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Vibriocidal efficacy of Bifidobacterium bifidum and Lactobacillus acidophilus cell-free supernatants encapsulated in chitosan nanoparticles against multi-drug resistant Vibrio cholerae O1 El Tor

BMC Infectious Diseases volume 24, Article number: 905 (2024) Cite this article

Abstract

Background

Cholera is a diarrheal disease recognized for being caused by toxin-producing Vibrio (V.) cholerae. This study aims to assess the vibriocidal and immunomodulatory properties of derived cell-free supernatants (CFSs) of Bifidobacterium (B.) bifidum and Lactobacillus (L.) acidophilus encapsulated in chitosan nanoparticles (CFSb-CsNPs and CFSa-CsNPs) against clinical multi-drug resistance (MDR) isolates of V. cholerae O1 El Tor.

Methods

We synthesized CFSb-CsNPs and CFSa-CsNPs using the ionic gelation technique. The newly nanostructures were characterized for size, surface zeta potential, morphology, encapsulation efficacy (EE), stability in different pH values and temperatures, release profile, and in vitro cytotoxicity. The antimicrobial and antibiofilm effects of the obtained nanocomposites on clinical MDR isolates (N = 5) of V. cholerae E1 Tor O1 were investigated by microbroth dilution assay and crystal violet staining, respectively. We conducted quantitative real-time PCR (qRT-PCR) to analyze the relative gene expressions of Bap, Rbmc, CTXAB, and TCP in response to CFSb-CsNPs and CFSa-CsNPs. Additionally, the immunomodulatory effects of formulated structures on the expression of TLR2 and TLR4 genes in human colorectal adenocarcinoma cells (Caco-2) were studied.

Results

Nano-characterization analyses indicated that CFSb-CsNPs and CFSa-CsNPs exhibit spherical shapes under scanning electron microscopy (SEM) imaging, with mean diameters of 98.16 ± 0.763 nm and 83.90 ± 0.854 nm, respectively. Both types of nanoparticles possess positive surface charges. The EE% of CFSb-CsNPs was 77 ± 4.28%, whereas that of CFSa-CsNPs was 62.5 ± 7.33%. Chitosan (Cs) encapsulation leads to increased stability of CFSs in simulated pH conditions of the gastrointestinal tract in which the release rates for CFSb-CsNPs and CFSa-CsNPs were reached at 58.00 ± 1.24% and 55.01 ± 1.73%, respectively at pH = 7.4. The synergistic vibriocidal effects observed from the co-administration of both CFSb-CsNPs and CFSa-CsNPs, as evidenced by a fractional inhibitory concentration (FIC) index of 0.57, resulting in a significantly lower MIC of 2.5 ± 0.05 mg/mL (p < 0.0001) compare to individual administration. The combined antibacterial effect of CFSb-CsNPs and CFSa-CsNPs on Bap (0.14 ± 0.05), Rbmc (0.24 ± 0.01), CTXAB (0.30 ± 0.09), and TCP (0.38 ± 0.01) gene expression was significant (p < 0.001). Furthermore, co-administration of CFSb-CsNPs and CFSa-CsNPs also demonstrated the potency of suppressing TLR 2/4 (0.20 ± 0.01 and 0.12 ± 0.02, respectively) gene expression (p = 0.0019) and reduced Caco-2 cells attached bacteria to 526,000 ± 51,46 colony-forming units/mL (11.19%) (p < 0.0001).

Conclusion

Our study revealed that encapsulating CFSs within CsNPs enhances their vibriocidal activity by improving stability and enabling a controlled release mechanism at the site of interaction between the host and bacteria. Additionally, the simultaneous use of CFSb-CsNPs and CFSa-CsNPs exhibited superior vibriocidal potency against MDR V. cholerae O1 El Tor strains, indicating these combinations as a potential new approach against MDR bacteria.

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Introduction

Cholera is a worldwide health issue that is of great concern in the medical field, particularly in developing countries like Iran [1]. While antibiotic treatment is typically the initial approach for treating bacterial infections, the emergence of multidrug-resistance (MDR) strains has restricted the effectiveness of antibiotics [2, 3].

Probiotics possess significant antimicrobial or antagonistic capabilities, which encompass the secretion of antimicrobial substances like bacteriocins, competitive displacement of pathogens, reinforcement of the intestinal barrier to resist pathogens, and bolstering the host’s immune system to effectively fight against infectious agents [4]. Clinical trials have underscored the success of probiotics in managing gut-related health issues, producing favorable results [5]. Probiotics are a broad category that includes many different kinds of bacteria and fungus; however, the most widespread probiotic strains are Lactobacillus and Bifidobacterium (B.) species [6]. Research indicates that numerous Bifidobacterium strains possess antibacterial properties effective against a range of pathogens, such as Escherichia (E.) coli, Salmonella (S.) typhi [7], Streptococcus (S.) mutans [8], Propionibacterium acnes [9], and Listeria (L.) monocytogenes [10]. Furthermore, investigations suggest that Bifidobacterium strains can be utilized against MDR microorganisms. Notably, a study found that probiotics, including certain Lactobacillus strains, were capable of inhibiting or dismantling the biofilm produced by MDR E. coli; notably, B. longum exhibited the most potent inhibition against the biofilm generated by E. coli IC2 [11].

Vibriocidal effects of probiotic bacteria were studied in some limited investigations; such as Leuconostoc mesenteroides and Bacillus subtilis natto, alone and in combination [12], Bacillus velezensis [13], and Lactobacillus reuteri [14].

Exposure of Vibrio (V.) cholerae to antibiotics creates a stressful environment, prompting the bacterium to initiate biofilm formation as a response to these circumstances [15]. During biofilm formation in V. cholerae, two crucial matrix proteins, RbmC and Bap, are secreted at different stages and serve distinct roles within the biofilm. Bap is responsible for anchoring the biofilm to the surface, while RbmC facilitates cell-to-cell connectivity [16]. Previous studies have indicated that the absence of RbmC has a minimal impact on the structure of the biofilm pellicle in V. cholerae. However, the loss of both Bap and RbmC results in a significant reduction in biofilm formation [17]. Furthermore, the TCP gene production promotes the aggregation of multiple V. cholerae cells, enabling the close association and clustering necessary for micro-colony formation. The coordinated activation of CTXAB genes enables the production and release of cholera toxin, which significantly contributes to the development of cholera and its associated symptoms [18]. On the other hand, once biofilms are established, they withstand both immune responses and antibiotic treatments, contributing to the recurring nature of the infection [19]. Antimicrobial probiotics with the ability to disperse biofilms show promise for improved clinical outcomes in treating diarrhea caused by Vibrio species, as they could potentially serve as single-agent therapies [20]. Moreover, cholera is described as an inflammatory disorder that specifically impacts the small intestine. The interaction between V. cholerae and Toll-like receptors (TLRs), particularly TLR2 and 4, plays a crucial role in the initiation and progression of the inflammatory response [21]. Cell-free supernatants (CFSs) from vibrant probiotic cultures, particularly those high in antimicrobial compounds including lactic acid bacteria (LAB), undoubtedly stand out as a highly effective natural antimicrobial agent. Previous studies investigated the antimicrobial effects of probiotic CFSs against pathogens such as E. coli [22], S. Typhi, S. Typhimurium [23], L. monocytogenes [24], and Staphylococcus (S.) aureus [25]. A study by Beristain-Bauza in 2015 found that the antimicrobial capabilities of the cell-free supernatant produced by Lactobacillus (L.) rhamnosus are largely attributed to the presence of lactic acid and a bacteriocin-like compound [26].

