Antagonistic Effects of Corynebacterium pseudodiphtheriticum 090104 on Respiratory Pathogens.

In previous studies, it was demonstrated that Corynebacterium pseudodiphtheriticum 090104, isolated from the human nasopharynx, modulates respiratory immunity, improving protection against infections. Here, the antagonistic effect of the 090104 strain on respiratory pathogens, including Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii, was explored. In a series of in vitro studies, the capacity of C. pseudodiphtheriticum 090104, its bacterium-like particles, and its culture supernatants to coaggregate, inhibit the growth, and change the virulent phenotype of pathogenic bacteria was evaluated. The results showed that the 090104 strain was able to exert a bacteriostatic effect on K. pneumoniae and S. pneumoniae growth. In addition, C. pseudodiphtheriticum 090104 coaggregated, inhibited biofilm formation, and induced phenotypic changes in all the respiratory pathogens evaluated. In conclusion, this work demonstrated that, in addition to its beneficial effects exerted by host-microbe interactions, C. pseudodiphtheriticum 090104 can enhance protection against respiratory pathogens through its microbe-microbe interactions. The mechanisms involved in such interactions should be evaluated in future research.

Comprehensive characterization of γ-aminobutyric acid (GABA) production by Levilactobacillus brevis CRL 2013: insights from physiology, genomics, and proteomics.

Introduction: Levilactobacillus brevis CRL 2013, a plant-derived lactic acid bacterium (LAB) with immunomodulatory properties, has emerged as an efficient producer of γ-aminobutyric acid (GABA). Notably, not all LAB possess the ability to produce GABA, highlighting the importance of specific genetic and environmental conditions for GABA synthesis. This study aimed to elucidate the intriguing GABA-producing machinery of L. brevis CRL 2013 and support its potential for safe application through comprehensive genome analysis. Methods: A comprehensive genome analysis of L. brevis CRL 2013 was performed to identify the presence of antibiotic resistance genes, virulence markers, and genes associated with the glutamate decarboxylase system, which is essential for GABA biosynthesis. Then, an optimized chemically defined culture medium (CDM) was supplemented with monosodium glutamate (MSG) and yeast extract (YE) to analyze their influence on GABA production. Proteomic and transcriptional analyses were conducted to assess changes in protein and gene expression related to GABA production. Results: The absence of antibiotic resistance genes and virulence markers in the genome of L. brevis CRL 2013 supports its safety for potential probiotic applications. Genes encoding the glutamate decarboxylase system, including two gad genes (gadA and gadB) and the glutamate antiporter gene (gadC), were identified. The gadB gene is located adjacent to gadC, while gadA resides separately on the chromosome. The transcriptional regulator gadR was found upstream of gadC, with transcriptional analyses demonstrating cotranscription of gadR with gadC. Although MSG supplementation alone did not activate GABA synthesis, the addition of YE significantly enhanced GABA production in the optimized CDM containing glutamate. Proteomic analysis revealed minimal differences between MSG-supplemented and non-supplemented CDM cultures, whereas YE supplementation resulted in significant proteomic changes, including upregulation of GadB. Transcriptional analysis confirmed increased expression of gadB and gadR upon YE supplementation, supporting its role in activating GABA production. Conclusion: These findings provide valuable insights into the influence of nutrient composition on GABA production. Furthermore, they unveil the potential of L. brevis CRL 2013 as a safe, nonpathogenic strain with valuable biotechnological traits which can be further leveraged for its probiotic potential in the food industry.

Bacterium-like Particles from Corynebacterium pseudodiphtheriticum as Mucosal Adjuvant for the Development of Pneumococcal Vaccines.

Previously, it was shown that intranasally (i.n.) administered Corynebacterium pseudodiphtheriticum 090104 (Cp) or CP-derived bacterium-like particles (BLPs) improve the immunogenicity of the pneumococcal conjugate vaccine (PCV). This work aimed to deepen the characterization of the adjuvant properties of Cp and CP-derived BLPs for their use in the development of pneumococcal vaccines. The ability of Cp and CP-derived BLPs to improve both the humoral and cellular specific immune responses induced by i.n. administered polysaccharide-based commercial pneumococcal vaccine (Pneumovax 23®) and the chimeric recombinant PSPF (PsaA-Spr1875-PspA-FliC) protein was evaluated, as well as the protection against Streptococcus pneumoniae infection in infant mice. Additionally, whether the immunization protocols, including Cp and CP-derived BLPs, together with the pneumococcal vaccines can enhance the resistance to secondary pneumococcal pneumonia induced after inflammatory lung damage mediated by the activation of Toll-like receptor 3 (TLR3) was assessed. The results showed that both Cp and CP-derived BLPs increased the immunogenicity and protection induced by two pneumococcal vaccines administered through the nasal route. Of note, the nasal priming with the PSPF T-dependent antigen co-administered with Cp or CP-derived BLPs efficiently stimulated humoral and cellular immunity and increased the resistance to primary and secondary pneumococcal infections. The CP-derived BLPs presented a stronger effect than live bacteria. Given safety concerns associated with live bacterium administration, especially in high-risk populations, such as infants, the elderly, and immunocompromised patients, BLPs emerge as an attractive mucosal adjuvant to improve the host response to pneumococcal infections and to enhance the vaccines already in the market or in development.

