Molecular and functional insights into probiotic interference with Salmonella typhimurium invA virulence gene expression

doi:10.21203/rs.3.rs-8276466/v1

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Molecular and functional insights into probiotic interference with Salmonella typhimurium invA virulence gene expression

https://doi.org/10.21203/rs.3.rs-8276466/v1

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Background

Salmonella enterica serovar Typhimurium (S. typhimurium) is a major foodborne pathogen increasingly complicated by antimicrobial resistance (AMR). The invA gene is a conserved virulence determinant essential for host epithelial invasion and widely used as a molecular marker. This study investigated the impact of Lactobacillus acidophilus and Bifidobacterium longum supplementation on S. typhimurium colonization and invA gene expression in a murine model.

Methods

Eighty- four poultry-derived samples (faeces, eggs, chicken meat) were screened for S. typhimurium isolation using selective enrichment and biochemical asssays. Forty female albino mice were assigned to five groups: negative control (administered phosphate-buffered saline only), positive control (S. typhimurium only), and three probiotic- treatment groups at low (250 mg/kg), medium (500 mg/kg), and high (750 mg/kg) doses. Probiotics were administered for 14 days; infection occurred on Day 7. Clinical signs were monitored, and liver and kidney tissues were analysed. Quantitative PCR (qPCR) targeting invA, normalized to GapA, evaluated virulence gene expression using the 2^−ΔΔCt method.

Results

Fifteen isolates of S. typhimurium (17.86%) were recovered, with highest prevalence in fecal samples (53.33%). Probiotic-treated mice maintained baseline activity and appearance, in contrast to the infected untreated mice showing lethargy, rough coats and heavy fecal discharge. Probiotic supplementation reduced bacterial load in fecal and intestinal samples, with the highest reduction at 750 mg/kg. qPCR revealed dose-dependent suppression of invA gene expression, with ΔCt ranging from – 8.05 (positive controls) to + 7.95 (highest- dose group).

Conclusion

Probiotic supplementation attenuates S. typhimurium virulence by suppressing invA expression in vivo, highlighting its potential application as a prophylactic strategy against Salmonellosis. This findings emphasize probiotic-based interventions as a promising adjunct approaches in AMR mitigation.

General Microbiology

Salmonella typhimurium

invA

probiotics

qPCR

virulence regulation

AMR

mice

Foodborne salmonellosis remains a global public health concern, responsible for over 94 million morbidity and 150,000 mortality per annum (Galán-Relaño et al., 2023). Salmonella enterica serovar Typhimurium (S. typhimurium) is one of the most prevalent serovars identified to causing both humans and animals disease. Over 2500 Salmonella serotypes have been identified to date, with over 60% grouped under Salmonella enterica, accounting for the majority of human infections (Crump et al., 2015).

High prevalence in food and water systems in Sub-Saharan Africa contributes significantly to the regional burden of gastroenteritis and invasive non-typhoidal salmonellosis, especially among immunocompromised individuals (Mahon and Fields, 2016). Clinical symptoms of Salmonella infections typically appear when the bacterial load reaches approximately 10⁷ − 10⁸ CFU, manifesting mainly as self-limiting gastroenteritis (Arya et al., 2017). Nonetheless, it can also cause severe extra-intestinal infections, particularly in hosts with compromised immune systems (Feasey et al., 2012).

According to the Centers for Disease Control and Preventions (CDC, 2021), salmonellosis is recognized as a clinical infection characterized initially by high fever (often exceeding 40°C), malaise and anorexia, which subsequently progress to diarrhea, vomiting, and abdominal discomfort. Clinical manifestations typically appear within 12 to 36 hours post- exposure and may continue for two to seven days. Also, in severe cases, dehydration develops as a secondary complications. The severity and duration of clinical symptoms depend on the number of infecting organisms, the immune status of the animal, and the presence of concurrent diseases.

The pathogen exhibits remarkable adaptability and invades host intestinal epithelial cells via the Type III secretion system (T3SS) to secrete flagellin, a key virulence factor that facilitates colonization by outcompeting commensal gut bacteria and inducting host inflammatory responses, in which invA plays a critical role (Lamichhane et al., 2024). Diagnosis routinely includes culture on selective media (Salmonella-Shigella Agar or Xylose Lysine Deoxycholate Agar), and molecular assay such as Real-time PCR (RT-PCR) (Odumeru and León-Velarde, 2012; Moosavy et al., 2015).