The application of nanoparticles (NPs) to encapsulate drugs for delivery has attracted considerable interest owing to its numerous benefits over conventional drug delivery techniques [27]. Nanocapsulation safeguards drugs against degradation within the gastrointestinal tract, thereby enhancing their absorption [28]. Chitosan nanoparticles (CsNPs), recognized for their mucoadhesive and biodegradable nature, along with their positively charged amine groups, interact effectively with the negatively charged elements of the bacterial cell wall [29]. CsNPs possess the capability to act as an absorption booster in the intestinal lining, extending the dwell time of delivery vehicles at absorption locations and easing the tight junctions of cellular membranes [30]. Incorporating antimicrobial agents into CsNPs represents a significant advancement in probiotic research. This approach not only fortifies the stability of CFSs but also leverages Cs’s natural antimicrobial features to markedly enhance antibacterial and antibiofilm effectiveness [31].

Herein, we aim to investigate vibriocidal and immunomodulatory properties of newly-synthesized CFSb-CsNPs (cell-free supernatant of B. bifidum encapsulated in CsNPs) and CFSa-CsNPs (cell-free supernatant of L. acidophilus encapsulated in CsNPs), particularly their combined action against MDR V. cholerae strains. The current study hypothesized that incorporating CFSb and CFSa within CsNPs increases the antibacterial potency via the controlled release profile of cargo at the specific host-bacteria interface, improves its stability in the gastrointestinal (GI) tract, and can introduce novel nanocomponent to tackle V.cholerae infection.

Materials and methods

Preparation of bacterial strains

The probiotic strains used in this study were L. acidophilus ATCC 4356 and B. bifidum ATCC 29,521. This research included clinical strains of V. cholerae (N = 5), sourced from the microbial archives of the bacteriology department at the Faculty of Science, Tarbiat Modares University, Tehran, Iran. These strains were collected during the most recent cholera outbreak in the country. The identification of V. cholerae strains was based on biochemical assays as well as PCR amplification of the 16–23 s rRNA intergenic region specific to V. cholerae [32]. The strains exhibited resistance to tetracycline, cotrimoxazole, and trimethoprim. For the current project, the strains were cultured on Brain Heart Agar (Sigma, USA) medium and incubated at 37 °C for 24 h, after which they were used in our study.

CFS preparation of B. Bifidum and L. Acidophilus

The CFSs from B. bifidum and L. acidophilus were prepared following the method outlined by Jeffrey et al. in 2020 [33], with a few modifications. The active overnight culture of B. bifidum and L. acidophilus strains was diluted in fresh de Man–Rogosa–Sharpe (MRS) broth medium (Ibresco, Iran) (OD600nm = 0.23), and incubated at 37 °C for 48 h in microaerobiosis. To prepare CFSs of probiotic strains following overnight culture, the cells were removed by centrifuging at 6,000×g/4°C (Microcentrifuge sigma 1-14k, Germany) for 15 min. The generated supernatants were meticulously harvested and processed through sterilization via 0.22 μm polyvinylidene fluoride (PVDF) filters (Millipore Sigma, USA) to eliminate any traces of live cells. Following this treatment, samples of the CFS from each probiotic strain were placed onto a solid MRS medium and incubated at 37 °C for 48 h to verify the complete eradication of viable cells. The sterilized supernatants were divided into smaller portions to facilitate storage and future use and stored at -20 °C.

Synthesis and confirmation of nanoparticles

The ionotropic gelation technique was employed in the fabrication of CFSb-CsNPs and CFSa-CsNPs utilizing tripolyphosphate (TPP; molecular weight: 367.86 g/mol, Sigma-Aldrich, USA), following a method previously outlined [34]. This method hinges on the ionic interaction between the primary amine groups present in the Cs solution, which carries a positive charge, and the phosphate groups of the TPP solution, which bear a negative charge. In short, low molecular weight (50–200 kDa, Sigma, USA) chitosan (Cs; 0.05% w/v) was dispersed in an aqueous acetic acid solution (1.0% v/v) and stirred using magnetic stirrers (IKA@RH basic 2, Germany) until complete dissolution occurred. The pH of the Cs solution was adjusted to 6 using 1.0 N sodium hydroxide (NaOH), which ensures that Cs remains soluble over the concentrations tested. An aqueous TPP solution was then formulated at a concentration of 0.2 mg/mL (pH = 4). A 0.45 μm pore filter (Millipore, Sigma) was used to filter both the Cs and TPP solutions. The spontaneous formation of CFSb-CsNPs and CFSa-CsNPs occurred through the dropwise addition method. This involved carefully measured quantities of TPP solutions, prepared with precise amounts of each CFSb and CFSa (200.0 mg/mL), being added to Cs solutions. The volume ratios for Cs and TPP were maintained at 1:10 for both types of nanoparticles. The process was carried out using a disposable insulin needle, allowing for a controlled dropping rate of 0.20 mL/min. Throughout this procedure, the mixture was kept under constant magnetic stirring conditions for 2 h (25 ̊C, 450 rpm).

The NPs suspensions were purified via centrifugation (4 °C, 24,000 × g, 30 min), then washed and redispersed in deionized water before undergoing freeze-drying to yield the final synthesized nanoparticles (Zirbus VaCo5, Germany). The clear supernatant, rich in unentrapped CFS, was retained for calculation of drug encapsulation efficiency (EE %) by an indirect method using the Pierce™ BCA Protein assay kit (Thermo Scientific, UK) following the instructions provided by the manufacturer. The EE% was determined according to the formulation described below.

$${\rm{EE}}\% \, = \, {{{\rm{Loaded }}\,{\rm{protein}}\,{\rm{in}}\,{\rm{CFSb}}\, - \,{\rm{CsNPs }}/{\rm{CFSa}}{\kern 1pt} \, - \,{\rm{CsNPs }}\, - {\rm{ }}\,{\rm{CsNPs }}} \over {{\rm{Total }}\,{\rm{amount }}\,{\rm{of }}\,{\rm{protein}}\,{\rm{ in}}\,{\rm{CFSb }}\, - \,{\rm{CsNPs}}/{\rm{CFSa}}\,{\rm{ }} - \,{\rm{CsNPs }}}}\, \times \,100$$

CS/TPP NPs were prepared using TPP solutions without CFS used as a control, following the same procedure.