Lacticaseibacillus rhamnosus CRL1505 Peptidoglycan Modulates the Inflammation-Coagulation Response Triggered by Poly(I:C) in the Respiratory Tract.

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.

Isolation, characterization, and immunomodulatory activity evaluation of probiotic strains from colostrum and canine milk.

Background: This study aimed to characterize potential probiotic strains for use in dogs to prevent infectious enteropathies. Lactic acid bacteria (LAB) isolated from canine milk and colostrum were characterized according to their functional properties, including their resistance to gastrointestinal conditions, inhibitory effect against pathogens, and intestinal adhesion. Methods: The immunomodulatory effects of the strains were also analyzed in in vitro and in vivo studies. Among the strains evaluated, two LAB strains (TUCO-16 and TUCO-17) showed remarkable resistance to pH 3.0, bile salts, and pancreatin, as well as inhibitory effects against pathogenic Escherichia coli, Salmonella sp., and Clostridium perfringens. Results: The TUCO-16 and TUCO-17 strains induced a significant increase in the expression of TNF-α, IL-8, and TLR2 in canine macrophages. The oral administration of TUCO-16 and TUCO-17 strains to mice significantly augmented their resistance to pathogenic E. coli or Salmonella intestinal infections. Both canine strains reduced intestinal damage and pathogen counts in the liver and spleen and avoided their dissemination into the bloodstream. These protective effects were related to the ability of TUCO-16 and TUCO-17 strains to differentially modulate the production of IFN-γ, IFN-ß, TNF-α, IL-6, KC, MCP-1, and IL-10 in the intestinal mucosa. Conclusion: Both strains, TUCO-16 and TUCO-17, are potential probiotic candidates for improving intestinal health in dogs, particularly for their ability to inhibit the growth of Gram-negative pathogens common in gastrointestinal infections and modulate the animal's immune response. Further studies are required to effectively demonstrate the beneficial effects of TUCO-16 and TUCO-17 strains in dogs.

Immunobiotic Ligilactobacillus salivarius FFIG58 Confers Long-Term Protection against Streptococcus pneumoniae.

Previously, we isolated potentially probiotic Ligilactobacillus salivarius strains from the intestines of wakame-fed pigs. The strains were characterized based on their ability to modulate the innate immune responses triggered by the activation of Toll-like receptor (TLR)-3 or TLR4 signaling pathways in intestinal mucosa. In this work, we aimed to evaluate whether nasally administered L. salivarius strains are capable of modulating the innate immune response in the respiratory tract and conferring long-term protection against the respiratory pathogen Streptococcus pneumoniae. Infant mice (3-weeks-old) were nasally primed with L. salivarius strains and then stimulated with the TLR3 agonist poly(I:C). Five or thirty days after the last poly(I:C) administration mice were infected with pneumococci. Among the strains evaluated, L. salivarius FFIG58 had a remarkable ability to enhance the protection against the secondary pneumococcal infection by modulating the respiratory immune response. L. salivarius FFIG58 improved the ability of alveolar macrophages to produce interleukin (IL)-6, interferon (IFN)-γ, IFN-ß, tumor necrosis factor (TNF)-α, IL-27, chemokine C-C motif ligand 2 (CCL2), chemokine C-X-C motif ligand 2 (CXCL2), and CXCL10 in response to pneumococcal challenge. Furthermore, results showed that the nasal priming of infant mice with the FFIG58 strain protected the animals against secondary infection until 30 days after stimulation with poly(I:C), raising the possibility of using nasally administered immunobiotics to stimulate trained immunity in the respiratory tract.

In Silico Comparative Genomic Analysis Revealed a Highly Conserved Proteolytic System in Lactobacillus delbrueckii.

