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Anti-biofilm potential of Lactobacillus casei and Lactobacillus rhamnosus cell-free supernatant extracts against Staphylococcus aureus
Navid Saidi1, Horieh Saderi2, Parviz Owlia1, Mohammad Soleimani3
1 Department of Microbiology, Faculty of Medicine, AJA University of Medical Sciences, Tehran, Iran
2 Molecular Microbiology Research Center, Faculty of Medicine, Shahed University, Tehran, Iran
3 Department of Microbiology, Faculty of Medicine; Infectious Diseases Research Center, AJA University of Medical Sciences, Tehran, Iran
| Date of Submission | 08-Jun-2021 |
| Date of Acceptance | 31-Jan-2022 |
| Date of Web Publication | 25-Feb-2023 |
Correspondence Address:
Dr. Mohammad Soleimani
Department of Microbiology, Faculty of Medicine, AJA University of Medical Sciences, Shahid Etemadzadeh Street, Tehran
Iran
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/abr.abr_156_21
Background: Biofilm production is an important virulence factor in Staphylococcus aureus. Most of the infections associated with biofilms of this bacterium are very difficult to treat using antibiotics. The present research studied the effects of the two probiotic Lactobacillus species L. casei and L. rhamnosus on S. aureus biofilm.
Materials and Methods: Cell-free supernatant (CFS) extracts of L. casei ATCC 39392 and L. rhamnosus ATCC 7469 culture were prepared. The effects of sub-minimum inhibitory concentrations of the CFS extracts on cell surface hydrophobicity (CSH), initial attachment, biofilm formation, and their ability in eradicating S. aureus ATCC 33591 biofilms were assessed. In addition, the effects of CFS extracts on expression of the genes involved in formation of S. aureus biofilms (cidA, hld, sarA, icaA, and icaR) were also evaluated through real-time polymerase chain reaction.
Results: CFSs of both Lactobacillus spp. significantly reduced CSH, initial attachment, and biofilm formation and eradicated the biofilms. The above findings were supported by scanning electron microscopy results. These two Lactobacillus CFSs significantly changed the expression of all studied biofilm-related genes. Expression levels of cidA, hld, and icaR genes significantly increased by 4.4, 2.3, and 4.76 fold, respectively, but sarA and icaA genes were significantly downregulated by 3.12 and 2.3 fold.
Conclusion: The results indicated that CFS extracts of L. casei and L. rhamnosus had desirable antagonistic and anti-biofilm effects against S. aureus. Consequently, carrying out further research enables us to prepare pharmaceuticals from these CFSs in order to prevent and treat infections caused by S. aureus biofilms.
Keywords: Biofilm, Lactobacillus casei, Lactobacillus rhamnosus, probiotic, Staphylococcus aureus
How to cite this article:
Saidi N, Saderi H, Owlia P, Soleimani M. Anti-biofilm potential of Lactobacillus casei and Lactobacillus rhamnosus cell-free supernatant extracts against Staphylococcus aureus. Adv Biomed Res 2023;12:50 |
How to cite this URL:
Saidi N, Saderi H, Owlia P, Soleimani M. Anti-biofilm potential of Lactobacillus casei and Lactobacillus rhamnosus cell-free supernatant extracts against Staphylococcus aureus. Adv Biomed Res [serial online] 2023 [cited 2023 Mar 31];12:50. Available from: https://www.advbiores.net/text.asp?2023/12/1/50/370360 |
| Introduction | |  |
Staphylococcus aureus is a Gram-positive bacterium and the causal agent of a broad spectrum of infections, including acute infections mostly caused by secretion of exoenzymes and chronic infections such as osteomyelitis, chronic wound infections, eye infections, chronic rhinosinusitis, and endocarditis that develop due to biofilms formed by this bacterium.[1] Biofilm is a collection of adhered microorganisms that is attached to a surface and covered by a matrix of polymeric extracellular matrix.[2] In S. aureus, two types of biofilms are produced: polysaccharide intercellular adhesin (PIA)-dependent biofilms and PIA-independent biofilms. In PIA-dependent biofilms, products of the ica gene locus that include an N-acetylglucosamine transferase gene (icaA and icaD), a PIA deacetylase gene (icaB), a PIA exporter gene (icaC), and a regulatory gene (icaR) are involved in biofilm biosynthesis. Expression of the ica locus can be suppressed by production of tcar and icaR, which leads to PIA downregulation and hence prevents biofilm formation. In PIA-independent biofilm formation, adhesive proteins such as surface-associated proteins including protein A, fibronectin-binding proteins (FnBPB and FnBPA), S. aureus surface proteins (SasG), biofilm-associated protein, and clumping factor B play parts as important components in attachment and development of biofilm matrix.[3],[4] These adhesive proteins are controlled by several regulatory systems. Activation of the accessory gene regulator (agr) system reduces expression of adhesive proteins and induces biofilm dispersal by increasing expression of detergent-like peptides and nucleases. On the other hand, the staphylococcal accessory regulator (sarA) system induces attachment and allows initial biofilm formation by suppressing extracellular proteolytic and nucleolytic enzymes.[5]