To verify the precision of the synthesis of CFSb-CsNPs and CFSa-CsNPs, measurements of size distribution and zeta potential charge were performed (HORIBA-SZ100, Japan). For further confirmation of the created nanostructures, the nanopowders were dispersed in a 1.0% acetic acid solution, and a droplet was deposited onto an aluminum foil, followed by gold coating for morphological characterization using SEM imaging (20 kV, VEGA3 TESCAN, Czech Republic).

In vitro release kinetics of CFSs from CFSb-CsNPs and CFSa-CsNPs

The release kinetics profile of CFSs from CFSb-CsNPs and CFSa-CsNPs was conducted in buffers at varying pH levels. To achieve this, nanoparticles were suspended in phosphate buffer solutions (PBS) with a pH of 7.2 inside two distinct dialysis bags (Sigma, molecular weight cutoff = 12.0 kDa). Each sealed dialysis tube was subsequently submerged in a 20.0 mL solution at different pH values ranging from 1.2 to 7.4. At predetermined intervals (2, 4, 6, 8, 12, 24, 48, and 72 h), 5.0 mL aliquots were withdrawn and replenished with an equal volume of fresh solution. The quantity of CFSs discharged from both types of nanoparticles was determined utilizing the Pierce™ BCA Protein assay kit (Thermo Scientific, UK), following the manufacturer’s guidelines for usage. Bare CFSa and CFSb were used as positive controls.

Stability study of CFSb-CsNPs and CFSa-CsNPs

The stability analysis of the synthesized CFSb, CFSa, CFSb-CsNPs, and CFSa-CsNPs involves the examination of their size distribution, surface charge, and physical appearance at specific intervals over a period of 10, 20, 30, 45, and 60 days. The assessment of stability is conducted under different temperatures, including − 20 °C ± 1.03, 4 °C ± 0.37, 25 °C ± 0.16, and 37 ̊C ± 2.23 in a dark sealed glass container. To assess the quantity of protein loaded from the CFS encapsulated within CsNPs, following the evaluation of the antimicrobial properties of the synthesized nanostructures against MDR V. cholerae isolates at predetermined time intervals, the protein content was measured using the Pierce™ BCA Protein Assay Kit. To explore the pH stability of synthesized nanostructures, specifically CFSb-CsNPs, and CFSa-CsNPs, these particles were immersed in PBS at various pH levels 1.2, 4.5, 5.5, 6.5, and 7.4 at 37 °C. The pH of the PBS solutions was adjusted using either HCl (37.0% v/v) or NaOH (50.0% w/v) solutions. After a 2 h incubation period, which allowed for the establishment of swelling equilibrium, the particle size distribution was determined.

Antibacterial activity evaluation using microbroth dilution

The microbroth dilution assay, according to the Clinical and Laboratory Standards Institute [35], was done to determine the minimal inhibitory concentrations (MIC) of the synthesized nanoparticles. Briefly, cationic adjusted Muller Hinton Broth medium (MHB) (Merk, Germany) was utilized to achieve a final volume of 200.0 µL in each well of the 96-well microplate (SPL, Korea). This involved adding the overnight culture of clinical isolates of V. cholerae (20.0 µL) at a concentration of 1 × 108 CFU/mL (colony-forming units), along with 2-fold reduced serial concentrations of CsNPs, CFSb, CFSa, CFSb-CsNPs, and CFSa-CsNPs (800, 400, 200, 100, 50, 25, 10, 5, and 2.5 mg/mL). In the experiment, broth media with equal bacterial counts were utilized as the positive control, whereas blank broth media served as the negative control. The plates were then sealed and incubated for 18–24 h in a shaking incubator set at 37 °C (IKA@KS, Sigma, USA). The MIC values were determined by assessing the optical density (OD) of bacterial growth in wells treated with nanostructures versus the control wells (OD620 nm). Further, 100.0 µL of the supernatant from each well was inoculated onto a Brain heart infusion (BHI) agar medium to enumerate the viable bacteria that had been exposed to the synthesized nanoparticles. Initially, the ODs of bacterial samples subjected to varying nanoparticle concentrations at a wavelength of 620 nm were recorded. These readings were subsequently adjusted by subtracting the OD of the blank well. Ciprofloxacin (5.0 µg, Mast Group Ltd.) was also used as a positive control, revealing that 97.7 ± 0.02% of the strains exhibited susceptibility, while 2.3 ± 0.41% displayed an intermediate level of susceptibility. The checkerboard investigation also was carried out to study vibriocidal effects of co-administration of CFSb-CsNPs and CFSa-CsNPs. The concentration level of each nanostructure in combination ranged from 1/4 times the MIC (1/4 × MIC) to 4× MIC. To quantify the effect of the combinations, the fractional inhibitory concentration (FIC) was determined for each nanocomposite in combination with the FIC index being the sum of the FIC values of CFSb-CsNPs and CFSa-CsNPs. Synergistic interactions were characterized by an FIC index of ≤ 0.5 [36].

Microtitre biofilm inhibition study with crystal violet assay

To assess the prevention of biofilm development, we employed techniques outlined earlier [37]. Briefly, 90.0 µL of a bacterial solution in adjusted cations MHB (OD600 = 0.05), achieved by diluting overnight culture of bacteria in tryptic soy broth (TSB) with 1.0% sucrose, was introduced into the inner compartments of a 96-well polystyrene microtiter plate. This plate also contained 10.0 µL of CsNPs, CFSb, CFSa, CFSb-CsNPs, and CFSa-CsNPs, arranged in a 2-fold serial concentration (800 to 2.5 mg/mL). Following overnight incubation of sealed plates at 37 °C, allowing for bacterial proliferation and biofilm development, the next steps involved measuring the bacterial growth at OD600 (Epoch Microplate Spectrophotometer, BioTek Instruments Inc., USA). Subsequently, the planktonic cells and the remaining liquid were removed, and the attached mass was washed three times with distilled water. To visualize the biofilm, the mass was treated with a 0.1% crystal violet (CV) solution for 20 min, after which it was again rinsed three times with distilled water to eliminate any unattached dye. The dye that remained bound was then mixed gently with 70.0% ethanol, and the absorbance at 595 nm was measured on the same plate. The level of biofilm suppression was determined based on the quantity of biofilm formed in the absence of CsNPs, CFSb, CFSa, CFSb-CsNPs, and CFSa-CsNPs (considered as 100% biofilm) and the sterility of the media (rated as 0% biofilm). The findings were derived from averaging at least three independent experiments.

Cell culture cytotoxicity study

To investigate the cytotoxicity concentration of prepared nanostructures and bare CFSs, Caco-2 (human colorectal adenocarcinoma cell line) cells (Iranian Biological Resource Center) were seeded in a 96-well plate (4 × 105/well) in RPMI 1640 culture medium (Roswell Park Memorial Institute) supplemented with 12% fetal bovine serum (FBS), 1% l-glutathione, and 1% 10,000 I.U./mL penicillin and 10,000 µg/mL streptomycin (BioIdea, Iran). Following 85% confluence, CFSb-CsNPs, CFSa-CsNPs, CFSb, CFSa, and CsNPs were introduced to cells, and incubation in a humidified incubator was continued for 24, 48, and 72 h (25 °C, 5% Co2). Caco-2 cells infected with V. cholerae without treatment were the positive control, whereas those treated with PBS were designated as the negative control. The cytotoxicity of synthesized nanostructures (1-100 mg/mL) on Caco-2 cells was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit (Yekta Tajhiz Azma, Iran) according to manufacturer’s instructions. Each treatment was performed in triplicate.