Lactobacillus delbrueckii, the type species of the genus Lactobacillus, is widely recognized as the primary starter culture in the dairy industry due to its proteolytic activity, which enables it to growth in milk. In this study, a comprehensive genomic analysis of the proteolytic system was conducted on L. delbrueckii strains. The analysis included 27 genomes of L. delbrueckii, with a specific focus on the key enzyme involved in this system, the cell envelope-associated proteinase (CEP). The amino acid sequences, as well as the protein-structure prediction of the CEPs, were compared. Additionally, syntenic analysis of the genomic locus related to the CEPs revealed high conservation in L. delbrueckii subsp. bulgaricus strains, while L. delbrueckii subsp. lactis strains exhibited greater variability, including the presence of insertion sequences, deletions, and rearrangements. Finally, the CEP promoter region and putative regulatory elements responsible for controlling the expression of the proteolytic system in lactobacilli were investigated. Our genomic analysis and in silico characterization of the CEPs contribute to our understanding of proteolytic activity and the potential applications of these lactic acid bacteria in the dairy industry. Further research in this area will expand our knowledge and potential practical uses of these findings.

Oral Administration of Lacticaseibacillus rhamnosus CRL1505 Modulates Lung Innate Immune Response against Klebsiella pneumoniae ST25.

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-1ß, 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.

Hypermucoviscous Carbapenem-Resistant Klebsiella pneumoniae ST25 Infect Human Intestinal Epithelial Cells and Induce Moderate Inflammation.

Klebsiella pneumoniae is an opportunistic pathogen that can produce moderate and severe infections in immunosuppressed hosts. In recent years, an increase in the isolation of hypermucoviscous carbapenem-resistant K. pneumoniae with sequence type 25 (ST25) in hospitals in Norwest Argentina was observed. This work aimed to study the virulence and inflammatory potential of two K. pneumoniae ST25 strains (LABACER01 and LABACER27) in the intestinal mucosa. The human intestinal Caco-2 cells were infected with the K. pneumoniae ST25 strains, and their adhesion and invasion rates and changes in the expression of tight junction and inflammatory factors genes were evaluated. ST25 strains were able to adhere and invade Caco-2 cells, reducing their viability. Furthermore, both strains reduced the expression of tight junction proteins (occludin, ZO-1, and claudin-5), altered permeability, and increased the expression of TGF-ß and TLL1 and the inflammatory factors (COX-2, iNOS, MCP-1, IL-6, IL-8, and TNF-α) in Caco-2 cells. The inflammatory response induced by LABACER01 and LABACER27 was significantly lower than the one produced by LPS or other intestinal pathogens, including K. pneumoniae NTUH-K2044. No differences in virulence and inflammatory potential were found between LABACER01 and LABACER27. In line with these findings, no major differences between the strains were found when the comparative genomic analysis of virulence factors associated with intestinal infection/colonization was performed. This work is the first to demonstrate that hypermucoviscous carbapenem-resistant K. pneumoniae ST25 infects human intestinal epithelial cells and induces moderate inflammation.

The Respiratory Commensal Bacterium Corynebacterium pseudodiphtheriticum as a Mucosal Adjuvant for Nasal Vaccines.

Previously, we demonstrated that nasally administered Corynebacterium pseudodiphtheriticum 090104 (Cp) or its bacterium-like particles (BLPs) increase the resistance of mice against bacterial and viral respiratory pathogens by modulating the innate immunity. In this work, we evaluated the ability of Cp and BLPs to stimulate alveolar macrophages, and to enhance the humoral immune response induced by a commercial vaccine against Streptococcus pneumoniae. In the first set of experiments, Cp or the BLPs were incubated with primary cultures of murine alveolar macrophages and the phagocytic activity, and the production of cytokines was evaluated. The results revealed that Cp and BLPs were efficiently phagocyted by respiratory macrophages and that both treatments triggered the production of TNF-α, IFN-γ, IL-6, and IL-1ß. In the second set of experiments, 3-week-old Swiss mice were intranasally immunized at days 0, 14, and 28 with the pneumococcal vaccine Prevenar®13 (PCV), Cp + PCV, or BLPs + PCV. On day 33, samples of bronco-alveolar lavages (BAL) and serum were collected for the study of specific antibodies. In addition, immunized mice were challenged with S. pneumoniae serotypes 6B or 19F on day 33 and sacrificed on day 35 (day 2 post-infection) to evaluate the resistance to the infection. Both Cp + PCV and BLPs + PCV groups had higher specific serum IgG and BAL IgA antibodies than the PCV control mice. In addition, the mice that were immunized with Cp + PCV or BLPs + PCV had lower lung and blood pneumococcal cell counts as well as lower levels of BAL albumin and LDH, indicating a reduced lung damage compared to the control mice. Improved levels of anti-pneumococcal antibodies were also detected in the serum and BAL samples after the challenges with the pathogens. The results demonstrated that C. pseudodiphtheriticum 090104 and its bacterium-like particles are capable of stimulating the respiratory innate immune system serving as adjuvants to potentiate the adaptive humoral immune response. Our study is a step forward in the positioning of this respiratory commensal bacterium as a promising mucosal adjuvant for vaccine formulations aimed at combating respiratory infectious diseases.