S. aureus can form biofilms on host tissues and medical implants. Biofilms increase bacterial resistance to host defense mechanisms, and treatment of their resulting infections with antibiotics is difficult. Moreover, S. aureus biofilms are considered a source for the spread of infections to the other parts of the body. This increased resistance plays a special role in the development of nosocomial infections.[6],[7]
Biofilm formation together with high prevalence of S. aureus-induced infections and emergence of antibiotic-resistant strains make necessary to conduct research for finding of new medications for eradicating strains that are resistant to common treatments and/or for eradicating biofilms or preventing their formation. In general, anti-biofilm drugs are based on three main strategies: preventing bacterial adhesion to biotic or abiotic surfaces to reduce the probability of their establishment and biofilm formation, disrupting biofilm structure during its maturation, and disturbing signaling pathways.[8]
One of the treatment methods proposed for drug-resistant or biofilm-forming S. aureus infections is to use probiotics such as probiotic Lactobacillus strains. Several studies have investigated the effects of some Lactobacillus strains in preventing S. aureus growth and eradicating its biofilms. Probiotics can exhibit their therapeutic effects in different ways. For example, suppression of S. aureus growth by lactobacilli has been attributed to acidification of the culture media by fermentation of lactic acid and production of H2O2 and bacteriocins.[9] The present study was carried out to investigate the effects of two probiotic Lactobacillus spp. (L. casei and L. rhamnosus) on S. aureus biofilms.
| Materials and Methods | | |
The bacterial strains
The standard S. aureus strain ATCC 33591 which is a strong biofilm former was used in the present research. This bacterium was kept in Tryptic soy broth (TSB) with 20% glycerol at −70°C. The probiotic lactobacillus strains L. casei ATCC 39392 and L. rhamnosus ATCC 7469 were kept in Man, Rogosa, and Sharpe (MRS) broth with 30% glycerol at −70°C. MRS agar was used to culture these strains. The colonies were regularly evaluated to make sure they were pure in order to reduce the probability of contamination and error in the experiments.
Preparation of cell-free supernatant extracts
Several colonies of the freshly cultured Lactobacillus strains were transferred to 10-mL MRS broth and incubated under microaerophilic conditions for 24 h at 37°C. This culture medium was used to inoculate 1000-mL MRS broth. The inoculated culture was incubated for 48 h at 37°C and then centrifuged at 12,000 rpm for 20 min to separate the supernatant. The supernatant was passed through a 0.45-μm filter to completely remove the cells and obtain cell-free supernatant (CFS). Some of the CFSs were used for studying its antimicrobial activity and the rest to prepare CFS extract. The method previously explained by Brosnan et al. was employed with some modifications to prepare the extract. The extraction process, in which ethyl acetate was used, lasted for 6 h. The CFS was mixed with ethyl acetate at a ratio of 5:1, and the ethyl acetate was replaced every hour. Finally, the ethyl acetate was removed using a rotary evaporator to obtain the dry matter.[10]
Antimicrobial activity
The agar well diffusion method was used to assess CFS antimicrobial activity of the Lactobacillus strains against S. aureus and to see whether this antimicrobial effect was dependent on organic acids or on bacteriocin production. This experiment was performed in two ways: once after neutralizing the pH of the CFS with NaOH and once after boiling the CFS for 5 min at 100°C. Briefly, the overnight culture of the S. aureus standard strain ATCC 33591 was used to prepare a half McFarland suspension that was cultured on Mueller–Hinton agar plates. A well of 3-mm diameter was punched in the center of each culture plate. Fifty microliters of the prepared CFSs was poured into the wells, and the plates were incubated at 37°C for 24 h. Finally, the diameters of the clearing zones in the plates in the two experiments were compared.[11]
Minimum inhibitory concentration and minimum bactericidal concentration
Broth microdilution was used to determine minimum inhibitory concentration (MIC).[12] Serial two-fold dilutions (0.25–16 mg/mL) of the CFS extracts were made and poured into the wells of the microplate. The wells were inoculated with a suspension prepared from a 24-h culture of S. aureus ATCC 33591. The final bacterial concentration of each well was 105 cfu/mL. The wells without the extract and without the bacteria were the positive and negative controls, respectively. After 24 h, the wells were examined with respect to growth and the MIC was determined. The number of colonies in the wells that showed no visible signs of turbidity was counted to determine the minimum bactericidal concentration (MBC).