In vitro gene expression evaluation

In vitro experimental groups and treatment conditions were established: (i) untreated Caco-2 cells (negative control), (ii) Caco-2 cells incubated with V. cholerae (positive control, MOI: 7), (iii) Caco-2 cells + V. cholerae + CFSb, (iv) Caco-2 cells + V. cholerae + CFSa, (v) Caco-2 cells + V. cholerae + CsNPs, (vi) Caco-2 cells + V. cholerae + CFSb-CsNPs, (vii) Caco-2 cells + V. cholerae + CFSa-CsNPs, and (viii) Caco-2 cells + V. cholerae + CFSb-CsNPs + CFSa-CsNPs. Following cytotoxicity evaluation of the designed nanosystem, according to half-inhibitory concentrations (IC50), CTXAB, Bap, RbmC, and TCP gene expression were measured by quantitative real-time PCR (qRT-PCR) at sub-MIC concentrations of CFSb-CsNPs, CFSa-CsNPs, CFSb, and CFSa in all MDR isolates of V. cholerae. To investigate TLR2 and 4 gene expression in Caco-2 cells infected with V.cholerae strains, following treatment with CFSb-CsNPs, CFSa-CsNPs, CFSb, and CFSa (37 ̊C, 24 h), RNA extraction and complementary DNA (cDNA) synthesis were performed according to the instructions by Total RNA extraction and cDNA synthesis kits (Yekta Tajhiz Azma, Iran). The qRT-PCR was done with applications of the 2−ΔΔCT method [38] with a total volume reaction of 25.0 µL, consisting of 2X Real Q plus SYBR Green Master Mix (Amplicon), 10.0 pmol of primers, and 1.0 µl of cDNA product obtained from reverse transcription (Table 1). All samples were triplicated by control gene expression (GAPDH and 16srRNA).

Table 1 Oligonucleotides sequences used in this study
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In vitro V. Cholerae isolates attachment and internalization study in the presence of CFSb-CsNPs and CFSa-CsNPs

5 × 106 Caco-2 cells were seeded in a 24-well polystyrene plate (SPL, Korea), and the cells were allowed to reach 80% confluence. At this point, the synthesized nanocomposites were introduced to the cells and incubated at 37 ̊C. The wells were washed three times with PBS and 2 × 107 CFU/mL of V. cholerae isolates (MOI:7) were introduced into the wells. To investigate the attached bacteria, 0.01% Triton-X 100 was added to each well and the lysates were then cultured on BHI agar.

Another triplicate set of infected cells was subjected to treatment with gentamicin (Sigma, USA) for a duration of 1 h. This treatment aimed to remove the attached bacteria. After the treatment, the cells were lysed using 0.01% Triton-X 100 in PBS. The lysates were then cultured on BHI agar at various dilutions. The average number of colonies obtained from the cultures represents the count of bacteria that invaded the cells. To observe the adhesion of V. cholerae strains to Caco-2 cells in the presence of CFSb-CsNPs and CFSa-CsNPs, and to enhance visualization, Giemsa staining was applied to infected cells that were fixed with cold isopropanol after being exposed to the nanoparticles for 3 h at 37 °C (LABOMED; Inc, USA).

Statistical analysis

Data were examined using the one-way ANOVA (Non-parametric tests) followed by the Sidaks test in GraphPad Prism 9.5.1 (GraphPad Software, Inc., La Jolla, CA, USA). A P-value less than 0.05 was considered significant. The mean ± standard deviation (Mean ± SD) of the replicate results was evaluated. All tests were performed in triplicate.

Results

Characterization of synthesized NPs

SEM images reveal the spherical and smooth morphology of the formulated nanostructures (Fig. 1A-B). DLS analysis indicates an optimal average size of 98.16 ± 0.763 nm for CFSb-CsNPs and 83.9 ± 0.854 nm for CFSa-CsNPs. The surface charge measurements also show a positive zeta potential of + 1.7 ± 0.427 mV for CFSb-CsNPs and + 3.3 ± 0.2 mV for CFSa-CsNPs (Fig. 1C- D). EE% of CFSs within CsNPs was found to be suitable at 77 ± 4.28% for CFSb-CsNPs and 62.5 ± 7.33% for CFSa-CsNPs.

Fig. 1
figure 1

Physicochemical characteristics of synthesize nanostructures; SEM imaging; A CsNPs B CFSb-CsNPs, C CFSa-CsNPs; size distribution and zeta potential measurement; D CFSb-CsNPs, and E CFSa-CsNPs. CFSb-CsNPs cell-free supernatant of Bifidobacterium bifidum encapsulated in chitosan nanoparticles, CFSa-CsNPs cell-free supernatant of Lactobacillus acidophilus encapsulated in chitosan nanoparticles

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In simulated GI system pH conditions and considering the typical transit times of orally administered drugs through various segments of the GI [39], we noted that over a maximum duration of 2 h post-stomach transit, approximately 23.33 ± 4.37% and 20.00 ± 1.15% of the drug was released for CFSb-CsNPs and CFSa-CsNPs, respectively, at pH = 1.2. The small intestine, the colonization site of V.cholerae, typically has a consistent transit time, averaging between 3 and 4 h, though this can range from 2 to 6 h among healthy individuals. Over a maximum 6 h period in the small intestine with a pH = 7.4, the release rates for CFSb-CsNPs and CFSa-CsNPs reached at 58.00 ± 1.24% and 55.01 ± 1.73%, respectively (Fig. 2A-B).

Fig. 2
figure 2

Release profile analysis at various pH condition; A CFSb-CsNPs, B CFSa-CsNPs, C mean size distribution of fabricated nanostructures in response to 2 h incubation in different pH, D viable colony count in response to 24 h treatment with various convention of CsNPs, CFSa, CFSb, CFSa-CsNPs, and CFSb-CsNPs on brain heart infusion agar, E effect of CsNPs, CFSa, CFSb, CFSa-CsNPs, and CFSb-CsNPs composites on biofilm inhibition of multi-drug resistance Vibrio cholerae O1 El Tor isolates with CV staining in microtiter plate; cell cytotoxicity study, F 24 h, G 48 h, H 72 h. CFSb-CsNPs cell-free supernatant of Bifidobacterium bifidum encapsulated in chitosan nanoparticles, CFSa-CsNPs cell-free supernatant of Lactobacillus acidophilus encapsulated in chitosan nanoparticles

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Stability assessment of CFSb-CsNPs and CFSa-CsNPs