Bioprotective Lactic Acid Bacteria and Lactic Acid as a Sustainable Strategy to Combat Escherichia coli O157:H7 in Meat.

Human infection by Enterohemorrhagic Escherichia coli (EHEC) constitutes a serious threat to public health and a major concern for the meat industry. Presently, consumers require safer/healthier foods with minimal chemical additives, highlighting the need for sustainable solutions to limit and prevent risks. This work evaluated the ability of two antagonistic lactic acid bacteria (LAB) strains, Lactiplantibacillus plantarum CRL681 and Enterococcus mundtii CRL35, and their combination in order to inhibit EHEC in beef (ground and vacuum sealed meat discs) at 8 °C during 72 h. The effect of lower lactic acid (LA) concentrations was evaluated. Meat color was studied along with how LAB strains interfere with the adhesion of Escherichia coli to meat. The results indicated a bacteriostatic effect on EHEC cells when mixed LAB strains were inoculated. However, a bactericidal action due to a synergism between 0.6% LA and LAB occurred, producing undetectable pathogenic cells at 72 h. Color parameters (a*, b* and L*) did not vary in bioprotected meat discs, but they were significantly modified in ground meat after 24 h. In addition, LAB strains hindered EHEC adhesion to meat. The use of both LAB strains plus 0.6% LA, represents a novel, effective and ecofriendly strategy to inactivate EHEC in meat.

The Ability of Postimmunobiotics from L. rhamnosus CRL1505 to Protect against Respiratory Syncytial Virus and Pneumococcal Super-Infection Is a Strain-Dependent Characteristic.

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-ß, TNF-α, OAS1, RNAseL and IL-27 genes in response to RSV infection, and IL-6, IFN-γ, IL-1ß, 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.

Respiratory Commensal Bacteria Increase Protection against Hypermucoviscous Carbapenem-Resistant Klebsiella pneumoniae ST25 Infection.

In a previous work, we demonstrated that nasally administered Corynebacterium pseudodiphtheriticum 090104 beneficially modulated the respiratory innate immune response and improved the protection against Respiratory Syncytial Virus and Streptococcus pneumoniae in mice. In this work, we aimed to evaluate whether the immunomodulatory 090104 strain was able to enhance the resistance against the respiratory infection induced by hypermucoviscous carbapenemase-producing (KPC-2) Klebsiella pneumoniae strains belonging to the sequence type (ST) 25. The nasal treatment of mice with C. pseudodiphtheriticum 090104 before the challenge with multiresistant K. pneumoniae ST25 strains significantly reduced lung bacterial cell counts and lung tissue damage. The protective effect of the 090104 strain was related to its ability to regulate the respiratory innate immune response triggered by K. pneumoniae challenge. C. pseudifteriticum 090104 differentially modulated the recruitment of leukocytes into the lung and the production of TNF-α, IFN-γ and IL-10 levels in the respiratory tract and serum. Our results make an advance in the positioning of C. pseudodiphtheriticum 090104 as a next-generation probiotic for the respiratory tract and encourage further research of this bacterium as a promising alternative to develop non-antibiotic therapeutical approaches to enhance the prevention of infections produced by microorganisms with multiple resistance to antimicrobials such as KPC-2-producing hypermucoviscous K. pneumoniae strains belonging to ST25.

Genomic and Immunological Characterization of Hypermucoviscous Carbapenem-Resistant Klebsiella pneumoniae ST25 Isolates from Northwest Argentina.

