Search Results (11)

Background/Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) remains a critical public health concern due to its multidrug resistance and capacity to form persistent infections, particularly in the context of implanted medical devices. Alternative therapeutic strategies that target bacterial virulence instead of viability are increasingly explored. This study aimed to evaluate the antimicrobial and antivirulence activity of an extract derived from Lacticaseibacillus rhamnosus CRL 2244 against two MRSA strains—USA300 and M86—and to elucidate its effects on bacterial physiology and gene expression under host-mimicking conditions. Methods: Antimicrobial activity was assessed using agar diffusion, MIC, and time-kill assays. Scanning electron microscopy of cells exposed to the extract confirmed decreased cellular density and morphological changes. Phenotypic assays evaluated biofilm formation, staphyloxanthin production, and adhesion to fibronectin. RT-qPCR analyzed transcriptional responses. Viability was assessed in the presence of human serum and type I collagen. Results: The CRL 2244 extract demonstrated bactericidal activity with up to 6-log₁₀ CFU/mL reduction at 1× MIC. In USA300, the extract reduced the expression of hla, lukAB, fnbA, and icaA, correlating with decreased staphyloxanthin levels. In M86, a significant reduction in biofilm formation and repression of lukAB, nucA, and fnbA were observed. Adhesion to fibronectin was impaired in both strains. The extract showed no cytotoxicity in human serum but reduced viability in collagen-enriched conditions. Conclusions: The Lcb. rhamnosus CRL 2244 extract modulates MRSA virulence in a strain-specific manner, targeting key regulatory and structural genes without inducing cytotoxic effects. Full article

Lacticaseibacillus (L.) rhamnosus CRL75 is a lactic acid bacterium (LAB) isolated from local dairy products, demonstrating significant adaptation in skimmed milk (FM75). In this context, CRL75 exhibited high microbial growth (3.63 ± 0.18 log CFU·mL⁻¹), strong acidification (9.20 ± 0.10 g·L⁻¹ lactic acid, and 2.40 ± 0.10 pH units), and increased viscosity in FM75 after 16 h of fermentation. Additionally, this LAB strain produces both capsular polysaccharides (CPS+) and extracellular polysaccharides (EPS75), contributing to a ropy phenotype (>10 cm). The purified EPS75 (70.70 ± 3.25 mg·L⁻¹) displayed low molecular weight (12.7 kDa), with galactose and glucose as its primary monomers in a 4:1 ratio. In aqueous environments, EPS75 exhibited an extended size (147 nm), a random coil structure, and macromolecular aggregation. Furthermore, vibrational spectroscopy confirmed the presence of a neutral EPS with high thermal stability. Additionally, EPS75 exhibited dose-dependent antioxidant activity, effectively reducing metal ions (Fe³⁺, Mo⁶⁺, and Mn⁷⁺) and stabilizing radicals (ABTS^•+, HO^•, O₂^•−, and HOO^•). The biopolymer also demonstrated immunostimulatory and anti-inflammatory effects in RAW 264.7 cells. In vivo assays using Balb/c mice indicated that both EPS75 and FM75 prevented constipation, suggesting their potential as natural and safe agents for constipation-related disorders. Due to its viscosifying and health-promoting attributes, CRL75 offers promising applications for functional dairy products. Full article