In the stability study over 60 days, the nanostructures maintained minimal variation in size distribution with no change in the appearance of the lyophilized powder (Table 2). However, their zeta potentials underwent alterations, influenced by temperature changes which impacted their antimicrobial actions. The study on the alterations in the dimensions and surface charge of CFSb-CsNPs and CFSa-CsNPs nanostructures across varying temperatures reveals significant effects at 37 °C and 25 °C on nanoparticle size. However, the maximum stability of the nanostructure was obtained at 4 °C and especially at -20 °C. The analysis of protein levels using a Pierce™ BCA Protein Assay Kit over a designated timeframe revealed that at lower temperatures (4 °C and − 20 °C), after 60 days, the protein content in CFSb-CsNPs and CFSa-CsNPs only decreased to 82.02 ± 1.40% (6347.0 ± 10.37 µg/mL) and 78.064 ± 0.85% (6930.0 ± 22.38 µg/mL) at 4 °C and 88.32 ± 2.00% (7069.0 ± 12.0 µg/mL) and 85.0 ± 16.04% (7504.0 ± 16.04 µg/mL) at -20 °C, highlighting the preservation of their vibriocidal properties under lower storage conditions. This observation underscores the stabilizing effect of CsNPs on the encapsulated CFSs. In contrast, after only 20 days, the protein concentrations of CFSa and CFSb decreased to 523.0 ± 7.54 µg/mL and 517.0 ± 9.24 µg/mL at -20 °C, and 411.0 ± 7.85 µg/mL and 426.0 ± 3.04 µg/mL at 4 °C, respectively, which show decreased vibriocidal potency to 32.01 ± 0.05% and 19.01 ± 0.62%, respectively (Table 3). The impact of pH on the average diameters of CFSb-CsNPs and CFSa-CsNPs, following exposure to different pH levels of PBS solutions over 2 h, demonstrated a notable increase in size as the pH level rose from 4.5 to 5.5. However, beyond a pH = 5.5 up to the pH = 7.4, the particles exhibited remarkable stability in their size (p = 0.02) (Fig. 2C).

Table 2 Stability evaluation of synthesized nanostructures during 60 days in -20 ̊C, 4 ̊C, 25 ̊C, and 37 ̊C by measuring size distribution (nm) and surface charge (mV). CFSb-CsNPs cell-free supernatant of Bifidobacterium bifidum encapsulated in Chitosan nanoparticles, CFSa-CsNPs cell-free supernatant of Lactobacillus acidophilus encapsulated in chitosan nanoparticles
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Table 3 Stability evaluation of synthesized nanostructures during 60 days in -20 ̊C, 4 ̊C, 25 ̊C, and 37 ̊C by measuring protein content using the Pierce™ BCA protein assay kit (Thermo Scientific, UK). CFSb-CsNPs cell-free supernatant of Bifidobacterium bifidum encapsulated in Chitosan nanoparticles, CFSa-CsNPs cell-free supernatant of Lactobacillus acidophilus encapsulated in chitosan nanoparticles, CFSb cell-free supernatant of Bifidobacterium bifidum, and CFSa-CsNPs cell-free supernatant of Lactobacillus acidophilus
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Antibacterial activity evaluation using microbroth dilution technique

The MIC value for CFSb-CsNPs was determined to be 10.56 ± 0.19 mg/mL, whereas that for CFSa-CsNPs was 48.27 ± 2.29 mg/mL. Interestingly, the MIC values for CFSb (476 ± 5.71 mg/mL), CFSa (512 ± 20.34 mg/mL), and CsNPs (110 ± 0.36 mg/mL) were all found to be greater than those for CFSb-CsNPs and CFSa-CsNPs (p < 0.001). The colony counts of viable V. cholerae in response to concentrations exceeding 5 ± 0.14 mg/mL of CFSb-CsNPs and above 25 ± 0.03 mg/mL of CFSa-CsNPs further demonstrates the antimicrobial efficacy of the formulated CFSs-Cs encapsulation (Fig. 2D). Additionally, the synergistic vibriocidal effects observed from the co-administration of both CFSb-CsNPs and CFSa-CsNPs, as evidenced by a fractional inhibitory concentration (FIC) index of 0.57, resulting in a significantly lower MIC of 2.5 ± 0.05 mg/mL. This MIC value is substantially lower than those of the individual CFSs, CFSb-CsNPs, and CFSa-CsNPs highlighting the enhanced antibacterial activity achieved through their combined use (p < 0.0001).

Microtitre biofilm inhibition study with crystal violet assay

The process of removing unattached bacteria from the microtiter plate and subsequently staining the adherent biomass with CV highlighted a significant correlation between the extent of biofilm development and the antibiofilm component of the culture medium used. In the study on MDR V.cholerae biofilms, it was observed that growth in TSB supplemented with 1.0% glucose led to significant adherence of bacteria (~ 100.0%). Further analysis showed that treating V.cholerae with varying concentrations of CsNPs, CFSb, CFSa, CFSb-CsNPs, and CFSa-CsNPs demonstrated that all these components, at concentrations exceeding 200 ± 4.23 mg/mL, were effective in inhibiting biofilm formation. Notably, the inhibitory concentration for the CFSb-CsNPs and CFSa-CsNPs was found to be lower, specifically at 10 ± 0.06 mg/mL (Fig. 2E). The findings indicate a significant synergistic effect between CsNPs and CFS in inhibiting bacterial biofilms (p ≤ 0.001).

Cell culture cytotoxicity study

Figure 2F-H displays the cytotoxicity data gathered over a 72-h period. The in vitro assessment of CFS cytotoxicity from both selected bacteria and CsNPs reveals an IC50 value exceeding 100.0 mg/ml. Similarly, the IC50 values for CFSb-CsNPs and CFSa-CsNPs surpassed 100.0 mg/mL after 48 h. By the end of the 72-h observation period, the IC50 values for CFSb-CsNPs and CFSa-CsNPs were calculated to be 25.33 ± 2.60 mg/mL and 50.73 ± 0.05 mg/mL, respectively (p ≤ 0.001). In contrast, the IC50 of CsNPs over 72 h remains above 100 mg/mL. These results indicate that at the MIC, the synthesized nanostructures pose no harm to Caco-2 cells.

In vitro gene expression evaluation

Upon addition of diverse compounds, including CFSb, CFSa, CsNPs, CFSb-CsNPs, CFSa-CsNPs, and CFSb-CsNPs + CFSa-CsNPs, to the Caco-2 cell medium, the TLR2 gene expression was determined to be 0.66 ± 0.18, 0.74 ± 0.04, 0.81 ± 0.91, 0.45 ± 0.02, 0.61 ± 0.46, and 0.20 ± 0.01, respectively. Similarly, the TLR4 gene expression was found to be 0.51 ± 0.08, 0.60 ± 0.02, 0.82 ± 0.14, 0.27 ± 0.16, 0.42 ± 0.05, and 0.12 ± 0.02, respectively. Significantly, there was a prominent difference in the downregulation of the TLR2 and TLR4 genes when treated with CFSb-CsNPs + CFSa-CsNPs compared to other compounds (p < 0.0001). Moreover, CFSb-CsNPs and CFSa-CsNPs demonstrated greater efficacy in inhibiting the expression of TLR2 and TLR4 genes compared to CFSb, CFSa, and CsNPs (Fig. 3A- B).

