; 2University of Baghdad, College of Veterinary Medicine, Iraq; 3University of Baghdad, Institute of Genetic Engineering and Biotechnology, Iraq.

Received | August 15, 2024; Accepted | November 24, 2024; Published | December 09, 2024

*Correspondence | Hassan A. Abdul-Ratha, Al-Farahidi university, College of Science, Iraq; Email: [email protected]

Citation | Abdul-Ratha HA, Hayyawi SM, AL-Jumaily EF (2024). Effect of acidocin purified from Lacotobacillus acidophilus and biofilm extract on the cell membrane of Staphylococcus aureus. J. Anim. Health Prod. 12(s1): 312-318.

DOI | https://dx.doi.org/10.17582/journal.jahp/2024/12.s1.312.318

ISSN (Online) | 2308-2801

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Staphylococcus aureus can readily form biofilm which enhances the drug- resistance, resultineg in life-threatening infections involving different organs (Yan et al., 2019; Xiying et al., 2024).

The biofilm development process can be didactically divided into the following four stages: (i) attachment and adhesion, (ii) aggregation with extracellular matrix synthesis and bacterial proliferation, (iii) biofilm structuring and maturation, and (iv) biofilm dispersion with cell detachment (Cheng et al., 2023).

Bacterial biofilm consists of organized slimy clusters of bacteria adhered to the inner surface area of the live organs like mucous membranes, blood vessel and lymphatic endothelium or the surface of medical and veterinary devices (Clutterbuck and Woods, 2007). Biofilm, also called a biological membrane, does not assume the form of amorphous sediment but resembles a highly structured complex, it contains slime and bacteria which surround themselves with the complex polymeric matrix they secrete, the slime helps the bacteria embedded in it to be protected from the attack of drugs as well as phagocytosis, it inhibits chemotaxis of granulocytes and opsonization process, thus affecting an inflammatory restrain (Bradley et al., 2007).

Lactobacilli play an important role in suppressing undesirable intestinal microflora, organic acids and hydrogen peroxide produced by Lactobacillus acidophilus has demonstrated broad spectrum inhibition, previous studies suggested that bacteriocin either mediate or facilitate inhibitory activity ,the crude lactocidin produced by Lactobacillus acidophilus demonstrated antimicrobial effect against Staphylococcus aureus criteria for bacteriocin identification include a little potential for broad spectrum inhibition restricted to closely related species, a bactericidal mode of action and proteinaceous nature (Abo-Amer, 2006).

Lactobacillus acidophilus has been demonstrated to prevent and alleviate disturbances and to normalize the cytokine profile which might be of an advantage for patients suffering from infection (Acido et al., 2015).

Staphylococcus aurueus is one of the most important causes of multiple diseases and is difficult to treat with antibiotics because it forms a biofilm. Therefore the study aim to determine the effect of acidocin (alone) or with biofilm extract on cell permeability and the mode of action on cell structure under scanning electron microscope (SEM).

MATERIALS AND METHODS

Staphylococcus aureus and Lactobacillus acidophilus isolates

Staphylococcus aureus which previously isolated and diagnosed from milk samples and produced higher thickness (3mm) of biofilm was used to extract biofilm and to study the effect of acidocin on its cell wall.

Lactobacillus acidophilus was isolated from healthy cow’s milk samples and from chicken crops.

Biofilm production by Staphylococcus aureus

A qualitative assessment of biofilm production by Staphylococcus aureus was determined as previously described by Christensen et al. (1982). A Biuret method was used to determine the concentration of protein in biofilm according to (Edward et al., 2013).

Extraction and purification of Acidocin

Basal growth media (MRS broth+ Tween 80), was incubated with initial number of bacterial isolate and incubated at optimal conditions for production of acidocin. Cells were harvested by centrifugation at 6000 rpm for 15 minutes, the cell-free supernatant was referred as crude acidocin extract then was heated at 80°C for 10 minutes, then cooled and centrifuged at 6000 rpm for 15 minutes (Powell et al., 2007).

The supernatant was mixed thoroughly with n-butanol at a ratio 1:1. The mixture was centrifuged at 4000 rpm for 10 minutes to achieve phase separation. The organic phase was evaporated at 80°C by rotary evaporator, then the sediment was re-suspended in 20 mM sodium citrate buffer (pH5) and referred to as partial purified acidocin (Abo-Amer, 2007a). The antimicrobial activity of acidocin and protein concentration were determined.

Gel filtration chromatography

Gel filtration chromatography by sepharose 6B was used as a second step for acidocin purification.

Effect of acidocin on cell lysis and viability of Staphylococcus aureus

The effectiveness of pure acidocin was tested against culture of S. aureus (5 × 105 CFU ml-1 ), which produce 3 mm thickness of biofilm, 0.1 ml of different concentration of pure acidocin (25, 12.5, 6.25, 3.125) mg ml-1 was added to the 10 ml of S. aureus broth culture.

