Pediococcus pentosaceus MZF16 Probiotic Strain Prevents in vitro Cytotoxic Effects of Pseudomonas aeruginosa H103 and Prolongs the Lifespan of Caenorhabditis elegans

1. Introduction

Fermented foods are produced through controlled microbial growth, and enzymatic conversion of food components. Historically, food fermentation was a common preservation method, used to maintain food integrity and reduce the risk of contamination with pathogenic microorganisms, likely due to the production of antimicrobial metabolites such as organic acids, ethanol and bacteriocins [1]. In recent years, fermented foods have surged in popularity, because of their health-promoting properties especially for gastrointestinal health. These benefits are attributed to various mechanisms, including bioactive peptides and phenolic compounds conversion to biologically active compounds, as well as the beneficial effects of constituent microorganisms [2]. Fermented foods such as dairy products, fermented meats and vegetables, contain relatively resilient and stable microbial ecosystems, predominantly composed of lactic acid bacteria (LAB) and their primary metabolites including lactic acid [3]. Some LAB strains have long been recognized as probiotics due to their ancestral use and classification as beneficial bacteria with Generally Recognized As Safe (GRAS) status. Emerging probiotics are also being studied to better understand their potential health benefits. Among GRAS probiotics, the most well-known and studied bacteria belong to the Lactobacillus genus, recently reclassified into 23 genera [4]. Notably, Lactobacillus probiotic species including Lacticaseibacillus casei, Lactobacillus acidophilus, Lactobacillus johnsonii, and Lactiplantibacillus plantarum. These species have demonstrated various health benefits, including anti-carcinogenic, anti-diabetic, antioxidant and anti-inflammatory and also anti-microbial effects against food-borne pathogens [5].

Despite their frequent presence in many fermented products (i.e. meat, fish, dairy, beer, wine, fruits, vegetables, and beverages)[6], probiotics from the genus Pediococcus have been less studied. This genus consists of several species, including P. acidilactici, P. pentosaceus, P. damnosus, P. parvulus, P. inopinatus, P. halophilus, P. dextrinicus, and P. urinaeequi [7]. Among these, P. acidilactici and P. pentosaceus probiotic strains are notable for their high production of bacteriocins (pediocins) and bacteriocin-like substances (BLS) [8]. Indeed, pediocins and other postbiotics metabolites (e.g. lactic acid, biosurfactants) from some Pediococcus species have exerted significant anti-bacterial and anti-virulence activities against several food spoilage pathogens, as Listeria monocytogenes, Salmonella enterica and also against the Multi-Drug Resistant (MDR) pathogen Pseudomonas aeruginosa [9,10,11,12,13,14].

P. aeruginosa is known to be a potential source of the spoilage of various foods, notably those with high-water content and nutrient-rich foods, such as milk, meats, and some fruits and vegetables [15]. It is also an opportunistic Gram-negative pathogen, widespread in the environment and responsible of health care-associated infections and foodborne diseases [16]. In some cases, this germ has been associated with diarrheal disease, systemic infections, enterocolitis and digestive cancers [17,18]. Its ability to cause severe acute and chronic life-threatening infections is attributed to its multiple virulence factors [19]. Moreover, the high versatility and adaptability of this pathogen are supported by its large genome (6 to 8 Mb), with over 8% dedicated to regulatory genes, enabling it to evade a broad spectrum of antimicrobial agents [20]. Thereby, alternative treatments, such as competitive probiotics, are urgently needed [21].

Some studies have explored the effects of postbiotic LAB metabolites, such as lactic acid, bacteriocins and bacteriocin-like substances produced by various Pediococcus species (P. pentosaceus and P. acidilactici …) on P. aeruginosa [11,12,14]. However, interactions between these two species and also with the human host remain poorly characterized.

P. pentosaceus MZF16 strain has been previously isolated in our laboratory from the artisanal Tunisian meat “Dried Ossban”, and investigated for its probiotic potential [22,23]. The aim of the present study was to examine the interactions between P. pentosaceus MZF16 and the pathogen P. aeruginosa H103, with a particular focus on MZF16 strain ability to modulate the virulence of the pathogen and its interactions with the host.

2. Materials and Methods

2.1. Bacterial strains and culture conditions

The bacterial strains used in this study were the pathogen P. aeruginosa H103, a prototroph derivative of PAO1 strain [24], and the probiotic P. pentosaceus MZF16. They were respectively grown at 37 °C under rotary shaking (180 rpm) in Luria Bertani (LB) medium and in static conditions in Man Rogosa Sharpe (MRS) medium.