Real-time PCR (RT-PCR) became the most preferred diagnostic tool in food microbiology due to its high sensitivity and rapid turnaround time (Day et al., 2009). The assay detects amplification in real time using fluorescence from DNA-binding dyes or hybridization probes (Hyeon et al., 2011). Several PCR assays have been developed targeting Salmonella- specific genes, including 16S rRNA, agfA, and viaB, and virulence plasmids, with the invA gene serving as the gold-standard marker for species identification (Malorny et al., 2004).

However, increasing antimicrobial resistance (MDR and XDR phenotypes) has limited therapeutic options (Shu et al., 2015), highlighting the need for safe, accessible alternatives (Elbediwi et al., 2021; Wei et al., 2023). Probiotics such as Lactobacillus acidophilus and Bifidobacterium longum possess immunomodulatory and pathogen-exclusion properties (Saini et al., 2009; Franz et al., 2010; Zhang et al., 2022), yet molecular level in vivo evidence of their influence on S. typhimurium virulence especially invA suppression remains limited (Medellín‑Peña et al., 2024).

This study evaluates the impact of probiotic supplementation (L. acidophilus and B. longum) on S. typhimurium colonization, and invA gene expression in mice using a validated qPCR approach.

2.1 Ethics Considerations

Ethical approval was obtained from the Osun State University Ethical Review Committee, College of Health Sciences (UNIOSUNHREC 2025 /002B).

Every effort was made to minimize discomfort to animals throughout the study. The animals were maintained in sanitized, well-ventilated cages with unrestricted access to food and safe water ad libitum. All sacrifices were performed via physical methods on the anaesthetized animals, to ensure that chemical agents would not interfere with results. Hence, cervical dislocation involving a rapid, forceful separation of the first cervical vertebra from the skull was considered (American Veterinary Medical Association). This method was chosen to minimize distress and is consistent with internationally accepted protocols for small rodents.

Informed consent was obtained from poultry vendors before sample purchase. Sample collection followed national biosafety and ethical guidelines; no additional permits were required for market-purchased poultry products as all samples were sourced from legally registered markets, and collection complied with national guidelines for handling food-origin materials. No endangered or protected species were involved.

2.2 Study Location

This study was conducted at the Department of Microbiology Laboratory and Animal house, Osun State University, Osogbo, Nigeria.

2.3 Sample Collection and Processing

Eighty-four (84) poultry-derived samples (28 feces, chicken meat, and egg samples each) were collected from five major markets in Osogbo metropolis: Sasa, Oluode, Igbona, Alekuwodo and Oja-Oba. Samples were placed in sterile zip-lock bags, transported on ice and processed within 6 hours of collection. Each specimen was pre-enriched by suspending 1 g in 9mL of buffered peptone water (BPW) and incubating at 37^oC for 18 - 24 hours. Thereafter, 0.1mL Aliquots were transferred into 10mL of Rappaport-Vassiliadis broth for selective enrichment at 42°C for 24 hours (Bawa et al., 2020). Loopfuls from the enriched broth were streaked on Salmonella-Shigella Agar (SSA) and Xylose Lysine Desoxycholate agar (XLD agar) and incubated at 37^oC for 24 hours. Typical colonies (red or colourless with black-centers) were sub-cultured, stocked and identified biochemically the procedures of Oliveira et al. (2003) including catalase, oxidase, H₂S production on TSI agar, fermentation tests, indole, motility test, methyl red and urease tests (Andrews et al., 2018).

Prevalence was calculated as:

Prevalence (P) = Number of positive isolates / Total samples collected x 100.

2.4 In vivo Experimental Design

2.4.1 Animal preparation

Forty (40) female albino mice aged four weeks were obtained from a certified breeder (high-sanitary-status farms from mothers serologically negative to Salmonella). The mice were acclimatized for two weeks under controlled laboratory conditions (25°C temperature; 60 to 65% humidity; 12 hours light/dark cycle). The animals were provided standard feed and water ad libitum and monitored daily for health and behavioural changes.

2.4.2 Grouping and treatments

Mice were randomly divided into 5 experimental groups (n = 8 per group):

Group I: Negative control – received phosphate-buffered saline only;
Group II: Positive control – challenged with Salmonella typhimurium only;
Group III: Low probiotics dose (250 mg/Kg) + infection;
Group IV: Medium probiotics dose (500 mg/Kg) + infection;
Group V: High probiotics dose (750 mg/Kg) + infection.

Each mouse received oral gavage (0.2mL per dose) daily from Day 0 –14. Infection occurred on Day 7, all groups except Group I were orally inoculated with S. typhimurium suspension (0.2mL; 4 × 10³ CFU/mouse).