Lacticaseibacillus rhamnosus CRL1505 and Lactiplantibacillus plantarum CRL1506 increase the resistance of mice to Gram-negative pathogens infections. In this work, we advanced the characterization of the CRL1505 and CRL1506 immunomodulatory properties by evaluating their effect on the Toll-like receptor 4 (TLR4)-triggered immune response in macrophages. We performed experiments in murine RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) to evaluate the transcriptomic changes induced by lactobacilli. These in vitro experiments were complemented with in vivo studies in mice to determine the effect of CRL1505 and CRL1506 strains on Peyer’s patches and peritoneal macrophages. Microarray transcriptomic studies and qPCR confirmation showed that the CRL1505 and CRL1506 strains modulated the expression of inflammatory cytokines and chemokines as well as adhesion molecules in LPS-challenged RAW macrophages, making the effect of L. rhamnosus CRL1505 more remarkable. Lactobacilli also modulate regulatory factors in macrophages. L. plantarum CRL1506 increased il10 and socs2 while L. rhamnosus CRL1505 upregulated il27, socs1, and socs3 in RAW cells, indicating a strain-specific effect. However, in vivo, both strains induced similar effects. Peyer’s patches and peritoneal macrophages from mice treated with lactobacilli produced higher levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-6, and colony stimulating factor (CSF)-3 after LPS stimulation. This effect would allow improved protection against pathogens. In addition, both lactobacilli equally modulated socs1 and socs2 expressions and IL-10 and IL-27 production in Peyer’s patches macrophages and socs3 and IL-10 in peritoneal cells. Furthermore, lactobacilli reduced the production of IL-1β, IL-12, CSF2, C-C motif chemokine ligand (CCL)-2, and CCL8 in LPS-challenged macrophages. This differential modulation of regulatory and inflammatory factors would allow minimal inflammatory-mediated tissue damage during the generation of the innate immune response. This work provides evidence that L. rhamnosus CRL1505 and L. plantarum CRL1506 modulate macrophages’ TLR4-mediated immunotranscriptomic response, helping to improve protection against Gram-negative bacterial infections. Full article

Orally administered immunomodulatory lactobacilli can stimulate respiratory immunity and enhance the resistance to primary infections with bacterial and viral pathogens. However, the potential beneficial effects of immunomodulatory lactobacilli against respiratory superinfection have not been evaluated. In this work, we showed that the feeding of infant mice with Lacticaseibacillus rhamnosus CRL1505 or Lactiplantibacillus plantarum MPL16 strains can reduce susceptibility to the secondary pneumococcal infection produced after the activation of TLR3 in the respiratory tract or after infection with RVS. The treatment of mice with CRL1505 or MPL16 strains by the oral route improved the production of interferons in the respiratory tract, differentially modulated the balance of pro- and anti-inflammatory cytokines, reduced bacterial replication, and diminished lung damage. Additionally, we demonstrated that orally administered lactobacilli confer longstanding protection against secondary Streptococcus pneumoniae infection and that this effect would be mediated by the stimulation of trained alveolar macrophages. This work contributes to revealing the mechanisms involved in the modulation of the gut–lung axis by beneficial microbes by demonstrating that specific lactobacilli strains, through the stimulation of the common mucosal immune system, would be able to support the development of trained alveolar macrophages that would confer longstanding protection against secondary bacterial challenges produced after a primary inflammatory event in the respiratory mucosa. Full article

Lacticaseibacillus rhamnosus CRL1505 possesses immunomodulatory activities in the gastrointestinal and respiratory tracts when administered orally. Its adhesion to the intestinal mucosa does not condition its beneficial effects. The intranasal administration of L. rhamnosus CRL1505 is more effective than the oral route at modulating immunity in the respiratory tract. Nonetheless, it has not yet been established whether the adherence of the CRL1505 strain to the respiratory mucosa is needed to provide the immune benefits to the host. In this study, we evaluated the role of adhesion to the respiratory mucosa of the mucus-binding factor (mbf) knock-out L. rhamnosus CRL1505 mutant (Δmbf CRL1505) in the context of a Toll-like receptor 3 (TLR3)-triggered innate immunity response. In vitro adhesion studies in porcine bronchial epitheliocytes (PBE cells) indicated that L. rhamnosus Δmbf CRL1505 adhered weakly compared to the wild-type strain. However, in vivo studies in mice demonstrated that the Δmbf CRL1505 also reduced lung damage and modulated cytokine production in the respiratory tract after the activation of TLR3 to a similar extent as the wild-type strain. In addition, the mutant and the wild-type strains modulated the production of cytokines and antiviral factors by alveolar macrophages in the same way. These results suggest that the Mbf protein is partially involved in the ability of L. rhamnosus CRL1505 to adhere to the respiratory epithelium, but the protein is not necessary for the CRL1505 strain to exert its immunomodulatory beneficial effects. These findings are a step forward in the understanding of molecular interactions that mediate the beneficial effects of nasally administered probiotics. Full article