2.2. P. aeruginosa H103-gfp and P. pentosaceus MZF16-mCherry construction

P. aeruginosa H103 was grown in LB medium until reaching an OD600nm of 0.5, then centrifuged at 7000 rpm for 10 min at 4°C. The pellet was washed and resuspended in 300 mM sucrose buffer at a concentration of 10¹⁰ CFU/mL. A 100 µL aliquot of electrocompetent P. aeruginosa was mixed with 50 ng of plasmid pHC60 (gfp, tetR)[25] and electroporated at 1.8 kV in a 0.2-cm cuvette. Immediately after electroporation, 1 mL of LB medium was added, and the mixture was incubated for 2 h et 37°C with shaking at 180 rpm. P. aeruginosa H103-gfp transformants were selected on LB agar supplemented with Tetracycline (100 µg/mL).

For P. pentosaceus MZF16-mCherry construction, electrotransformation was performed using plasmid pRCR12 (mCherry, CmR), provided by Pr Paloma Lopez (Madrid, Spain), and following the method of Pérez-Ramos et al., (2018) [26]. Briefly, the bacterial pellet was washed and resuspended in 1 mL of lysozyme (2,000 U/ mL) and incubated for 20 min at 37 °C. Subsequently, 50 μL of electrocompetent cells were mixed with 5 μL of pRCR12 plasmid (0.5 μg) and electroporated at 2.5 kV in a 0.2-cm cuvette. After electroporation, 500 µL of MRS medium was immediately added, and the cells were incubated for 2 h at 37°C. P. pentosaceus MZF16-mCherry transformants were selected on MRS-agar supplemented with Chloramphenicol (10 μg/mL).

2.3. Caco-2/TC7 cell culture and infection

Human colon adenocarcinoma Caco-2/TC7 cells provided by Chantret (INSERM, Paris) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Lonza, Basel, Switzerland) supplemented with 20 % heat-inactivated fetal bovine serum (FBS, Sigma Aldrich) and penicillin/streptomycin (100 µg/mL). The cells were cultivated and incubated at 37 °C in 5% CO2 atmosphere, and the medium was regularly changed. Cells were passed after reaching approximately 90% of confluence.

For adhesion, invasion, cytotoxicity assays and IL-8 quantification, cells were seeded in 24-well plates treated for tissue culture and grown to confluence. For bacterial translocation assay and transepithelial resistance (TER) measurements, cells were seeded on inserts (6.4 mm diameter, 3 μm pore size, Falcon) until full differentiation (19-21 days).

Bacterial suspensions of P. aeruginosa H103 and its mixture with P. pentosaceus MZF16 (1:100) were prepared in DMEM without FBS and antibiotics. These suspensions were added to confluent or differentiated Caco-2/TC7 cells and incubated at 37 °C in 5 % CO2 for 3 h for adhesion and invasion assays, and overnight for the other experiments.

2.4. Cytotoxicity assay and Interleukin-8 quantification

Cytotoxicity and inflammation assays were performed on confluent Caco-2/TC7 cells grown in 24-well culture plates. Following overnight infection with bacterial suspensions, the supernatants were collected from Caco-2/TC7 monolayers. Then, cytotoxicity was assessed by measuring lactate dehydrogenase (LDH), a cytoplasmic enzyme released upon cell death, using an enzymatic assay (Cyquant Kit, Invitrogen). The concentration of IL-8 cytokine in the supernatants was quantified using the IL-8 Human ELISA Kit (Invitrogen).

2.5. In vitro adhesion and invasion assay

Bacterial adhesion and invasion into intestinal Caco-2/TC7 cells were assessed as follows. For the adhesion assay, Caco-2/TC7 cells were infected with suspensions of P. aeruginosa H103 and mixture with P. pentosaceus MZF16 (1:100 ratio) for 3 h. After incubation, cells were washed twice with Phosphate Buffer Saline (PBS) to remove non-associated bacteria and then lysed with 0.1% Triton X100. The lysates were serially diluted and plated on Cetrimide agar to quantify adherent pathogenic bacteria.

For the invasion assay, after 3 h of infection, adherent and non-internalized bacteria were eliminated by treating the cells with 300 μg/mL of gentamycin for 1 h. Following treatment, the cells were lysed, and internalized bacteria were quantified by plating.