2.4.3 Clinical monitoring

Probiotic administration continued for another 7 days post-infection. Animals were observed daily for changes in activity, clinical symptoms, and feed intake. On Day 15, mice were humanely sacrificed, and blood, liver and kidney tissues were harvested for microbiological, and molecular analysis (Chen et al., 2023).

2.5 Probiotic Preparation

Commercial probiotic formulations containing Lactobacillus acidophilus ATCC 4356, and Bifidobacterium longum BB536 (Nature Field, USA) were used. The probiotic blend was freshly reconstituted in sterile normal saline prior to each administration. Probiotic and pathogen inoculant were prepared following Chen et al. (2023), ensuring viable counts of 4×10³ CFU/mouse for the bacterial challenge.

2.6 Quantitative PCR (qPCR) Analysis

Quantitative real-time PCR (qPCR) were employed to detect and quantify the expression of the invA gene using synthesized primers (Forward primers: 5’- TTGTCACCGTGGTCCAGTTT-3’ and Reverse primer: 3’- TACCGGGCATACCATCCAGA-5’) virulence gene of S. typhimurium in liver and kidney tissues of infected mice. The GapA was used as the internal control to normalize expression levels (Siala et al., 2017). All reagents, methods and equipment were obtained from Applied Biosystems.

2.6.1 DNA extraction

Approximately, 100 mg of kidney and liver tissue was homogenized in 1 mL Tris EDTA (TE) buffer (10 Mm Tris-HCl, 1 mM EDTA, pH 8.0). A 520 μL aliquot was transferred into sterile microtubes, and 15 μL of 20% SDS with 3 μL of Proteinase K (20 mg/mL) was added. After incubation at 37 ºC for 1 hour, 100 μL of 5 M NaCl and 80 μL of a 10% CTAB solution in 0.7 M NaCl were added and votexed. The suspension was incubated for 10 minutes at 65ºC and cooled for 15 minutes. DNA was extracted with chloroform: isoamyl alcohol (24:1), precipitated with isopropanol (1: 0.6) at –20ºC overnight and pelleted by centrifugation. The DNA was washed with 70% ethanol, air-dried, and dissolved in 50 μL of TE buffer (Siala et al., 2017).

2.6.2 RNA treatment

To remove residual DNA, 20 ng of RNA was treated with NEB DNase 1 (M0303) according to manufacturer’s guidelines. Following incubation at 37°C for 10 minutes, DNase was inactivated by adding 1 µL of 0.5 M EDTA and heating at 75°C for 10 minutes. Treated samples were stored at -20^oC until use (Siala et al., 2017).

2.6.3 Gene quantification

Expression levels of the invA virulence gene were quantified using Luna® Universal qPCR Master Mix Protocol (M3003). Reaction mixtures (20 µL) contained 10 µL master mix, 0.5 µL each of forward primer and reverse primer, 0.5 - 1 µL reverse transcriptase and 2 µL of RNA template.

Thermal cycling conditions were: Reverse transcriptase: 42^oC for 10 minutes, initial denaturation: 95°C for 60 seconds, 40-45 cycles of denaturation at 95°C for 15 seconds, extension and plate reading at 60°C for 30 seconds, and Final extension: 72^oC for 10 minutes.

Amplification was performed on a Bio-Rad CFX96 Real-Time PCR system, and specificity confirmed by melt-curve analysis. GapA served as the reference gene for normalization.

Expression changes were calculated using:

∆Ct = Ct of Target gene (Ct_{inv A}) – Ct of the Reference gene (Ct_{Gap A})
∆∆Ct = ∆Ct (sample) - ∆Ct (calibrator)
Fold change = 2^–∆∆Ct

Positive detection threshold: Ct ≤ 35 (Heymans et al., 2018; Xue et al., 2025).

3.1 Prevalence of S. typhimurium isolated

A total of 15 (fifteen) S. typhimurium were recovered, from 84 poultry- derived samples with prevalence (P) of 17.86%. The highest number of S. typhimurium (n = 8; P = 53.33%) was from faecal samples, followed by chicken meat samples (n = 5; P = 33.33%); while egg samples yielded the least (n = 2; P = 13.33%). The study identified fourteen Gram-negative, rod-shaped and three Gram positive cocci isolates that formed distinctive colonies on selective media (Plate 1 and 2). These isolates showed colonies with characteristic black centers on Salmonella-Shigella Agar (SSA), and on Xylose Lysine Deoxycholate (XLD) agar, the colonies were red or pink, also presenting with black centers (Plate 1 and 2).