Cell surface hydrophobicity
Microbial adhesion to solvent (MATS) assay was employed to study the effect of sub-MICs of the CFS extracts of the Lactobacillus strains on cell surface hydrophobicity (CSH) of S. aureus.[13] The bacteria were cultured on TSB at ½, ¼, and ⅛ MICs of CFS extracts for 24 h. The bacterial cells were then centrifuged at 7000 rpm for 5 min. The bacterial culture without the CFS extract was used as the control. The cells were washed once to completely remove the culture medium, and a suspension with OD600 = 0.3 was prepared (OD1) by adding the necessary volume of PBS. Hexadecane (0.8 mL) was then added to 5.2 mL of the microbial suspension and vortexed to form an emulsion. This mixture was let stand for 20 min, and two phases were separated; then, the OD of the aqueous phase was measured at 600 nm (OD2). CSH was calculated using the following equation:
CSH% =1 – (OD2/OD1) ×100
Anti-adhesion assay
In each well of a 96-well microplate, 200 μL of each CFS extract at ½, ¼, and ⅛ MICs was poured. The well containing PBS was the control. The microplate was incubated at 4°C for 18 h, and the wells were then emptied. A suspension with OD600 = 0.3 was prepared from a 24-h culture of S. aureus, 200 μL of which was poured into each inoculated well, and the plate was incubated at 37°C for 4 h. The wells were then emptied, washed with PBS, and stained with crystal violet. The crystal violet was dissolved by adding 33% acetic acid. Finally, the OD was measured at 570 nm.[14]
Anti-biofilm formation assay
The microtiter plate assay was used for this purpose. The bacteria were first cultured on TSB for 24 h. Suspensions (108 cfu/mL) from this culture were inoculated in TSBGlc (TSB + 1% glucose) containing ½, ¼, and ⅛ MICs of the CFS extracts. This suspension (200 μL) was transferred to the wells of a 96-well microplate. The well without the extract was the positive control, and the well without the bacteria was the negative control. The microplate was incubated at 37°C for 24 h. The wells were then emptied, washed with PBS, and stained with crystal violet. The crystal violet was dissolved by adding 33% acetic acid, and the OD of the solution was measured at 570 nm. The OD of negative control well was recorded as ODc, and the OD of tested wells as ODt. [Table 1] is used to determine the degree of biofilm formation.[15]
Biofilm dispersal assay
The microtiter plate assay was also used in this experiment with the difference that the biofilms were formed first and then the effect of CFS extracts on them was studied. The 24-h culture of S. aureus was diluted with TSBGlc medium at a ratio of 1:100, and 200 μL of this suspension was poured in the wells of the microplate. The microplate was incubated at 37°C for 24 h for biofilm formation. The wells were then emptied and washed with PBS, and each one was filled with 200 μL of TSBG1c medium containing ½, ¼, and ⅛ MICs of the Lactobacillus strains' CFS extracts, and the microplate was incubated at 37°C for 24 h. The well containing only TSBGlc was the control. As in the previous experiment, staining was carried out with crystal violet and OD was measured. The same method was employed to determine minimum biofilm eradication concentration (MBEC) with the difference that the extracts were assessed at concentrations of 1–4 times higher than the MIC.[16]
Scanning electron microscopy of biofilm formation
Overnight culture of S. aureus was inoculated in TSBGlc containing ½ MIC of CFS extracts so that the final bacterial concentration was 108 cfu/mL. This suspension was transferred to a 6-well microplate. A glass coverslip was placed in each well. The plate was incubated at 37°C for 24 h for biofilm formation. The glass coverslips were then removed and washed three times with PBS and placed in glutaraldehyde for 2 h for the samples to be fixed. In the next stage, serial dilutions of ethanol (50%–100%) were used to dehydrate the samples. The surface of each plate was coated with a thin layer of gold to impart electrical conductivity. Finally, the samples were studied using a scanning electron microscope (MIRA3, TESCAN, Czech Republic).[17]
Effects of cell-free supernatants on expression of genes involved in biofilm formation
Overnight culture of S. aureus was inoculated in TSBGlc medium containing ½ MIC of the Lactobacillus strains' CFSs so that the final concentration of the bacteria was 108 cfu/mL. This suspension was transferred to a 6-well microplate that was incubated at 37°C for 24 h. The cells were then collected and their total RNA was extracted. To this end, TE buffer containing 40 mg/mL lysozyme and 100 μg/mL lysostaphin was added to the pellet to lyse the cells. Total RNA was extracted using RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's instructions. RNA concentration and purity were assessed with NanoDrop (NanoDrop One, Thermo Fisher, USA), and the QuantiTect Reverse Transcription Kit (Qiagen, Germany) was employed to remove genomic DNA contamination and synthesize cDNA. Real-time polymerase chain reaction (PCR) was used to compare the expression levels of the genes involved in biofilm formation in the samples treated with CFSs and the control sample. [Table 2] lists the primers that were used (the sequences of the genes of interest were extracted from the GenBank sequence database and the primers were designed using AlleleID 6). Real-time PCR was run in triplicates using QuantiTect SYBR Green PCR Kits. Expression of the genes was normalized using the 16S rRNA gene. The relative expression levels of the genes were determined using the ΔΔCT method.[18],[19] | Table 2: Sequences and characteristics of the primers used in the experiments
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Statistical analysis
All experiments were performed in triplicate. The results of the treatments were compared with those of the control using one-way ANOVA and the Tukey's multiple comparison test. Significant differences were evaluated at P < 0.05. GraphPad Prism 8 (GraphPad Software, San Diego, California, United States) was used for these analyses.