For the visualization of bacterial adhesion, Caco-2/TC7 cells were seeded on glass-bottom 24-well plates (Sensoplate, Greiner) and infected with fluorescent strains P. aeruginosa H103-gfp and P. pentosaceus MZF16-mCherry for 3 h. Afterward, Caco-2/TC7 cells were stained with DAPI (4',6-diamidino-2-phenylindole) and visualized using Confocal Laser Scanning Microscope (CLSM).

2.6. Bacterial translocation and Transepithelial Electrical Resistance (TER) measurement

After an overnight infection of Caco-2/TC7 differentiated cells cultured on inserts, aliquots from the basolateral compartment were collected. The number of translocated P. aeruginosa that crossed the epithelial monolayers was determined by serial dilution and plating on Cetrimide agar.

To assess the integrity of the epithelial barrier, the transepithelial electrical resistance (TER) of infected and non-infected monolayers was measured using the Millicell Electrical Resistance System (Millipore Corp, Bedford, MA).

2.7. Antimicrobial activity test

The antagonistic activity of P. pentosaceus MZF16 against P. aeruginosa H103 was assessed using the disk diffusion method. Overnight cultures of both strains were centrifuged at 8500 rpm for 5 min. Supernatants of P. pentosaceus MZF16 were either pH-adjusted (pH=7) or not before being filtered (0.22 µm membrane).

A suspension of P. aeruginosa H103 at a concentration of 10⁶ CFU/mL was spread on Muller Hinton Agar plate. Disks impregnated with Cell Free Supernatants (CFS) of MZF16 were placed on the agar surface, and the plates were incubated at 37 °C for 24h.

2.8. Bacterial aggregation assay

Auto and coaggregation assays were performed as described previously [27] with minor modifications. Briefly, overnight cultures of H103 and MZF16 were centrifuged at 10 000 g for 10 min. The pellets were washed twice and resuspended in sterile phosphate buffer saline (PBS). The bacterial suspensions were adjusted to an OD₆₀₀ of approximately 0.3.

For the coaggregation assay, equal volumes of the standardized bacterial suspensions were mixed. Both individual and mixed suspensions were aliquoted into hemolysis tubes (4 mL) and incubated at room temperature without agitation.

The OD₆₀₀ of the suspensions was measured at the start of the experiment (A₀) and after 3 h of incubation at 37 °C (At_=3h). The auto-aggregation percentage was calculated as:

$$Autoaggregation\left(\%\right)=\left[1-\left({\displaystyle \frac{\mathrm{A}\mathrm{t}=3\mathrm{h}}{\mathrm{A}0}}\right)\right]\times 100$$

The coaggregation percentage was determined using the formula:

$$Coaggregation\left(\%\right)=\left[\left({\displaystyle \frac{\left(\mathrm{A}\mathrm{H}103+\mathrm{M}\mathrm{Z}\mathrm{F}16\right)-\left(\mathrm{A}\mathrm{m}\mathrm{i}\mathrm{x}\right)}{(\mathrm{A}\mathrm{H}103+\mathrm{M}\mathrm{Z}\mathrm{F}16)}}\right)\right]\times 100$$

where A_H103 and A_MZF16 are the OD₆₀₀ values of the mixed bacterial suspension at time 0, and A_mix is the OD₆₀₀ of the mixed suspension at t=3 h.

2.9. Pyoverdine production measurement

Pyoverdine, a key siderophore and virulence factor of P. aeruginosa. Its production was quantified by spectrophotometry at A_{405 nm.} Bacterial suspensions of P. aeruginosa H103 and the mix with the probiotic P. pentosaceus MZF16 were grown in colorless DMEM medium supplemented with 20% Fetal bovine serum (FBS). After incubation, pyoverdine production was measured from collected supernatants. Results are expressed as the relative pyoverdine levels (mean ± SEM) compared to the control, P. aeruginosa H103 culture.