Lacticaseibacillus rhamnosus CRL1505 beneficially modulates the inflammation-coagulation response during respiratory viral infections. This study evaluated the capacity of the peptidoglycan obtained from the CRL1505 strain (PG-Lr1505) to modulate the immuno-coagulative response triggered by the viral pathogen-associated molecular pattern poly(I:C) in the respiratory tract. Adult BALB/c mice were nasally treated with PG-Lr1505 for two days. Treated and untreated control mice were then nasally challenged with poly(I:C). Mice received three doses of poly(I:C) with a 24 h rest period between each administration. The immuno-coagulative response was studied after the last administration of poly(I:C). The challenge with poly(I:C) significantly increased blood and respiratory pro-inflammatory mediators, decreased prothrombin activity (PT), and increased von Willebrand factor (vWF) levels in plasma. Furthermore, tissue factor (TF), tissue factor pathway inhibitor (TFPI), and thrombomodulin (TM) expressions were increased in the lungs. PG-Lr1505-treated mice showed significant modulation of hemostatic parameters in plasma (PT in %, Control = 71.3 ± 3.8, PG-Lr1505 = 94.0 ± 4.0, p < 0.01) and lungs. Moreover, PG-Lr1505-treated mice demonstrated reduced TF in F4/80 cells from lungs, higher pro-inflammatory mediators, and increased IL-10 compared to poly(I:C) control mice (IL-10 in pg/mL, Control = 379.1 ± 12.1, PG-Lr1505 = 483.9 ± 11.3, p < 0.0001). These changes induced by PG-Lr1505 correlated with a significant reduction in lung tissue damage. Complementary in vitro studies using Raw 264.7 cells confirmed the beneficial effect of PG-Lr1505 on poly(I:C)-induced inflammation, since increased IL-10 expression, as well as reduced damage, production of inflammatory mediators, and hemostatic parameter expressions were observed. In addition, protease-activated receptor-1 (PAR1) activation in lungs and Raw 264.7 cells was observed after TLR3 stimulation, which was differentially modulated by PG-Lr1505. The peptidoglycan from L. rhamnosus CRL1505 is able to regulate inflammation, the procoagulant state, and PAR1 activation in mice and macrophages in the context of the activation of TLR3 signaling pathways, contributing to a beneficial modulation of inflammation-hemostasis crosstalk. Full article

The design of probiotic hygiene products for daily use is considered an adequate alternative for the restoration of the vaginal microbiome, maintaining health, and/or preventing infections of the female urogenital tract. Most of these probiotic products are available on the world market, but their efficacy and safety are not sufficiently documented. One of the requirements to transfer novel probiotic formulas/products to the productive sector is to demonstrate their innocuity and the absence of adverse or collateral effects on the host, mainly assayed in experimental models. The inclusion of beneficial lactobacilli in nanofibers by electrospinning technique has shown promising application possibilities, and the immobilization of Lacticaseibacillus rhamnosus CRL1332 in nanofibers with and without bioprotective substances and their characterization were previously performed by our research group. In this work, the safety of the intravaginal (i.va.) administration of these functional nanofibers in a murine experimental model was evaluated. L. rhamnosus CRL1332 immobilized in different nanofibers was intravaginally inoculated into mice (seven daily doses). Vaginal washes were taken for microbiological (cultivable lactobacilli) and cytological techniques, and the vagina was used for histological and morphological-ultrastructural evaluation. Our results demonstrated that the intravaginal administration of L. rhamnosus CRL1332 immobilized in nanofibers is safe in murine models, given the absence of an inflammatory response at the cytological and histological levels, with minor modifications at the ultrastructural level, and also related to the normal cultivable vaginal microbiota. On the other hand, the number of cultivable lactobacilli increased in the vagina of mice receiving L. rhamnosus CRL1332 nanofibers. The results indicate the safety of lactobacilli-functional nanofibers and support their inclusion in the design of vaginal probiotic products to prevent/treat urogenital infections and reconstitute the women’s vaginal microbiota. Full article