| Results | | |
Antimicrobial activity
To study the antimicrobial activity of the Lactobacillus strains' CFSs using the agar well diffusion method, their pH was neutralized and they were heated. The results showed that the CFS of neither of the Lactobacillus strains was able to form clearing zones when its pH was neutralized, whereas the CFSs of both strains formed clearing zones when their pH was not neutralized. The clearing zone diameter for L. casei ATCC 39392 was 9 mm and that of L. rhamnosus ATCC 7469 was 9.4 mm. Moreover, heating the CFSs to 100°C did not change the clearing zone diameters. Consequently, none of the Lactobacillus species produced active bacteriocin against S. aureus, and their antimicrobial activity was probably due to production of organic acids.
Minimum inhibitory concentrations and minimum bactericidal concentrations of cell-free supernatants
Using broth microdilution method, it was shown that the MICs and MBCs of CFS extracts for both Lactobacillus strains against S. aureus ATCC 33591 were 4 mg/mL and 8 mg/mL, respectively. Therefore, concentrations 0.5, 1, and 2 mg/mL of CFSs were used as sub-MICs in the rest of the experiments.
Results of the cell surface hydrophobicity assay
The results of the MATS assay revealed that CFS extracts of both Lactobacillus strains at ½ and ¼ MICs significantly reduced surface hydrophobicity of S. aureus cells (P < 0.0001). At ⅛ MIC, the CFS of L. rhamnosus significantly decreased surface hydrophobicity (P = 0.003), whereas that of L. casei did not cause any significant differences (P = 0.7). This reducing effect was dose dependent; i.e., increases in CFS concentrations further reduced surface hydrophobicity of the cells. [Figure 1] demonstrates CSH changes in S. aureus ATCC 33591 under the influence of various concentrations of the L. casei and L. rhamnosus CFS extracts. | Figure 1: Changes in the cell surface hydrophobicity of Staphylococcus aureus ACTT 33591 in ½, ¼, and ⅛ minimum inhibitory concentrations of the Lactobacillus casei and Lactobacillus rhamnosus cell-free supernatants. Significant differences with the control (**P < 0.01, ****P < 0.0001)
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Effects of CSFs on initial attachment
The results of the experiment on the effects of L. casei ATCC 39392 and L. rhamnosus ATCC 7469 CFSs on the initial attachment ability of S. aureus ATCC 33591 to surfaces indicated that ½, ¼, and ⅛ MICs of CFS extracts significantly reduced attachment of S. aureus cells (P < 0.001). This attachment inhibitory effect was considerable, so that the L. rhamnosus and L. casei CFSs at ½ MIC (2 mg/mL) reduced the initial attachment of S. aureus by 100% and 98.9%, respectively. [Figure 2] presents these results. | Figure 2: Initial attachment of Staphylococcus aureus ATCC 33591 cells in ½, ¼, and ⅛ minimum inhibitory concentrations of the Lactobacillus casei and Lactobacillus rhamnosus cell-free supernatants. Significant differences with the control (****P < 0.0001)
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Effects of the cell-free supernatants on biofilm formation
Microtiter plate assay was employed to evaluate the effects of Lactobacillus strains' CFSs on S. aureus biofilm formation. The ODs of the control and the samples treated with ½, ¼, and ⅛ MICs of CFS extracts showed that all concentrations of both extracts significantly reduced biofilm formation (P < 0.0001). S. aureus ATCC 33591 produced strong biofilms. These biofilms changed to weak ones under the influence of ½ MIC of the CFS extract of L. casei. The other concentrations of this CFS extract and all concentrations of the L. rhamnosus CFS extract led to the formation of moderate type biofilms. The complete results are listed in [Figure 3]. | Figure 3: Formation of Staphylococcus aureus ATCC 33591 biofilm in ½, ¼, and ⅛ MICs of the Lactobacillus casei and Lactobacillus rhamnosus CFSs. Significant differences with the control (****P < 0.0001)
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Ability of cell-free supernatants to eradicate biofilms