2.10. Extraction and quantification of AHLs and HAQs molecules

N-acyl-homoserine lactones (AHLs) and 2-alkyl-4-quinolones (HAQ, PQS system) signaling molecules were extracted from P. aeruginosa H103 cultures and co-cultures with P. pentosaceus MZF16 (1:100), grown in DMEM medium, following the protocol described by Fletcher et al., (2007)[28]. For quantification, Escherichia coli carrying plasmid pSB401 (luxRI′::luxCDABE) and P. aeruginosa PAO1 ΔpqsA CTX-lux::pqsA strain were used as biosensors for AHLs and HAQs, respectively. The assay was performed using a combined spectrophotometer/luminometer approach. Briefly, overnight cultures of the biosensor strains were adjusted to an OD_{580 nm} = 1 and then diluted to 1:50 and 1:100 ratios. Crude extracts of quorum-sensing (QS) molecules were diluted in LB medium and added to a 1:50 dilution of the biosensors. The 3-oxo-C12-HSL, C4-HSL, HHQ, and PQS synthetic standards (Sigma-Aldrich, Saint-Louis, MO) at final concentrations of 5 μM were added to a 1:100 dilution of the biosensor strains as positive controls.

The assay was performed in 96-well microtiter plates (white-sided, clear-bottom). Bioluminescence (relative light units, RLU) and absorbance (A5₈₀) were measured every 15 min for 24 h at 37 °C using a Spark 20M multimode microplate reader (Tecan, Männedorf, Switzerland). The bioluminescence data were normalized to the A580 values.

2.11. In vivo Caenorhabditis elegans killing assay

The C. elegans slow killing assay was performed as previously described by Tan et al [29]. C. elegans wild-type Bristol strain N2 worms were grown at 22°C on nematode growth medium (NGM) agar plates using Escherichia coli OP50 strain as the nutrient. P. aeruginosa culture and its coculture with P. pentosaceus MZF16 were spread onto 35 mm NGM solidified agar plates before an overnight incubation at 37°. The plates were cooled to room temperature for 4 h. Then 20 to 30 L4-synchronized worms prepared as described by Blier et al [30] were plated and incubated at 22°C in a humid environment to prevent plate drying. Worm survival was scored daily for 16 days using an optical microscope (Leica CME, Switzerland). The worms were considered dead when they remained static without grinder movements for 20 s or did not respond to light flashes. Results are expressed as a percentage of living worms. The results are the average of at least three independent assays. The nematode survival was calculated using the Kaplan–Meier method, and survival differences were tested for significance using the log–rank test (GraphPad Prism version 4.0).

2.12. Statistical analysis

Statistical analysis of the data was performed using GraphPad Prism software version 10.2.2 with two-sample unpaired test. All tests were conducted at least three times, and statistical significance was considered at p < 0.05, p < 0,01 and p < 0,001.

3. Results

3.1. Autoaggregation and coaggregation

The autoaggregation and coaggregation abilities of P. pentosaceus MZF16 and P. aeruginosa H103 were measured in PBS after 3 h incubation at room temperature (Table 1). P. pentosaceus MZF16 demonstrated a high capacity for autoaggregation (44.7%) and coaggregation with P. aeruginosa H103 (45.7%).

3.2. Antimicrobial activity test

The cell-free supernatant of P. pentosaceus MZF16 exhibited no antibacterial activity against P. aeruginosa H103 in our conditions, suggesting the absence of an anti-Pseudomonas bacteriocin or bacteriocin-like metabolite in our strain.

3.3. Cytotoxicity assay and Interleukin-8 quantification

The cytotoxicity and pro-inflammatory activity of P. aeruginosa H103 towards the intestinal Caco-2/TC7 cells were evaluated after overnight incubation in the presence or absence of P. pentosaceus MZF16. The results demonstrated that MZF16 slightly decreased the level of lactate dehydrogenase (LDH) produced by the eukaryotic cells in response to the cytotoxic effect of the pathogen, with a reduction of about 20% (Figure 1A). In addition, MZF16 significantly reduced the level of IL-8 by more than 25% (Figure 1B).

3.4. Adhesion and invasion

The competitive exclusion of pathogen adhesion and invasion to Caco-2 cells by P. pentosaceus MZF16 was evaluated over 3 h by spreading cell lysates on Cetrimide agar. The simultaneous addition of P. pentosaceus MZF16 with P. aeruginosa H103 to intestinal Caco-2/TC7 cells resulted in a decrease of 39±8 % in the number of adhering P. aeruginosa H103 (Figure 2A), and a reduction of 85±4 % in invasive bacteria number (Figure 2B). Visualization of bacterial adhesion to Caco-2 cells using confocal laser scanning microscopy (CLSM) showed that MZF16-mCherry formed a layer on the cells. In contrast, H103-gfp appeared more scattered in the different stacks, likely due to its mobility and invasive ability (Figure 2C).