Fig. 3
figure 3

Effect of CFSb, CFSa, CsNPs, CFSb-CsNPs, CFSa-CsNPs, and CFSb-CsNPs + CFSa-CsNPs on gene expression analysis by quantitative real-time PCR, A TLR2, B TLR4, C CTXAB, D TCP, E Bap, and F RbmC genes, non-parametric ANOVA (Sidaks test) was done for comparisons analysis (p˂0.05). CFSb-CsNPs cell-free supernatant of Bifidobacterium bifidum encapsulated in chitosan nanoparticles, CFSa-CsNPs cell-free supernatant of Lactobacillus acidophilus encapsulated in chitosan nanoparticles, Bap Biofilm associated protein, RbmC Rugosity and Biofilm Modulators, TCP Toxin-coregulated pilus, TLR Toll-like receptor

Full size image

The expression levels of CTXAB and TCP genes in V. cholerae isolates were examined in response to treatments, including CFSb, CFSa, CsNPs, CFSb-CsNPs, CFSa-CsNPs, and CFSb-CsNPs + CFSa-CsNPs. The results showed a reduction in CTXAB gene expression by 1.38 ± 0.05, 1.28 ± 0.03, 1.09 ± 0.06, 1.92 ± 0.18, 1.61 ± 0.01, and 3.33 ± 0.09, respectively. The gene expression of TCP was reduced by 1.44 ± 0.03, 1.23 ± 0.04, 1.12 ± 0.13, 2.08 ± 0.05, 1.36 ± 0.07, and 2.63 ± 0.01, respectively (p < 0.001) (Fig. 3C-D).

The expression of Bap gene was found to be 0.80 ± 0.01, 0.78 ± 0.33, 0.91 ± 0.49, 0.61 ± 0.02, 0.74 ± 0.64, and 0.14 ± 0.05 fold changes, respectively, while for RbmC it was 0.73 ± 0.04, 0.78 ± 0.91, 0.83 ± 0.41, 0.61 ± 0.04, 0.72 ± 0.16, and 0.24 ± 0.01 respectively, in response to CFSb, CFSa, CsNPs, CFSb-CsNPs, CFSa-CsNPs, and CFSb-CsNPs + CFSa-CsNPs (p < 0.001). The combination of CFSb-CsNPs and CFSa-CsNPs demonstrated a winner nanocomponent that significantly attenuated the expression of all CTXAB, TCP, Bap, and RbmC genes in MDR clinical isolates of V.cholerae (Fig. 3E- F).

In vitro V. Cholerae isolates attachment and internalization study in the presence of CFSb-CsNPs and CFSa-CsNPs

According to the results in Fig. 4A, V. cholerae demonstrated a strong ability to attach to and form a robust biofilm on the surface of Caco-2 cells. However, when CFSb-CsNPs were applied (Fig. 4B), the number of attached bacteria decreased by 27.71 ± 0.03% (p < 0.0001). Similarly, when CFSa-CsNPs were applied (Fig. 4C), the number of attached bacteria decreased by 42.18 ± 0.14% (p < 0.0025). Co-administration of CFSb-CsNPs and CFSa-CsNPs resulted in a further reduction of attached bacteria to 526,000 ± 51,46 (11.19%) colony-forming units (CFU) (p < 0.0001) in the presence of 2 × 107 CFU/mL bacteria (Fig. 4D- F).

Fig. 4
figure 4

Vibrio(V.) cholerae adhesion and internalization to monolayer Caco-2 cells (MOI: 7 V. cholerae /100 Caco-2 cells) observed after Giemsa staining using light microscopy ×40 in the presence of synthesized nanostructures, A positive control, B CFSb-CsNPs, C CFSa-CsNPs, D CFSb-CsNPs + CFSa-CsNPs, and E negative control; diagrams representing F attached V.cholerae and G bacterial internalization rates in Caco-2 cells in present of nanostructures; comparisons were analyzed with non-parametric ANOVA (Sidaks test) ****˂0.0001, ***˂0.0004, **˂0.0025, and *˂0.05. CFSb-CsNPs cell-free supernatant of Bifidobacterium bifidum encapsulated in chitosan nanoparticles, CFSa-CsNPs cell-free supernatant of Lactobacillus acidophilus encapsulated in chitosan nanoparticles, Caco-2 cells Human colorectal adenocarcinoma cells

Full size image

The presence of CFSb-CsNPs also led to a bacterial internalization rate of approximately 20 ± 1.00 CFU/mL. In contrast, the presence of CFSa-CsNPs resulted in a higher internalization rate of approximately 31.67 ± 2.96 CFU/mL. However, when CFSb-CsNPs and CFSa-CsNPs were combined, a significant reduction in bacterial internalization was observed, with an internalization rate of approximately 4.66 ± 1.45 CFU/mL (Fig. 4G).

Discussion

V. cholerae, specifically serogroup O1, is accountable for the majority of cholera cases worldwide and has been subjected to treatment with multiple classes of antibiotics throughout the years [40]. Our investigation focuses on evaluating the antibacterial, antibiofilm, and immunomodulatory properties of CFSb-CsNPs and CFSa-CsNPs, against clinical isolates of MDR V. cholerae. Additionally, our research underscores the importance of CsNPs in boosting the stability and managing the release of CFSs under simulated GI pH levels and varying temperatures. Numerous literature studies have shown prominent antibacterial effects of probiotic bacteria and their CFSs. Recent in vitro investigations revealed antimicrobial effect of lactobacilli strains against various Vibrio species, including V. parahaemolyticus [41, 42] and V. cholerae [43]. Other study demonstrated the bactericidal or bacteriostatic effects of CFSs derived from four lactobacillus strains, namely L. acidophilus, L. delbrueckii, L. plantarum, and L. johnsonii, with doses ranging from 18 to 22%, 20–22%, 46–48%, and 50–54%, respectively, against Pseudomonas (p) aeruginosa strains [44]. The CFS of L. reuteri AN417 also exhibited antibacterial activity against three oral pathogens: Porphyromonas gingivalis, Fusobacterium nucleatum, and S. mutans [45]. M. K. Al-Malkey et al. (2017) [46] reported that bacteriocins produced by L. acidophilus and L. rhamnosus exhibited promising results as potential bio-therapeutic alternatives to combat multi-drug resistant P. aeruginosa. The largest inhibitory zones were observed with free cell supernatants, underscoring their significant antimicrobial potential. This finding aligns with the work of Daba and Saidi (2015) [47] who investigated the inhibitory effects of bacteriocin-producing LAB against P. aeruginosa and E.coli using CFSs and cell diffusion methods, reinforcing the role of these probiotics in combating antibiotic-resistant pathogens. Consistent with the aforementioned studies, our findings demonstrated that CFSb and CFSa exhibited MIC values of 476 ± 5.71 mg/mL and 512 ± 20.34 mg/mL, respectively, against MDR isolates of V. cholerae.