The ability of L. casei ATCC 39392 and L. rhamnosus ATCC 7469 CFS extracts in eradicating S. aureus ATCC 33591 biofilms was investigated. The results revealed that the CFSs of both Lactobacillus strains facilitated eradication of S. aureus biofilms. All concentrations of these CFSs significantly decreased the strength of the biofilms (P < 0.0001). At ½ MIC (2 mg/mL), both CFSs were able to turn strong biofilms into weak ones. Moreover, the strong biofilms were changed into moderate ones at the other MICs. [Figure 4] presents these results. At the 4 MIC (16 mg/mL), CFSs of both Lactobacillus species were able to completely eradicate S. aureus biofilms. Therefore, this concentration was determined as the MBEC. | Figure 4: Eradication of Staphylococcus aureus ATCC 33591 biofilm in ½, ¼, and ⅛ MICs of the Lactobacillus casei and Lactobacillus rhamnosus CFSs. Significant differences with the control (****P < 0.0001)
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Scanning electron microscopy of the biofilms
The effects of the L. casei ATCC 39392 and L. rhamnosus ATCC 7469 CFS extracts on S. aureus biofilm formation were studied using scanning electron microscopy (SEM). As shown in [Figure 5], thick biofilms at high cell density were formed in the control sample, whereas the number of biofilm cells considerably decreased in the samples treated with the Lactobacillus strains' CFSs. | Figure 5: Scanning electron microscopy images of Staphylococcus aureus biofilms: (a) The control sample, (b) The sample treated with cell-free supernatant (CFSs) from the cultures of Lactobacillus casei, (c) The sample treated with CFSs from the cultures of Lactobacillus rhamnosus. Left: ×5000; Right: ×10,000
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Results of real-time polymerase chain reaction
Using real-time PCR, the effects of L. casei and L. rhamnosus CFS extracts at 2 mg/mL on expression of genes involved in S. aureus biofilm formation were studied. The studied genes were cidA, hld, sarA, icaA, and icaR. Some of them are necessary for biofilm formation; i.e., increases in their expression enhance biofilm formation. However, the other genes are expressed under planktonic conditions and their expression decreases formation of biofilms or eradicates them. The CFSs of both Lactobacillus species changed the expression levels of all five studied genes: they increased expression of the cidA, hld, and icaR genes but decreased the expression of the sarA and icaA genes. [Figure 6] shows variations in the expression of the studied genes. | Figure 6: Changes in the expression of genes involved in the formation of Staphylococcus aureus ATCC 33591 biofilms in ½ MIC of Lactobacillus casei and Lactobacillus rhamnosus cell-free supernatant (CFSs). Significant differences with the control (*P < 0.05, ***P < 0.001, ****P < 0.0001)
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| Discussion | | |
Although S. aureus is may exist as a nonpathogenic microorganism in some parts of the body, especially in the nose and on skin, it can be the causal agent of a broad spectrum of mild to life-threatening infections.[1],[2] Considering the high prevalence of S. aureus and the difficult treatment of infections caused by this bacterium, especially by its biofilm-forming isolates, it is necessary to conduct research in order to find new treatment methods,[20] which can be either as biofilm formation prevention or as biofilm eradication. Although antibiotics play an important role in modern treatment methods, emergence of antibiotic-resistant strains necessitates constant search for more potent and efficient antibiotics. Among the suggested treatments that have attracted interest is the use of probiotics that have antagonistic properties against other microorganisms. Many studies have demonstrated that probiotics have considerable therapeutic potential for localized and systemic bacterial infections.[21] Several reports have been published on the effects of probiotics on Staphylococcus, but few studies have investigated their effects on the steps in biofilm formation and the CSH in S. aureus. Consequently, the present research investigated the antimicrobial and anti-biofilm effects of two standard strains (L. casei ATCC 39392 and L. rhamnosus ATCC 7469) on S. aureus ATCC 33591.