The effect of pure acidocin on cell permeability

Ten ml of 18-hour old broth culture of Staphylococcus aureus was harvested by centrifugation at 3000 rpm for 15 min, the cells washed twice with sterile buffer (pH 6.5), then re-suspended in 10 ml of the same buffer. Pure Acidocin was added (3.125 mg ml-1) to the washed cells at a ratio of 0.1:1.0, after 1 hour of incubation at 37°C, the cells were harvested by centrifugation at 3000 rpm for 15 minutes. DNA concentration was determined by optical density reading at 260 nm. Cells suspended in buffer (pH 6.5), without Acidocin and in the same buffer containing acidocin, but without cells, served as controls (Todorov et al., 2006).

Preparation of bacteria for scanning electron microscope

This methods was preapared according to Aebi and Pollard (1987). The first sample was taken from growth culture 5×105 CFU ml-1 of Staphylococcus aureus not treated with acidocin.

The second sample was taken from Staphylococcus aureus isolates 5×105 CFU ml-1 treated with 3.125 mg ml-1 acidocin.

Third sample 5×105 CFU ml-1 of Staphylococcus aureus was treated with combination of acidocin 3.125 mg ml-1 with biofilm 4.5 mg ml-1 protein concentration.

The different treatment of bacterial suspension was centrifugated at 3000 rpm for 15 min. The pellets were washed with phosphate buffer saline for 3 times. The pellet treated with chemical fixation, mixing of 20% glutaraldehyde and 4% formaldehyde in PBS for 30 min Dehydration with Isopropanol at 10, 30, 50, 70, 80, 90, 100 % for 20 sec critical point drying were chemically fixed in mixing of formaldehyde and glutaraldehyde at concentrations between 1% (vol/vol) and 10% (vol/vol) in phosphate buffered saline, PBS (Dulbecco’s PBS, H31-002 from PAA, Pasching, Austria), by adding fixative solution to 1 mL aliquots. Samples were then centrifuged (5,000 g), Supernatant removed, and pellet resuspended in 200 µl of PBS and pipetted on SEM grids (holey carbon coated, 200 meshes). After transfer to the grids, they were washed twice with PBS and twice with bi- distilled water, dehydrated either in microtiter plate wells or directly in droplets of dehydrating solvent on an inert parafilm surface in subsequent steps of increasing organic solvent (ethanol, acetone or isopropanol) concentrations (0, 15, 30, 50, 70, 80, 90, 96, 100, 100, 100% vol/vol) and transferred to the critical point dryer Bal-Tec CPD 030. The solvent was replaced by liquid CO2 at a pressure of 50 bars and a temperature of 8 oC. After complete replacement of the organic solvent by CO2, the chamber was heated resulting in an increased pressure due to evaporating CO2. Two minutes after reaching the critical point, the pressure was released very slowly, resulting in a dry sample surrounded by CO2 at ambient pressure and temperature. Dry samples were mounted on aluminum stubs using double-sided carbon tape. For enhanced electrical conductivity, the edges of the SEM grids were painted with conductive silver paste coated in a Balzers sputter coater SCD 40 (Bal-Tec, Balzers, Liechtenstein) with Au/Pd (90%/10% w/w) under an angle of 45 oC. The coating thickness was approximately 20 nm (determined in focused Ion Beam cross-sections and by a surface texture analyzer, results not shown). Cryo-immobilized samples were Tungsten coated unidirectionally in the Bal-Tec cryo-sputter coater SCD 500 with a layer thickness of 5nm.

RESULTS AND DISCUSSION

The result showed antibacterial effect with a reduction of cells counts of the treated sample after a ½ hour, 1 hour, 1 ½ hour, 2 hours, 2½ hours, 3 hours,4 hour, and 18 hours of incubation Table 1.

 

Table 1: The effectiveness of different concentrations of pure acidocin at different incubation times against log viable counts cfu ml-1 of Staphylococcus aureus isolates.