In recent years, an increase in the prevalence hypermucoviscous carbapenem-resistant Klebsiella pneumoniae with sequence type 25 (ST25) was detected in hospitals of Tucuman (Northwest Argentina). In this work, the virulence and the innate immune response to two K. pneumoniae ST25 strains (LABACER 01 and LABACER 27) were evaluated in a murine model after a respiratory challenge. In addition, comparative genomics was performed with K. pneumoniae LABACER01 and LABACER27 to analyze genes associated with virulence. Both LABACER01 and LABACER27 were detected in the lungs of infected mice two days after the nasal challenge, with LABACER01 counts significantly higher than those of LABACER27. Only LABACER01 was detected in hemocultures. Lactate dehydrogenase (LDH) and albumin levels in bronchoalveolar lavage (BAL) samples were significantly higher in mice challenged with LABACER01 than in LABACER27-infected animals, indicating greater lung tissue damage. Both strains increased the levels of neutrophils, macrophages, TNF-α, IL-1ß, IL-6, KC, MCP-1, IFN-γ, and IL-17 in the respiratory tract and blood, with the effect of LABACER01 more marked than that of LABACER27. In contrast, LABACER27 induced higher levels of IL-10 in the respiratory tract than LABACER01. Genomic analysis revealed that K. pneumoniae LABACER01 and LABACER27 possess virulence factors found in other strains that have been shown to be hypervirulent, including genes required for enterobactin (entABCDEF) and salmochelin (iroDE) biosynthesis. In both strains, the genes of toxin-antitoxin systems, as well as regulators of the expression of virulence factors and adhesion genes were also detected. Studies on the genetic potential of multiresistant K. pneumoniae strains as well as their cellular and molecular interactions with the host are of fundamental importance to assess the association of certain virulence factors with the intensity of the inflammatory response. In this sense, this work explored the virulence profile based on genomic and in vivo studies of hypermucoviscous carbapenem-resistant K. pneumoniae ST25 strains, expanding the knowledge of the biology of the emerging ST25 clone in Argentina.

Topical Administration of Lactiplantibacillus plantarum Accelerates the Healing of Chronic Diabetic Foot Ulcers through Modifications of Infection, Angiogenesis, Macrophage Phenotype and Neutrophil Response.

This work aimed to evaluate the adjuvant treatment to surgical debridement using topical applications of Lactiplantibacillus plantarum ATCC 10241 cultures in complicated diabetic foot ulcers as compared to diabetic foot ulcers receiving surgical wound debridement. A randomised controlled trial was performed involving 22 outpatients with complicated diabetic foot ulcers that either received surgical debridement (SuDe, n = 12) or surgical debridement plus topical applications of L. plantarum cultures (SuDe + Lp, n = 10) every week during a 12 week treatment period. Compared to patients receiving SuDe, patients treated with SuDe + Lp exhibited significantly increased fibroplasia and angiogenesis, as determined by Masson's trichrome staining and the study of CD34 cells, α-smooth muscle actin to semi-quantify vascular area, number of vessels and endothelial cells. In addition, a promotion of the polarisation of macrophages from M1 (CD68) to M2 (CD163) phenotype was observed in SuDe + Lp patients with remarkable differences in the tissue localisation. Bacterial counts were significantly diminished in the SuDe + Lp group compared to the SuDe group. Ex vivo assays, using polymorphonuclears isolated from peripheral blood of patients with diabetes and healthy individuals and challenged with Staphylococcus aureus demonstrated that the addition of L. plantarum supernatants significantly improved the phagocytosis of these cells. L. plantarum-secreted components increased the neutrophils bactericidal activity and regulated the netosis induced by S. aureus. At day 49, the average wound area reduction with SuDe + Lp was 73.5% compared with 45.8% for SuDe (p < 0.05). More patients progressed to closure with SuDe + Lp compared with SuDe treatment, indicating the ability of L. plantarum to accelerate the healing. At day 60, 60% of patients treated with SuDe + Lp achieved 100% of wound area reduction compared with 40% for SuDe. We propose that SuDe + Lp could be an effective adjuvant to surgical debridement when SuDe is not satisfactory for patients with complicated diabetic foot ulcers. The treatment is cheap and easy to apply and the product is easy to obtain.

Lactiplantibacillus plantarum Strains Modulate Intestinal Innate Immune Response and Increase Resistance to Enterotoxigenic Escherichia coli Infection.