The results showed that CFSs of both Lactobacillus spp. were able to inhibit S. aureus growth. In the broth microdilution method, the MIC of both Lactobacillus spp. (L. casei and L. rhamnosus) for S. aureus ATCC 33591 was 4 mg/mL, their MBC was 8 mg/mL, and their MBC/MIC ratio was 2. Since MBC/MIC of ≤4 and >4 indicates that the antimicrobial effect is bactericidal and bacteriostatic, respectively.[22] Therefore, CFSs of both studied Lactobacillus spp. had bactericidal effect on S. aureus. These results conform to those of previous studies conducted on other lactobacilli (Koohestani et al. and Melo et al.). In the research by Koohestani et al. in which the agar spot method and the broth microdilution method were used, L. acidophilus LA5 and L. casei 431 formed clearing zones in S. aureus ATCC 25923 (diameters of clearing zones of 50 mm and 37 mm for L. acidophilus and L. casei, respectively) and MIC of 40 mg/mL for both supernatants.[23] In the study carried out by Melo et al., in which the broth microdilution method was employed, the MICs of L. plantarum TCUESC02 and L. fermentum TCUESC01 CFSs for S. aureus CCMB262 were 2.5 and 20 mg/mL, respectively.[17]
In the present research, comparison of antimicrobial activity of the CFSs prior to and following pH neutralization and heating indicated that neither of the studied Lactobacillus spp. produced active bacteriocin against S. aureus and their antimicrobial activity was probably due to production of organic acids. L. casei and L. rhamnosus are among heterofermentative lactic acid bacteria and can produce various metabolites including different organic acids in the hydrocarbon fermentation process.[24] Hu et al. studied the CFSs of L. plantarum strains and identified five different organic acids (lactic, acetic, tartaric, citric, and malic acids). They also showed that CFSs of these strains had antimicrobial activity against different pathogens including S. aureus.[25] It seems that, at identical pH values, organic acids exhibit greater antimicrobial activity compared to mineral acids. Minor et al. reported that acidification of the culture (to pH 4.6) using lactic acid decreased biomass of S. aureus by 99% compared to the control (the culture medium without lactic acid). They also observed that acidification of the culture medium to pH 5 using acetic acid and to pH 4.5 employing citric acid had similar effects and the antimicrobial effects of these organic acids were stronger than the mineral acids (phosphoric and hydrochloric acids), because these two mineral acids were able to exert similar effects at pH values of 4.1 and 4, respectively.[26]
Staphylococci are known as the most prevalent infectious agents associated with biofilms. S. aureus can form biofilms in host tissues and on medical catheters and implants.[2],[7] The performance of antimicrobial agents in treating infections does not only depend on their bactericidal and bacteriostatic effects but also on their ability to suppress production of virulence factors.[20] The present research investigated the effects of the L. casei and L. rhamnosus CFSs on factors influencing S. aureus biofilms. For this purpose, CSH, initial attachment, biofilm production and eradication, and also expression of genes involved in biofilm formation were assessed. Bacterial adhesion is the first step in biofilm formation, which depends on environmental conditions, characteristics of the surface on which the biofilm is formed, and the extracellular polymers produced by the bacteria. However, biofilm formation is mainly managed by physicochemical properties such as electrostatic and van der Waals interactions and CSH. In fact, CSH plays the main role in bacterial colonization on biotic and abiotic surfaces. In addition, medical equipment such as catheters, mechanical heart valves, and artificial pacemakers are all made of hydrophobic materials and hydrophobic microorganisms have a propensity to adhere to such surfaces.[27] In this study, sub-MICs of the CFSs of both studied Lactobacillus spp. significantly decreased CSH in S. aureus (P < 0.0001). This result conforms to those of previous studies. Walencka et al. reported in their research that the biosurfactant extracted from L. acidophilus reduced CSH in S. aureus, thereby decreasing its biofilm formation.[28] Melo et al. used the microtiter plate assay and SEM in their study and observed that sub-MICs of L. fermentum were able to significantly reduce biofilm formation by S. aureus. Moreover, they noticed that expression of the biofilm formation of the icaA gene declined and that of the icaR gene involved in regulation of biofilm formation increased.[17] The gene products of the icaADBC locus are responsible for PIA biosynthesis. PIA is the main molecule responsible for intercellular adhesion in S. aureus. Expression of the icaR gene suppresses the expression of this gene locus.[29] In the present research also, similar results were obtained. CFSs of both studied Lactobacillus spp. significantly reduced biofilm formation. This reducing effect was confirmed by the scanning electron microscope and the microtiter plate assay. Moreover, both CFSs increased expression of the icaR suppressor gene and decreased expression of the icaA gene.