Pure acidocin conc.mg/ml/ Time of incubation (hour)	25	12.5	6.25	3.125
	Mean of bacterial count Log cfu/ml	Mean of bacterial count Log cfu/ml	Mean of bacterial count Log cfu/ml	Mean of bacterial count Log cfu/ml
½	4.5300±0.0481	4.5580±0.0335	4.7297±0.0443	4.6807±0.0557
	3	5	7	1
	Ac	Ac	Aa	Ab
1	4.2540±0.0693	4.4543±0.0110	4.4417±0.1987	4.5983±0.0690
	5	5	7	2
	Bc	Bb	Bb	Ba
1½	3.5870±0.0529	3.6617±0.1079	3.8183±0.0292	3.9233±0.0311
	2	0	4	8
	Cc	Cc	Cb	Ca
2	3.0637±0.0636	3.4233±0.1089	3.5650±0.0807	3.7840±0.0769
	3	8	3	7
	Dd	Dc	Dd	Da
2½	2.6947±0.0129	2.7277±0.0912	2.7557±0.0716	2.9123±0.0213
	1	2	3	6
	Ec	Eb	Eb	Ea
3	1.9910±0.0052	2.2523±0.0271	2.4013±0.0095	2.4580±0.0060
	0	7	3	8
	Fc	Fb	Fa	Fa
4	0.5923±0.0639	0.8937±0.0642	1.0777±0.0208	1.1700±0.0509
	8	6	0	0
	Gd	Gc	Gb	Ga
18	-	-	-	-

 

Acidocin demonstrated a bacteriolytic mode of action as immediate decrease of b statistical analysis

The results data were analyzed statistically by using the Microsoft Program (SPSS). Statistical analysis of data was performed on the basis of Analysis of Variance (ANOVA) and specific group differences were determined using least significant differences (LSD), as described by Snedecor and Cochran (1980).

Bacterial counts measured by Mils and Misra technique from cultured of S. aureus that treated with 0.1 ml of different concentration of pure acidocin that indicate cell lyses, these concentrations of pure acidocin gave inhibition zone 34 mm, against S. aureus isolates that produced 3 mm biofilm thickness as measured by well diffusion methods after incubated 18 hours at 37°C., so these result depended on these concentrations of acidocin to determines the dose of injection for the nursing mice as a laboratory animal design.

Data shown in Table 1 indicate that there were significant differences between pure acidocin concentration 25 mg/ml during the incubation time in which the count in a half an hour has the superior value as compared with other times, while the lowest value of viable count was recorded after 4 hours incubation (0.592) log cfu/ml. Table 1 shows high significant differences between pure acidocin concentration (12.5, 6.25, 3.125 mg) ml-1 in a half hour, While lower significant differences in four hour. The best action of pure acidocin concentration was 6.25mg ml-1, in half hour and in three hours, while the acidocin concentration 3.125 mg ml-1 was the best at the time of one hour , one and a half, two, two and a half, three, and four hours. The best action of acidocin was in concentration 6.25mg ml-1, in a half hour and in three hours while the concentration 3.125 mg ml-1, was superior action in one ,one and a half, two, two and a half ,three, and four hours. The result showed the significant activities of pure acidocin concentrations against S. aureus produced thick biofilm resulted a strong decrease in rate of bacterial count, the most effective in low concentration was 3.125 mg ml-1 after 4 hours that complete killing of S. aureus cells.

The effect of pure Acidocin on cell permeability

The results showed the lyses due to acidocin activity 25 mg ml-1, also showed that the treated S.aureus isolates with acidocin gave reading 2.640, while untreated culture was 0.461, and acidocin only gave 0.492 (Table 2).

The results showed that the treated S. aureus isolates with acidocin gave reading in spectrophotometer as 2.640, while untreated culture was 0.461, and acidocin only gave 0.492. These results indicated the effect of treatment with acidocin that may rupture the cell membrane and made DNA leaked from the cell of S. aureus bacteria due to the cell lysis.

 

Table 2: The level of DNA recoded at 260 nm after treatment of S. aureus isolates treatment with pure acidocin.

Treatment	Absorbance at 260 nm
	S. aureus
Treated cells	2.640
Untreated cells	0.461
Acidocin (no cells)	0.492

 

These results gave an idea or conclusions that cationic antimicrobial peptides disrupt bacterial membranes, thus allowing free exchange of intracellular and extracellular contents of the target cells (Zhao and Lacasse, 2008).

Many peptides and proteins interact strongly with amphiphilic molecules and these interactions are of vast importance, not only in vivo but also in technical applications, however there are exceptions, pore formation leads to the disruption of the proton motive force (PMF) and the efflux of intracellular material, bacteriocin can have a narrow or abroad spectrum of activity, Class Ia bacteriocins, or lantibiotics, use lipid II as target for their mode of action and kill sensitive bacteria by pore formation or by hampering cell wall formation, lantibiotic can also inhibit the germination of spores, class IIA, or pediocin- like bacteriocins, mainly form pores in the membranes of sensitive cells by disruption the PMF, cells cannot secrete antibiotics via transport systems located in their cell membranes (Heunis et al., 2011).

Scanning electron microscopy (SEM) results

The results of Scanning electron microscopy showed S. aureus cells without any treatment as smooth and undamaged cell in the control group Figure 1.

 

After treating the bacteria with pure acidocin, the results showed the alteration in the S. aureus cells morphology, Figure 2.