In the study conducted by Raafat et al., the impact of Cs on bacterial cells was explored, revealing its ability to modify the bacterial and indicated antimicrobial properties associated with Cs [48]. In a separate investigation, the incorporation of L. helveticus CFS into CsNPs resulted in notable antimicrobial activity against Bacillus cereus, Bacillus sciuri, E. coli, and P. aeruginosa, surpassing the efficacy of free CFS. However, the enhanced antimicrobial effect of CFS loaded into CsNPs was observed specifically against Salmonella enterica, reducing the MIC by 47% compared to free CFS [49]. We examined the vibriocidal properties of ionic gelation synthesized CFSb-CsNPs and CFSa-CsNPs, which were found to have optimal sizes and a positive surface charge. These synthesized nanostructures, due to their spherical shape, were efficiently taken up by bacteria through endocytosis, which significantly boosted their ability to kill bacteria. The MIC value of both CFSb-CsNPs and CFSa-CsNPs against MDR V. cholerae strains was substantially reduced to 10.56 ± 0.19 mg/mL and 48.27 ± 2.29 mg/mL, respectively, compared to Cs-free CFS (p < 0.0001). Additionally, the combined administration of CFSb-CsNPs and CFSa-CsNPs resulted in a notable increase in vibriocidal activity (FIC = 0.57).

The stability of the antibacterial properties of CFS is crucial for their commercial viability. Ideally, CFS should be kept at room temperature to minimize the extra expenses linked to refrigeration or freezing. However, their performance at 37 °C, which corresponds to the body temperature in vivo models, warrants consideration. Cardost et al. conducted a study on the stability of Enterococcus. faecalis DBFIQ E24 CFS at various temperatures (4 °C, 25 °C, 37 °C, and − 20 °C) over 5 months. The results showed that the CFS retained its antimicrobial effectiveness for only a month, after which a noticeable decline in antimicrobial activity was observed at all tested temperatures [50]. In contrast, Nakamura et al. found that concentrated CFS from L. gasseri LA39 exhibited good stability, with high bacteriocin activity observed for 2 months at 37 °C and up to 9 months at 4 °C. Similarly, stable results (3 months) were reported for CFS from L. plantarum YML007 when stored at room temperature [51].

To evaluate the benefits of nano-formulation compared to unformulated CFSs, our study employed two approaches: examining the stability of CFSs versus bare-CFSs, where we observed minimal changes in size distribution and no alteration in the appearance of the lyophilized powder of CFSb-CsNPs and CFSa-CsNPs over 60 days. Additionally, we noted the superior antimicrobial effectiveness of CFSb-CsNPs and CFSa-CsNPs relative to standalone CFSs, achieved through high protein loading capacity over a specified duration, and emphasized the retention of their vibriocidal properties even under less stringent storage conditions.The pH-responsive characteristics of CFSb-CsNPs and CFSa-CsNPs can be attributed to the protonation of the primary amino groups within the Cs backbone. This process enhances the electrical charge and the repulsive forces among the cross-linked chitosan chains [52]. Our findings suggest that the particle size is highly reactive to variations in the pH of the surrounding aqueous medium, implying that the surface concentration of protonated amino groups and the extent of protonation are inversely proportional to changes in solution pH. This research focuses on understanding the swelling and shrinking dynamics associated with fluctuating pH levels, aiming to develop intelligent nanoparticle systems suitable for targeted drug delivery. Specifically, we have shown that CsNPs exhibit pH sensitivity through a reversible cycle of particle expansion and contraction, with particle size diameters varying from approximately 150 nm to around 90 nm. Consistent with our results in the study by R. S. Tığlı Aydın et al. (2012) [53], the pH responsiveness of the 5FU-CsNPs was examined, revealing a notable swelling reaction at pH = 5. Previous literature reviews do not report on investigations into the stability of CFS at varying pH levels. However, in a study conducted by Wala’a and Nibras in 2013, they explored the stability of bacteriocins derived from L. acidophilus CFS against Serratia marcescens. Their findings indicated that bacteriocins from L. acidophilus were effective against Serratia marcescens. Specifically, the bacteriocin from L. acidophilus retained stability at pH = 4, with half of its activity diminished at pH = 8, and completely lost at other pH values [46].

The results of our study showed that the synthesized nanomedicine, with a focus on its vibriocidal property, exhibited controlled drug release profiles under both acidic and physiological conditions. These pH conditions were chosen to simulate the acidic environment of the GI, ranging from the mouth to the intestinal lumen. The present findings indicated that the optimal conditions for drug delivery were observed at pH = 7.2. This suggests that the nanostructures effectively retain their cargo until they reach the intended target in the intestinal lumen, where they are expected to interact with pathogens. Similar to our study, Sarveswari et al. conducted an in vivo cholera model and reported findings that align with our results. They observed a burst release of the drug at pH = 7, followed by a controlled and linear release in alkaline pH conditions [54]. Saberpour et al. conducted a study in which they investigated the release behavior of mesenchymal stem cells-derived conditioned media from Cs nanostructures under two different pHs 3.2 and 7.2. In contrast to our findings, they reported no initial burst release within the first few hours. Instead, they observed a gradual release of approximately 40% of the cargo for 12–48 h at pH 7.2 [55]. In line with these studies and our release profile of cargo, the prolonged release pattern of the cargo enables a consistent and uniform drug concentration to be maintained over an extended period. This ensures the effectiveness of the drug while minimizing the risk of side effects associated with rapid release. Our research specifically indicates that during the transition across the small intestine, the site of colonization for V. cholerae, CFSb-CsNPs and CFSa-CsNPs discharged their contents at rates of 35.01 ± 1.03% and 33.41 ± 0.24%, respectively.

To evaluate the primary inhibitory effects of CFSb-CsNPs and CFSa-CsNPs on MDR V. cholerae, we investigated the expression of virulence genes in the selected isolates. In our study, we observed that the combination of CFSb-CsNPs and CFSa-CsNPs had an impact on the growth environment of V. cholerae, leading to changes in the expression of genes involved in the bacteria’s pathogenesis. The analysis of gene expression revealed that CFSb-CsNPs, CFSa-CsNPs, and CFSb-CsNPs + CFSa-CsNPs exhibited attenuating effects on CTXAB and TCP genes expression in compare to Cs-free CFSs (p˂0.003). A recent study also reported L. rhamnosus GG and B. longum were both shown to be capable of removing 68% and 59%, respectively, of the cholera toxin from aqueous solutions over 18 h of incubation at 37 °C. By altering the toxin receptor through an enzymatic mechanism, probiotics have been used in several ways to interfere with the quorum-sensing function of the toxin receptor. Other proposed pathways include the inhibition of toxin synthesis, the lowering of gut pH, and the abatement of pathogenicity [56]. The findings of the study by Alamdary et al. [57] revealed that L. acidophilus is a safe supplement that has a protective effect on human epithelial colorectal cells and is potent enough to be utilized as an additional treatment for attenuating toxin production in acute infectious diarrhea brought on by V. cholerae.