No studies concerning the effects of supernatants and/or organic acids produced by lactobacilli on expression of the other genes involved in S. aureus biofilms were found in a search within the reliable websites. However, some studies had investigated the effects of other metabolites produced by lactobacilli. Yan et al. employed the microtiter plate assay and SEM to study the effects of the biosurfactant produced by L. plantarum on initial attachment and biofilm formation in S. aureus. They also assessed the effects of a number of the genes (icaA, sarA, srtA, and cidA) involved in biofilm formation through real-time PCR. The L. plantarum biosurfactant significantly reduced initial attachment and biofilm formation and decreased expression of some of the genes involved in biofilm formation (sarA and cidA).[14] In this research, the L. casei and L. rhamnosus CFSs reduced the expression of the sarA gene in S. aureus. It should be mentioned that sarA induces attachment and allows initial biofilm formation by suppressing the extracellular proteolytic and nucleolytic enzymes. Consequently, its reduced expression prevents initial attachment of S. aureus cells.[30] In the present study also, the L. casei and L. rhamnosus CFSs increased the expression of the hld and cidA genes. The hld gene is expressed when the agr quorum-sensing system is activated. Therefore, increased expression of the hld gene is a sign of improved activity of the agr quorum-sensing system. Researchers have shown that the agr system downregulates the genes encoding adhesion factors and biofilm formation. This can reduce adhesion and hence indirectly decrease initial biofilm formation. In addition, we know that agr upregulates expression of detergent-like peptides and nucleotides that seem to increase biofilm separation.[30] Activity of the cid operon induces autolysis in S. aureus cells. Programmed cell death involves cidA, and its expression is necessary for biofilm formation because autolysis of a number of bacterial cells provides the eDNA required in biofilm formation.[31] In the present research, expression of the cidA gene in the samples treated with the L. casei and L. rhamnosus CFSs increased several folds (3.48 and 4.4 fold) compared to the control sample. This indicated extensive autolysis and death of S. aureus cells.
| Conclusion | | |
In summary, the results of this study indicated that L. casei ATCC 39392 and L. rhamnosus ATCC 7469 had desirable antagonistic effects against the standard strain of S. aureus ATCC 33591. CFSs of these two Lactobacillus spp. in sub-MIC concentration significantly reduced surface hydrophobicity, initial attachment, and biofilm formation and eradicated biofilms. Moreover, significant changes were observed in expression of all studied genes involved in biofilm formation. Consequently, it is hoped that conducting further research, especially on effective compounds in the CFSs of the studied lactobacilli, will make it possible to produce drugs for preventing/treating infections caused by S. aureus biofilms.
Acknowledgments
This study was supported by the AJA University of Medical Sciences in the form of an approved plan code 97000812 and the Molecular Microbiology Research Center at Shahed University.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
| References | | |
| 1. | Arciola CR, Campoccia D, Montanaro L. Implant infections: Adhesion, biofilm formation and immune evasion. Nat Rev Microbiol 2018;16:397-409.  |
| 2. | Martins N, Rodrigues CF. Biomaterial-related infections. J Clin Med 2020;9:722. |
| 3. | Singh AK, Prakash P, Achra A, Singh GP, Das A, Singh RK. Standardization and classification of in vitro biofilm formation by clinical isolates of Staphylococcus aureus. J Glob Infect Dis 2017;9:93-101. |
| 4. | Maikranz E, Spengler C, Thewes N, Thewes A, Nolle F, Jung P, et al. Different binding mechanisms of Staphylococcus aureus to hydrophobic and hydrophilic surfaces. Nanoscale 2020;12:19267-75. |
| 5. | Jenul C, Horswill AR. Regulation of Staphylococcus aureus Virulence. Microbiol Spectr 2019;7:1-34. |
| 6. | Del Pozo JL. Biofilm-related disease. Expert Rev Anti Infect Ther 2018;16:51-65. |
| 7. | Otto M. Staphylococcal Biofilms. In: Romeo T, editor. Bacterial Biofilms Berlin, Heidelberg: Springer; 2008. p. 207-28. |
| 8. | Suresh MK, Biswas R, Biswas L. An update on recent developments in the prevention and treatment of Staphylococcus aureus biofilms. Int J Med Microbiol 2019;309:1-12. |
| 9. | Bhola J, Bhadekar R. Invitro synergistic activity of lactic acid bacteria against multi-drug resistant staphylococci. BMC Complement Altern Med 2019;19:70. |
| 10. | Guimarães A, Santiago A, Teixeira JA, Venâncio A, Abrunhosa L. Anti-aflatoxigenic effect of organic acids produced by Lactobacillus plantarum. Int J Food Microbiol 2018;264:31-8. |