 

The results of the group which treated with pure acidocin showed leakage of cell membrane compound as pure formation and complete destruction of cell membrane (Figure 3).

 

The results of the third group that treated with combination effect of pure acidocin and biofilm showed the existence of gaps with vacuole within the cell and crash inner cell cytoplasmic components and destruction of cell, (Figure 4).

 

Effect of acidocin on cell lysis and viability of Staphylococcus aureus

The antibacterial effect with a reduction of cells count of the treated sample after different times of incubation, showed that the most important finding on these results were the bacteriolytic mode of action of acidocin due to the immediate decrease of bacterial count measured from cultured of S. aureus that treated with different concentration of pure acidocin that indicate cell lyses, this result was came in agreement with Al-Gharbawee (2012) who showed that the acidocin produced from L. acidophilus was exhibited bactericidal mode of action with cell lysis. Acidocin effectiveness as a bactericidal on sensitive microorganisms by inhibiting the transport of amino acid and causing the leakage of essential compounds by the formation of pores in the cytoplasmic membrane (Ahmed et al., 2010), and alternative of the enzymatic activity with inactivation of anionic carriers through the formation of selective and non-selective pores (Van Belkum and Stiles, 2000), then lysis the bacterial cell. This result agrees with Abdul-Ratha et al. (2012) who noted the acidocin effect against enteropathogenic E. coli, in addition the results are in agreement with Ali (2010) who noted that the bacteriocin (plantaracin) exhibited bactericidal mode of action with cell lysis. This result agrees with Abdul-Ratha et al. (2012) who noted the acidocin effect against enteropathogenic E. coli, in addition the results are in agreement with Ali (2010) who noted that the bacteriocin (plantaracin) exhibited bactericidal mode of action with cell lysis.

Scanning electron microscopy (SEM) results

The results showed the antibacterial effect of acidocin was damaging the S.aureus cell with separation of cell wall that agrees with Al-Garbawee (2012) who treated E. coli cell with acidocin, in addition it agrees with Khalil et al. (2009) who showed that the mode of bactericidal action may probably been due to pore formation after damaging cell surfaces, in addition to an alternated in the cell morphology. The cell membrane with certain morphology exhibits degradation of membrane structure of S. aureus during treatment and a significant increase in permeability, leaving the bacterial cells incapable of properly regulating transport through the plasma membrane and finally causing cell death (Sondi and Sondi, 2004). The main mechanism of antibacterial agents was as the destruction of bacterial cell wall, interference on bacterial cell membrane, inhibition of protein synthesis, In this study, the cell surface morphology and internal structure of S.aureus changed after acidocin treatment, It could be speculated that the cell membrane permeability of S. aureus was changed by the effect of acidocin in many of them were accumulated and combined specifically to molecules with cell wall synthesis function, so the cell wall synthesis of S. aureus was affected and resulted in deformation and collapse of cell wall, with various depressions appearing (Al-Jumaily et al., 2015).

There were Lysis of cytoplasm, nucleic acid destruction, disappearance, and vacuolization in S. aureus cells after treatment with acidocin. It could be speculated that acidocin could cause the organelle damage of S. aureus ribosoms and then affect to the synthesis and expression of protein and DNA, finally resulting in transferring interruption of material, energy, and information, and the cell turned to death, besides the bacteriocin could attach to the surface of S. aureus cell wall by electrostatic attraction, the enzyme synthesis of cell wall was inhibited and the cell wall structure was destroyed, so depression, defect, and fracture could be found in the surface of cell wall, also bacteriocin could result in the decrease of hydrophobicity in the cell surface, the cell membrane permeability, and ultrastructure changes (Lee et al., 2013).

Mode of action studies indicated that pore formation leading to ATP efflux is important for the bactericidal activity against biofilm cells, the results suggested that bacteriocins that form stable pores on biofilm cells are highly potent for the treatment of S. aureus biofilm infections (Okuda et al., 2013).

ACKNOWLEDGEMENTS

We want to dedicate thanks and appreciations to Microbiology Department, College of Veterinary Medicine, Baghdad University, Iraq and to the colleagues in Biotechnology Research Center at Al-Nahrain University, Iraq for their support during the course of this work.

Novelty Statement

The current manuscript work was accomplished in cooperation between authors and through their own efforts with the assistance of specialized laboratories at the university of Baghdad and the university of AL- Nahrain.

Author’s Contribution

H.A.Abdul-Ratha: conceived of the presented idea ,investigate and supervised the findings of this work and supervised the project.

Sahar Mahdi Hayyawi: conceived of the presented idea,verified the analytical methods ,carried out the experiment .

Essam F.AL-Jumaily: conceived of the presented idea,assistant supervisor for this project.

Conflict of interest

The authors have declared no conflict of interest.

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