In the exploration of the bactericidal abilities of probiotic bacteria and the CFSs they produce, several studies have concentrated on their antibiofilm properties. In an investigation in 2023, the CFS obtained from Lactobacillus spp. isolated from fecal samples of healthy children exhibited a remarkable preformed antibiofilm activity against V. cholerae, reducing it by 90% [58]. Other study demonstrated that the CFS derived from seven strains of Lactobacillus, isolated from infant feces, effectively inhibited the formation of biofilms by P. aeruginosa in burn wounds [59]. Zamani H., et al. [60] also revealed the CFS obtained from L.plantarum isolated from Siahmazgi cheese demonstrated significant anti-adhesive potential, inhibiting the adhesiveness of P.aeruginosa, S. aureus, and E.coli by 76.8%, 58.6%, and 52.2%, respectively [60]. However, our study revealed that while free CFSs did not have notable antibiofilm effects, CFSb-CsNPs and CFSa-CsNPs exhibited effective antibiofilm activity against MDR V.cholerae isolates. The application of CFSb-CsNPs resulted in a downregulation of Bap and RbmC genes in V. cholerae isolates (p = 0.0421). The substantial reduction in the number of Caco-2 cells attached bacteria, in comparison to the positive control (3,396,000 ± 205,066), offers additional proof of the inhibitory impact exerted by co-administration of CFSb-CsNPs + CFSa-CsNPs on the attachment of V. cholerae isolates to the cell surface. This finding further supports the antibiofilm properties of the synthesized nanostructures (p < 0.0001).

Cs and CsNPs, depending on their molecular weight [61], have been noted for their ability to bind to the bacterial membrane. This binding facilitates the deeper penetration of antibacterial agents into biofilms and alters their hydrophobic characteristics. This interaction is believed to contribute to the observed inhibition of adhesion [62, 63]. This is vital because biofilms create a physical obstacle that restricts the reach of antibiotics to the bacterial cells beneath [64]. The diminutive size and positive charge of CsNPs enable them to traverse the intricate structure of biofilms, accessing bacteria that would typically be shielded. Recent research findings have underscored the efficacy of Cs-nano-capsulation as a potent bactericide, demonstrating superior biofilm disruption capabilities. Additionally, these studies highlight the critical role of delivery systems in boosting antibacterial effectiveness [65,66,67,68,69,70,71,72,73].

Caco-2 cells are a suitable model for studying drug absorption, transport, and metabolism in the intestine due to their resemblance to human intestinal epithelial cells. They possess functional characteristics like microvilli and tight junctions, which are important for simulating the intestinal barrier [74]. Some studies have shown that V. cholerae induces an inflammatory response through TLR and NOD receptors [75], which can assist in the development of targeted drugs for regulating inflammation. Our study demonstrates that CFSa-CsNPs and CFSb-CsNPs effectively reduce the expression of TLR2 and TLR4 genes in Caco-2 cells. Notably, the reduction in gene expression is more pronounced when these probiotic components (CFSs) are combined (CFSa-CsNPs + CFSb-CsNPs) compared to their individual effects. These findings are consistent with previous research that highlights the synergistic benefits of combined interventions.

Consistent with our findings, previous research has examined the CFSs of various probiotics, including L. acidophilus, L. casei, L. lactis, L. reuteri, and Saccharomyces boulardii. The metabolites produced by these probiotics demonstrated the ability to reduce the expression of PGE-2 and IL-8 in human colon epithelial HT-29 cells. Furthermore, probiotic supernatants exhibited varying effects on the production of IL-1β, IL-6, TNF-α, and IL-10 by human macrophages, indicating their distinct anti-inflammatory activities [76].

Conclusion

In conclusion, this research indicates that the CFSs derived from probiotic bacteria L.acidophilus and B.bifidum possess vibriocidal capabilities. Furthermore, the synergy achieved by encapsulating selected probiotic CFSs within Cs in nanoscale exhibited enhanced antimicrobial and antibiofilm activities. This enhancement was attributed to the exceptional stability of the synthesized nanoparticles across various temperatures and pH levels. Importantly, the study highlights the superior vibriocidal therapeutic effects of combining CFSa-CsNPs and CFSb-CsNPs compared to their individual applications. While this study highlights the antimicrobial and antibiofilm properties of CFSb-CsNPs and CFSa-CsNPs, further research utilizing a cholera animal model may yield promising results regarding the effectiveness of probiotic CFSs in treatment. Additionally, the investigation and purification of active compounds within the CFS can open new avenues for treating bacterial infections and preventing the emergence of antibiotic-resistant strains.

Data availability

The datasets generated during and/or analyzed during the current study are available and/or analyzed during the current work are available from the corresponding authors upon reasonable request.

Abbreviations

V. cholerae :

Vibrio cholerae

MDR:

Multi-drug resistance

Cs:

Chitosan

NPs:

Nanoparticles

CsNPs:

Chitosan nanoparticles

B. bifidum :

Bifidobacterium bifidum

L. acidophilus :

Lactobacillus acidophilus

CFSb-CsNPs:

Cell-free supernatant of Bifidobacterium bifidum encapsulated in chitosan nanoparticles

CFSa-CsNPs:

Cell-free supernatant of Lactobacillus acidophilus encapsulated in chitosan nanoparticles

TPP:

Sodium tripolyphosphate

PBS:

Phosphate-buffered saline

DLS/Zeta:

Dynamic light scattering/zeta potential

SEM:

Scanning electron microscopy

Bap :

Biofilm associated protein

RbmC :

Rugosity and Biofilm Modulators

TCP :

Toxin-coregulated pilus

TLR:

Toll-like receptor

Caco-2 cells:

Human colorectal adenocarcinoma cells

EE:

Encapsulation efficacy

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Acknowledgements

The authors wish to thank the Research Council of Tarbiat Modares University for developing the project.

Funding

The study was supported by the Research Council of Tarbiat Modares University (Code Number: IR.MODARES.AEC.1401.036) and the Iran National Science Foundation (INSF) (Grant Number: 4012567).

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Authors and Affiliations

  1. Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

    Mozhgan Derakhshan-sefidi & Bita Bakhshi

  2. Department of Biostatistics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

    Aliakbar Rasekhi

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  1. Mozhgan Derakhshan-sefidi
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Contributions

MDS conceptualization, performed the experiments, and drafted the manuscript. BB conceptualization, supervised the work, and revised the manuscript. AR contributed to data interpretation and validated the results. All authors reviewed and approved the final manuscript.

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Correspondence to Bita Bakhshi.

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Ethics approval and consent to participate

The study was reviewed and approved by the Medical Ethics Committee of Tarbiat Modares University (Code: IR.TMU.REC.1397.092) before the study began.

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The authors declare no competing interests.

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Derakhshan-sefidi, M., Bakhshi, B. & Rasekhi, A. Vibriocidal efficacy of Bifidobacterium bifidum and Lactobacillus acidophilus cell-free supernatants encapsulated in chitosan nanoparticles against multi-drug resistant Vibrio cholerae O1 El Tor. BMC Infect Dis 24, 905 (2024). https://doi.org/10.1186/s12879-024-09810-2

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BMC Infectious Diseases

ISSN: 1471-2334

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