Probiotic formula for the vaginal tract must contain high numbers of viable beneficial bacteria that maintain their characteristics during the production and further storage, in order to exert the claimed probiotic effect. Four probiotic strains—Lactobacillus gasseri CRL1320, Limosilactobacillus reuteri CRL1324, Ligilactobacillus salivarius CRL1328 and Lacticaseibacillus rhamnosus CRL1332—originally isolated from vagina of healthy women exhibiting beneficial properties were evaluated. Therefore, the optimization of growth parameters and dry biomass production with high viability while preserving probiotic features of strains is a great challenge. Different growth conditions in MRS medium were set up in a laboratory bioreactor; two initial pH and agitation were recorded speeds during static or controlled fermentations. Production conditions of 37 °C with controlled pH 6.5 and 5.5-MRS with 150 and 75 rpm stirring speeds were used for CRL1329 and CRL1332 strains, respectively, while static and free pH MRS for CRL1324 and CRL1328 probiotics allowed us to obtain maximal cell viability counts. However, during 12 h of fermentation, biomass yields of 19.3, 16.2 and 15.2 g/L were achieved when CRL1329, CRL1328 and CRL1324 probiotic strains were grown in static and free pH MRS. The highest biomass yield for CRL1332 strain was produced under controlled MRS-initial pH 6.5 and 75 rpm fermentation conditions. To preserve probiotic high biomass viability, freeze-drying was carried out in the presence of different cryoprotective agents. Thus, the highest viable numbers (10.9–11.8 log CFU/g) with survival rates between 91.3 and 95.6% were attained in the presence of 10% trehalose (L. reuteri and L. salivarius), lactose (L. rhammosus) and lactose + trehalose + sucrose mix (L. gasseri). When stability during post-freeze-drying storage was evaluated, probiotic strains showed a remarkably higher viability recovery when stored at 4 °C than at 25 °C for 12 and 3 months, respectively. In addition, surface characteristics of vaginal probiotics were affected to different extents during storage depending on the strain, protective agent and storage time/temperature. Critical factors for growth conditions, drying process and storage stability of probiotic lactobacilli strains were optimized in view to preserve cell high viability and surface features for the design of vaginal probiotic formula. Full article

Orally administered Lacticaseibacillus rhamnosus CRL1505 enhances respiratory immunity, providing protection against respiratory viruses and Streptococcus pneumoniae. However, the capacity of the CRL1505 strain to improve respiratory immunity against Gram-negative bacterial infections has not been evaluated before. The aim of this work was to evaluate whether the Lcb. rhamnosus CRL1505 was able to beneficially regulate the respiratory innate immune response and enhance the resistance to hypermucoviscous KPC-2-producing Klebsiella pneumoniae of the sequence type 25 (ST25). BALB/c mice were treated with the CRL1505 strain via the oral route and then nasally challenged with K. pneumoniae ST25 strains LABACER 01 or LABACER 27. Bacterial cell counts, lung injuries and the respiratory and systemic innate immune responses were evaluated after the bacterial infection. The results showed that K. pneumoniae ST25 strains increased the levels of TNF-α, IL-1β, IL-6, IFN-γ, IL-17, KC and MPC-1 in the respiratory tract and blood, as well as the numbers of BAL neutrophils and macrophages. Mice treated with Lcb. rhamnosus CRL1505 had significantly lower K. pneumoniae counts in their lungs, as well as reduced levels of inflammatory cells, cytokines and chemokines in the respiratory tract and blood when compared to infected controls. Furthermore, higher levels of the regulatory cytokines IL-10 and IL-27 were found in the respiratory tract and blood of CRL1505-treated mice than controls. These results suggest that the ability of Lcb. rhamnosus CRL1505 to help with the control of detrimental inflammation in lungs during K. pneumoniae infection would be a key feature to improve the resistance to this pathogen. Although further mechanistic studies are necessary, Lcb. rhamnosus CRL1505 can be proposed as a candidate to improve patients’ protection against hypermucoviscous KPC-2-producing strains belonging to the ST25, which is endemic in the hospitals of our region. Full article