(a)Plate 1: Images of Salmonella on SSA Agar (b) Plate 2: Images of Salmonella on XLD Agar

Examining the morphology more closely, 47% of the isolates exhibited an irregular shape, while 52% were circular. A smooth and shiny surface was observed in 58% of the isolates, while 47% displayed a rough and shiny appearance. The colonies also exhibited diverse edge characteristics, including undulate, entire, curled, filamentous, and rhizoid forms. The predicted accuracy of the bacterial species identification ranged between ≥ 80.20% and ≤ 99.0%.

3.2 Animal experiment

a. Clinical observations

Probiotics treated mice maintained better physical appearance and activity levels as it show gradual improvement in activity and appearance over the 14 days period compared to the infected untreated group (positive control), appeared weak, less active, and show ruffled fur, lethargy and heavy fecal droppings and the negative control group maintained a normal activity level.

b. Re-isolation of Salmonella from the infected mice

Following experimental infection and subsequent re-isolation from the faecal samples of infected mice, the presence of S. typhimurium was confirmed. A total of 15 isolates were identified, comprising 13 (87%) Gram-negative rods and 2 (13.3%) Gram-positive cocci. Further characterization through biochemical tests showed that all isolates were catalase-positive, urease-positive, citrate-positive, and oxidase-negative. These result’s consistency with the expected morphology of S. typhimurium, and strongly indicated a successful infection. In contrast, the majority of isolates recovered from infected mice that had received probiotic treatment were identified as Gram-positive rods.

c. Effect of probiotics supplementation on Salmonella typhimurium colonization in mice

The Salmonella load counts in faecal samples, and intestinal tissues was measured to assess the colonization levels. Mice infected with Salmonella typhimurium showed significantly higher bacterial counts faecal (5.2 x 10⁶ CFU/mL) and intestinal tissues (4.8 x 10⁶ CFU/mL) samples compared to the negative control group (not detected) (Fig. 4.3). However, probiotic-treated groups exhibited a reduction in bacterial load count, with the highest decrease observed in the group receiving the optimal probiotic dosage for faecal (1.8 × 10⁶ CFU/mL) and, intestinal tissues (1.5 × 10⁶ CFU/mL) (Table 1).

Table 1: Salmonella load counts (CFU/ml) in treatment groups

d. Determination of Optimal Probiotic Dosage

To determine the most effective probiotic dose, bacterial reduction were analysed across different dosage groups. The high-dose probiotic group exhibited the most significant decrease (p < 0.05), in bacterial load for faecal (1.8 × 10⁶ CFU/mL) and intestinal tissues (1.5 × 10⁶ CFU/mL) (Fig. 1).

e. Expression of invA virulence gene

Infected mice showed high expression levels, whereas probiotic treatment significantly reduced gene expression (Ct = 20.80), with the high-dose probiotic group showing the most reduction (Ct = 33.41). The positive control group (Group B) recorded the highest invA gene expression, as indicated by very low ΔCt values of – 8.05 in tissue samples. This confirmed strong pathogen colonization and effective infection. In contrast, the negative control group (Group A) had no detectable amplification of the invA gene, with Ct values undetermined or beyond detection, indicating absence of infection (Fig. 2).

(a.) (b.)

For the probiotic-treated groups, a progressive reduction in invA expression was reported. Group C recorded mild expression with ΔCt values of – 0.61, reflecting partial suppression of invA. Group D showed lower expression with a ΔCt of + 3.42, while Group E, which received the highest probiotic dose, exhibited the lowest detected expression, with a ΔCt of + 7.95 and a late Ct value of 33.41 (Table 2).

Table 2: Quantitative PCR analysis of invA Gene expression

S. typimurium was isolated from poultry-derived samples (egg, meat and poultry faecal droppings), with an overall prevalence of 17.86% (15/84). This agree with Olorunjuwon et al., (2016), who reported varying Salmonella prevalence from healthy animals and animal products in Nigeria; 6.4% in chicken, 14.1% in raw meat, 39% in slaughtered animals and 2% in raw beef respectively. In the present study, faecal samples showed the highest detection rate (n = 8; 53.33%), followed by meat samples (n = 5; 33.33%); and eggs (n = 2; 13.33%). The low recovery from eggs aligns with the report of Chousalkar and Robert (2012), who recorded minimal Salmonella contamination on eggshells and internal contents.