3.5. Transepithelial electric resistance (TER) and translocation

The transepithelial resistance (TER) and the bacterial translocation of P. aeruginosa H103 were evaluated on differentiated Caco-2/TC7 cells after an overnight infection. Results showed that P. pentosaceus MZF16 prevented the reduction of TER provoked by P. aeruginosa H103, which was about 30% (Figure 3A). Additionally, P. pentosaceus MZF16 significantly reduced the translocation of the pathogen across the Caco-2 cells by three times (Figure 3B).

3.6. Quorum-sensing and pyoverdine production

N-acyl-homoserine lactones (AHL) production was assessed using the biosensor strain E. coli harboring the plasmid pSB401 (luxRI′::luxCDABE), able to detect short (C4) and long (C12) HSL chains produced by P. aeruginosa. The production of 2-alkyl-4-quinolones (HAQ) signaling molecules (HHQ and PQS) was assessed using the P. aeruginosa PAO1 ΔpqsA CTX-lux::pqsA biosensor strain, which detects HAQ derivatives produced by P. aeruginosa. Both molecules production was examined in presence and absence of MZF16 strain. Results showed that P. pentosaceus MZF16 significantly reduced (of about 35%) the production of HAQs molecules by P. aeruginosa H103 (Figure 4A), but had no effect on HSLs (Figure 4B). Additionally, the effect of MZF16 on P. aeruginosa H103 pyoverdine production was studied, showing a slight decrease in the presence of the probiotic (Figure 4C).

3.7. In vivo virulence test on C. elegans

A slow killing assay was monitored over 16 days (Figure 5). The results showed that in the presence of P. pentosaceus MZF16, the lifespan of C. elegans was significantly prolonged compared to when the worms were exposed only to the pathogen P. aeruginosa H103. Especially, on day 8 of the test, only 10% of the worms infected by H103 survived, while in the presence of P. pentosaceus MZF16, the survival rate of C. elegans infected by H103 was about 40%.

4. Discussion

Pseudomonas aeruginosa is a major opportunistic pathogen responsible for both acute and chronic infections, especially in immunocompromised individuals [31]. The rise of multidrug resistant P. aeruginosa strains has led to an urgent need for alternative treatments to antibiotics, such as probiotics.

Pediococcus pentosaceus MZF16 strain, isolated from Tunisian dried meat (Ossban), exhibits probiotic properties, including resistance to the harsh gastrointestinal conditions and the high ability of adhesion to intestinal cells, without any risks for human health [22].These traits underscore its potential in fighting pathogens.

In the present work, we evaluated the capacity of P. pentosaceus MZF16 to compete with P. aeruginosa H103 and reduce its virulence. For this, undifferentiated intestinal Caco-2/TC7 cells were treated simultaneously with both bacteria and the cytotoxicity, inflammatory potential, and adhesion/invasion of H103 have been investigated. Our study demonstrated that P. pentosaceus MZF16 can mitigate P. aeruginosa H103-induced cytotoxicity and inflammation in intestinal Caco-2/TC7 cells. Specifically, MZF16 reduced both LDH release (necrosis marker) and IL-8 secretion. These findings align with previous research showing similar protective effects of probiotics against various pathogens. For example, P. acidilactici SPM138 increased the human intestinal epithelial cells HT-29 cells survival rate from 20% to 98% and reduced by more than 60% IL-8 levels released upon Clostridium difficile infection [32]. Similarly, P. acidilactici TMAB26 demonstrated immunomodulatory effects by alleviating Klebsiella pneumoniae endotoxin-induced gut inflammation [12].

P. pentosaceus MZF16 also reduced the adhesion, invasion and translocation of P. aeruginosa H103 across undifferentiated/differentiated Caco-2/TC7 cells, preserving epithelial barrier integrity as evidenced by TEER measurements. Similar anti-adherence effects have been reported for P. acidilactici IAH-5 against S. Typhimurium [33]. In another study, P. pentosaceus L1 isolated from pickled radish enable also the decrease of adhesion of an E. coli enterotoxigenic strain to porcine intestinal epithelial cells [34].

The observed reduction in adhesion/invasion and cytotoxicity may result from direct probiotic-pathogen interaction or a postbiotic effect mediated by secreted factors. In fact, autoaggregation is stated to be a prerequisite for probiotic bacteria adhesion to intestinal epithelium, whereas their coaggregation abilities with pathogens enable forming an effective barrier, preventing epithelium colonization by harmful bacteria. Our strain P. pentosaceus MZF16 exhibited high autoaggregation (44.7%) and coaggregation abilities with H103 (45.7%), supporting its role in forming a protective barrier. This coaggregation capacity aligns with previous findings for P. pentosaceus DHR005 which showed lower autoaggregation (28,6%) and coaggregation (22%) against Salmonella Typhimurium PTCC1609 [33].