Both viable and non-viable orally administered Lacticaseibacillus rhamnosus CRL1505 modulate immunity in local (intestine) and distal (respiratory) mucosal sites. So, intestinal adhesion and colonization are not necessary for this probiotic strain to exert its immunomodulatory effects. In this work, a mucus-binding factor knockout CRL1505 strain (ΔmbfCRL1505) was obtained and the lack of binding ability to both intestinal epithelial cells and mucin was demonstrated in vitro. In addition, two sets of in vivo experiments in 6-week-old Balb/c mice were performed to evaluate ΔmbfCRL1505 immunomodulatory activities. (A) Orally administered ΔmbfCRL1505 prior to intraperitoneal injection of the Toll-like receptor 3 (TLR3) agonist poly(I:C) significantly reduced intraepithelial lymphocytes (CD3⁺NK1.1⁺CD8αα⁺) and pro-inflammatory mediators (TNF-α, IL-6 and IL-15) in the intestinal mucosa. (B) Orally administered ΔmbfCRL1505 prior to nasal stimulation with poly(I:C) significantly decreased the levels of the biochemical markers of lung tissue damage. In addition, reduced recruitment of neutrophils and levels of pro-inflammatory mediators (TNF-α, IL-6 and IL-8) as well as increased IFN-β and IFN-γ in the respiratory mucosa were observed in ΔmbfCRL1505-treated mice when compared to untreated control mice. The immunological changes induced by the ΔmbfCRL1505 strain were not different from those observed for the wild-type CRL1505 strain. Although it is generally accepted that the expression of adhesion factors is necessary for immunobiotics to induce their beneficial effects, it was demonstrated here that the mbf protein is not required for L. rhamnosus CRL1505 to exert its immunomodulatory activities in local and distal mucosal sites. These results are a step forward towards understanding the mechanisms involved in the immunomodulatory capabilities of L. rhamnosus CRL1505. Full article

Previously, we demonstrated that the non-viable strain Lacticaseibacillus rhamnosus CRL1505 (NV1505) or its purified peptidoglycan (PG1505) differentially modulated the respiratory innate antiviral immune response triggered by Toll-like receptor (TLR)-3 activation in infant mice, improving the resistance to primary respiratory syncytial virus (RSV) infection and secondary pneumococcal pneumonia. In this work, we evaluated the effect of other non-viable L. rhamnosus strains and their peptidoglycans on the respiratory immune response and their impact on primary and secondary respiratory infections. In addition, the duration of the protective effect induced by NV1505 and PG1505 as well as their ability to protect against different Streptococcus pneumoniae serotypes were evaluated. Our results showed that among the five selected L. rhamnosus strains (CRL1505, CRL498, CRL576, UCO25A and IBL027), NV1505 and NVIBL027 improved the protection against viral and pneumococcal infections by modulating the respiratory immune response. Of note, only the PG1505 presented immunomodulatory activities when compared with the other purified peptidoglycans. Studies on alveolar macrophages showed that NV1505 and PG1505 differentially modulated the expression of IL-6, IFN-γ, IFN-β, TNF-α, OAS1, RNAseL and IL-27 genes in response to RSV infection, and IL-6, IFN-γ, IL-1β, TNF-α, CCL2, CXCL2, CXCL10 and IL-27 in response to pneumococcal challenge. Furthermore, we demonstrated that NV1505 and PG1505 treatments protected mice against secondary pneumococcal pneumonia produced by different serotypes of S. pneumoniae until 30 days after stimulation with poly(I:C). This work advances the characterization of the protective effect of NV1505 and PG1505 by demonstrating that they increase resistance against the pneumococcal serotypes 3, 6B, 14 and 19F, with an effect that lasts up to 30 days after the primary viral inflammation. The results also confirm that the immunomodulatory properties of NV1505 and PG1505 are unique and are not shared by other members of this species, and suggest the existence of a capacity to stimulate trained immunity in alveolar macrophages. Full article