Experimental infection in mice produced classical salmonellosis symptoms such as diarrhoea, weight loss, and anorexia, consistent with earlier findings by Feary and Hassel (2006), and Habrun et al. (2006). In contrast, probiotic- supplemented mice maintained normal weight and feeding behaviour, supporting the findings of Bibi et al. (2024), who reported that probiotics consumption enhance gastrointestinal stability. Similarly, Remes-Troche et al. (2020) reported that L. acidophilus alleviated digestive symptoms in infected mice, while Kim et al. (2021) and Zhang et al. (2022) documented that L. acidophilus and B. longum mitigated intestinal inflammation in murine colitis models via increased acetate and lactate production.

Gene expression analysis revealed a progressive, dose-dependent suppression of invA expression in probiotic-treated groups. The positive control exhibited the lowest ΔCt and highest fold change values, indicating intense pathogen colonization, while the negative control showed no amplification, validating assay specificity. Probiotic treatment significantly increased ΔCt values, with the highest dose group (Group E) demonstrating the strongest inhibition of invA gene expression. This suggests that probiotic supplementation down regulates Salmonella virulence in a concentration-dependent manner.

These results are consistent with earlier studies demonstrating that probiotics attenuate virulence gene expression in Salmonella and related pathogens. Liu et al. (2022) and Chen et al. (2023) reported that L. johnsonii and other LAB strains reduced S. typhimurium virulence by modulating oxidative stress and inflammatory responses. Likewise, Lee et al. (2016) observed that LAB strains significantly decreased invA transcription and host inflammation during epithelial infection. Furthermore, Adetoye et al. (2018) demonstrated anti-Salmonella activity of LAB isolates from cattle, while Wang et al. (2021) and Yan and Polk (2019) provided evidence that probiotics could enhance host immunity and indirectly suppress pathogen colonization and virulence. Overall, these findings reinforce the therapeutic promise of probiotic supplementation as a natural strategy for mitigating Salmonella pathogenicity and antibiotic resistance.

Limitations include the use of a single infection dose, limited molecular targets (only invA gene), and absence of immunological profiling (e.g., cytokine responses). Future work should bridge gaps on transcriptomic profiling and multi-gene virulence analysis.

The findings from this study demonstrate that probiotic supplementation using L. acidophilus and B. longum effectively reduced S. typhimurium colonization in mice and significantly down-regulated the expression of the virulence gene invA validating the specificity of the infection model and molecular detection approach. Importantly, mice treated with probiotics demonstrated a dose-dependent reduction in invA expression, with the highest probiotic blend group showing the most pronounced suppression.

This study also provides molecular evidence that probiotic supplementation can modulate bacterial virulence, potentially reducing the severity of salmonellosis. The study highlights the practical potential of probiotics as complementary interventions to antibiotics for managing salmonellosis, contributing to the broader goal of reducing antimicrobial resistance risks associated with foodborne pathogens.

Ethical approval and consent to participate

All animal procedures were approved by the Osun State University Ethical Review Committee (UNIOSUNHREC 2025/002B). Animals weren’t anesthetized but euthanized via physical method (cervical dislocation). This method was chosen to minimize distress and is consistent with internationally accepted protocols for small rodents.

Informed consent

was obtained from poultry product vendors prior to sample collection. Also, market-based sample collection required no additional permits under Nigerian biosafety regulations, as all samples were sourced from legally registered markets, and collection complied with national guidelines for handling food-origin materials. No endangered or protected species were involved.

Clinical Trial Number

Not applicable.

Conflict of Interest

The authors declare that there is no conflict of interest.

Consent to Publication

Not applicable.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not- for- profit sectors.

Author Contributions

AUA conceptualized and designed the study, performed laboratory experiments, analyzed data and drafted the manuscript.

Acknowledgements

The authors gratefully acknowledge the Department of Microbiology, Osun State University, Osogbo, Nigeria, Multidisciplinary Research Laboratory, Osun State University, Osogbo, Nigeria and Inqaba Biotech, Ibadan, Nigeria, for providing laboratory facilities and support throughout the study.

Availability of data and materials

All datasets generated or analysed during this study are included in this article and its supplementary files. Raw qPCR Ct values and microbiological datasets are available from the corresponding author on request.

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Plate 1 and 2 are available in the Supplementary Files section.

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Molecular and functional insights into probiotic interference with Salmonella typhimurium invA virulence gene expression

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Abstract

Background

Methods

Results

Conclusion

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1. Introduction

2. Methods

3. Results

3.1 Prevalence of S. typhimurium isolated

4. Discussion

5. Conclusion

Declarations

Ethical approval and consent to participate

Clinical Trial Number

Conflict of Interest

Funding

Author Contributions

Acknowledgements

Availability of data and materials

References

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Additional Declarations

Supplementary Files

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