P. pentosaceus MZF16 is able to produce lactic acid and coagulin bacteriocin [22], we investigated if the protective effect of the probiotic was related to one of these aspects. Interestingly, despite the ability of P. pentosaceus MZF16 to produce antibacterial metabolites, no direct antimicrobial activity was detected against P. aeruginosa H103 under our experimental conditions. Whereas, in other studies, bacteriocins from some P. acidilactici and P. inopinatus strains exerted antimicrobial activities against P. aeruginosa [10,14]. This lack of activity may be due to culture conditions, as bacteriocin production can vary based on time of exposition and media composition. Nonetheless, the ability of MZF16 to co-exist with H103 cocultured in DMEM medium (data not shown) suggests that in our experimental conditions, the protective effect of MZF16 was not directly linked to the production of lactic acid and/or the bacteriocin, but it may be related to a competitive mechanism for nutrients or epithelial binding sites [35].

Furthermore, other studies have shown that some Pediococcus species are able to produce exopolysaccharides and biosurfactants with a high ability to disrupt P. aeruginosa biofilms and display anti-microbial, anti-adhesive and anti-virulence properties [11,26].

The pathogenicity of P. aeruginosa involves the production of several virulence factors [36] such as pyoverdine and pyocyanin, regulated by the cell density-dependent signaling mechanism « quorum sensing (QS) » [37]. In P. aeruginosa, two main QS systems are defined, the system mediated by N-acylhomoserine lactones (AHLs) known as Las and Rhl, which are responsible of the production of homoserine lactone signal molecules, and the Pqs system depending on 2-alkyl-4-quinolones (HAQ) and which is interlinked with the Las and Rhl systems. These systems are involved in the pathogenesis of P. aeruginosa and the synthesis of some virulence factors such as rhamnolipid, pyocyanin, pyoverdine and the production of enzymes like proteases and lipases and biofilm formation [38,39].

The inhibition of this signalization can attenuate QS-related bacterial virulence [40,41]. Thus, anti-QS may be a promising strategy to fight against some pathogens including P. aeruginosa. In this context; we aimed to evaluate the effect of MZF16 on P. aeruginosa H103 QS. Interestingly, we found that the production of HSL molecules and PQS level were notably reduced in presence of MZF16. Pyoverdine is a siderophore and an important virulence factor in P. aeruginosa, enabling iron sequestration from host to improve the bacterial establishment during the infection. Our results showed that P. pentosaceus MZF16 decreased the pyoverdine level released in the coculture media, suggesting that P. pentosaceus MZF16 may be able to modulate the QS of P. aeruginosa H103. In agreement with our findings, some strains of Pediococcus, i.e P. acidilactici M7 have shown a quorum quenching activity, inhibiting short-chain HSL production, motility, and virulence factors production (elastase, protease, pyocyanin, and biofilm production) of Pseudomonas aeruginosa isolates [42]. Another study on P. acidilactici HW01 bacteriocin showed a decrease of the production of virulence factors such as QS-regulated pyocyanin, protease, and rhamnolipid [10].

Finally, the slow killing assay on C. elegans in vivo model confirmed the anti-virulence effect of P. pentosaceus MZF16 against P. aeruginosa H103. Indeed, the presence of MZF16 has significantly enhanced the survival of C. elegans infected with P. aeruginosa H103. This result is consistent with studies showing that probiotics like P. acidilatici DM9 and P. pentosaceus SDL1409 extended remarkably C. elegans lifespan during P. aeruginosa PA14 infection [9].

5. Conclusions

This study highlights the potential of Pediococcus pentosaceus MZF16 as an effective probiotic against Pseudomonas aeruginosa H103. MZF16 is able to mitigate in vivo and in vitro cytotoxicity, decreases inflammation levels, and pathogen adhesion and invasion in intestinal models, preserves epithelial cells integrity, and modulates quorum-sensing pathways, reducing virulence factor production. These findings underscore the potential of using probiotics as adjunct therapies to combat multidrug-resistant pathogens. Further research is needed to decipher molecular mechanisms involved in these bacterial interactions.