| 11. | Shokri D, Khorasgani MR, Mohkam M, Fatemi SM, Ghasemi Y, Taheri-Kafrani A. The inhibition effect of lactobacilli against growth and biofilm formation of Pseudomonas aeruginosa. Probiotics Antimicrob Proteins 2018;10:34-42. |
| 12. | CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard-11 th Edition. CLSI DOCUMENTE M07-A11. Wayne, PA: Clinical and Laboratory Standards Institute; 2018. |
| 13. | Mirani ZA, Fatima A, Urooj S, Aziz M, Khan MN, Abbas T. Relationship of cell surface hydrophobicity with biofilm formation and growth rate: A study on Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. Iran J Basic Med Sci 2018;21:760-9. |
| 14. | Yan X, Gu S, Cui X, Shi Y, Wen S, Chen H, et al. Antimicrobial, anti-adhesive and anti-biofilm potential of biosurfactants isolated from Pediococcus acidilactici and Lactobacillus plantarum against Staphylococcus aureus CMCC26003. Microb Pathog 2019;127:12-20. |
| 15. | Saidi N, Owlia P, Marashi SM, Saderi H. Inhibitory effect of probiotic yeast Saccharomyces cerevisiae on biofilm formation and expression of α-hemolysin and enterotoxin A genes of Staphylococcus aureus. Iran J Microbiol 2019;11:246-54. |
| 16. | Merghni A, Dallel I, Noumi E, Kadmi Y, Hentati H, Tobji S, et al. Antioxidant and antiproliferative potential of biosurfactants isolated from Lactobacillus casei and their anti-biofilm effect in oral Staphylococcus aureus strains. Microb Pathog 2017;104:84-9. |
| 17. | Melo TA, Dos Santos TF, de Almeida ME, Junior LA, Andrade EF, Rezende RP, et al. Inhibition of Staphylococcus aureus biofilm by Lactobacillus isolated from fine cocoa. BMC Microbiol 2016;16:250. |
| 18. | Lv X, Miao L, Ma H, Bai F, Lin Y, Sun M, et al. Purification, characterization and action mechanism of plantaricin JY22, a novel bacteriocin against Bacillus cereus produced by Lactobacillus plantarum JY22 from golden carp intestine. Food Sci Biotechnol 2018;27:695-703. |
| 19. | Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 2001;25:402-8. |
| 20. | Sharifi A, Mohammadzadeh A, Zahraei Salehi T, Mahmoodi P. Antibacterial, antibiofilm and antiquorum sensing effects of Thymus daenensis and Satureja hortensis essential oils against Staphylococcus aureus isolates. J Appl Microbiol 2018;124:379-88. |
| 21. | Sharifi-Rad J, Rodrigues CF, Stojanović-Radić Z, Dimitrijević M, Aleksić A, Neffe-Skocińska K, et al. Probiotics: versatile bioactive components in promoting human health. Medicina (Kaunas) 2020;56:E433. |
| 22. | Ginting EV, Retnaningrum E, Widiasih DA. Antibacterial activity of clove ( Syzygium aromaticum) and cinnamon ( Cinnamomum burmannii) essential oil against extended-spectrum β-lactamase-producing bacteria. Vet World 2021;14:2206-11. |
| 23. | Koohestani M, Moradi M, Tajik H, Badali A. Effects of cell-free supernatant of Lactobacillus acidophilus LA5 and Lactobacillus casei 431 against planktonic form and biofilm of Staphylococcus aureus. Vet Res Forum 2018;9:301-6. |
| 24. | Maske BL, de Melo Pereira GV, da S Vale A, de Carvalho Neto DP, Karp SG, Viesser JA, et al. A review on enzyme-producing lactobacilli associated with the human digestive process: From metabolism to application. Enzyme Microb Technol 2021;149:109836. |
| 25. | Hu CH, Ren LQ, Zhou Y, Ye BC. Characterization of antimicrobial activity of three Lactobacillus plantarum strains isolated from Chinese traditional dairy food. Food Sci Nutr 2019;7:1997-2005. |
| 26. | Taylor TM, Doores SX. Organic acids. In: Antimicrobials in Food. 4 th ed. Boca Raton: CRC Press; 2020. p. 133-90. |
| 27. | Ganji-Azad E, Javadi A, Jahanbani Veshareh M, Ayatollahi S, Miller R. Bacteria cell hydrophobicity and interfacial properties relationships: A new MEOR approach. Colloid Interfac 2021;5:49. |
| 28. | Walencka E, Rózalska S, Sadowska B, Rózalska B. The influence of Lactobacillus acidophilus-derived surfactants on staphylococcal adhesion and biofilm formation. Folia Microbiol (Praha) 2008;53:61-6. |
| 29. | Nguyen HT, Nguyen TH, Otto M. The staphylococcal exopolysaccharide PIA – Biosynthesis and role in biofilm formation, colonization, and infection. Comput Struct Biotechnol J 2020;18:3324-34. |
| 30. | Archer NK, Mazaitis MJ, Costerton JW, Leid JG, Powers ME, Shirtliff ME. Staphylococcus aureus biofilms: Properties, regulation, and roles in human disease. Virulence 2011;2:445-59. |
| 31. | Sugimoto S, Sato F, Miyakawa R, Chiba A, Onodera S, Hori S, et al. Broad impact of extracellular DNA on biofilm formation by clinically isolated Methicillin-resistant and -sensitive strains of Staphylococcus aureus. Sci Rep 2018;8:2254. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]
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