Currently, probiotic bacteria with not transferable antibiotic resistance represent a sustainable strategy for the treatment and prevention of enterotoxigenic Escherichia coli (ETEC) in farm animals. Lactiplantibacillus plantarum is among the most versatile species used in the food industry, either as starter cultures or probiotics. In the present work, the immunobiotic potential of L. plantarum CRL681 and CRL1506 was studied to evaluate their capability to improve the resistance to ETEC infection. In vitro studies using porcine intestinal epithelial (PIE) cells and in vivo experiments in mice were undertaken. Expression analysis indicated that both strains were able to trigger IL-6 and IL-8 expression in PIE cells in steady-state conditions. Furthermore, mice orally treated with these strains had significantly improved levels of IFN-γ and TNF-α in the intestine as well as enhanced activity of peritoneal macrophages. The ability of CRL681 and CRL1506 to beneficially modulate intestinal immunity was further evidenced in ETEC-challenge experiments. In vitro, the CRL1506 and CRL681 strains modulated the expression of inflammatory cytokines (IL-6) and chemokines (IL-8, CCL2, CXCL5 and CXCL9) in ETEC-stimulated PIE cells. In vivo experiments demonstrated the ability of both strains to beneficially regulate the immune response against this pathogen. Moreover, the oral treatment of mice with lactic acid bacteria (LAB) strains significantly reduced ETEC counts in jejunum and ileum and prevented the spread of the pathogen to the spleen and liver. Additionally, LAB treated-mice had improved levels of intestinal IL-10 both at steady state and after the challenge with ETEC. The protective effect against ETEC infection was not observed for the non-immunomodulatory TL2677 strain. Furthermore, the study showed that L. plantarum CRL1506 was more efficient than the CRL681 strain to modulate mucosal immunity highlighting the strain specific character of this probiotic activity. Our results suggest that the improved intestinal epithelial defenses and innate immunity induced by L. plantarum CRL1506 and CRL681 would increase the clearance of ETEC and at the same time, protect the host against detrimental inflammation. These constitute valuable features for future probiotic products able to improve the resistance to ETEC infection.

Lactobacillus delbrueckii CRL 581 Differentially Modulates TLR3-Triggered Antiviral Innate Immune Response in Intestinal Epithelial Cells and Macrophages.

Lactobacillus delbrueckii subsp. lactis CRL 581 beneficially modulates the intestinal antiviral innate immune response triggered by the Toll-like receptor 3 (TLR3) agonist poly(I:C) in vivo. This study aimed to characterize further the immunomodulatory properties of the technologically relevant starter culture L. delbrueckii subsp. lactis CRL 581 by evaluating its interaction with intestinal epithelial cells and macrophages in the context of innate immune responses triggered by TLR3. Our results showed that the CRL 581 strain was able to adhere to porcine intestinal epithelial (PIE) cells and mucins. The CRL 581 strain also augmented the expression of antiviral factors (IFN-α, IFN-ß, Mx1, OAS1, and OAS2) and reduced inflammatory cytokines in PIE cells triggered by TLR3 stimulation. In addition, the influence of L. delbrueckii subsp. lactis CRL 581 on the response of murine RAW macrophages to the activation of TLR3 was evaluated. The CRL 581 strain was capable of enhancing the expression of IFN-α, IFN-ß, IFN-γ, Mx1, OAS1, TNF-α, and IL-1ß. Of note, the CRL 581 strain also augmented the expression of IL-10 in macrophages. The results of this study show that the high proteolytic strain L. delbrueckii spp. lactis CRL 581 was able to beneficially modulate the intestinal innate antiviral immune response by regulating the response of both epithelial cells and macrophages relative to TLR3 activation.

Novel LysM motifs for antigen display on lactobacilli for mucosal immunization.

We characterized two LysM domains of Limosilactobacillus fermentum, belonging to proteins Acglu (GenBank: KPH22907.1) and Pgb (GenBank: KPH22047.1) and bacterium like particles (BLP) derived from the immunomodulatory strain Lacticaseibacillus rhamnosus IBL027 (BLPs027) as an antigen display platform. The fluorescence protein Venus fused to the novel LysM domains could bind to the peptidoglycan shell of lactobacilli and resisted harsh conditions such as high NaCl and urea concentrations. Acglu with five LysM domains was a better anchor than Pgb baring only one domain. Six-week-old BALB/c mice were nasally immunized with the complex Venus-Acglu-BLPs027 at days 0, 14 and 28. The levels of specific serum IgG, IgG1 and IgG2a and the levels of total immunoglobulins (IgT) and IgA in broncho-alveolar lavage (BAL) were evaluated ten days after the last boosting. Venus-Acglu-BLPs027, nasally administered, significantly increased specific BAL IgT and IgA, and serum IgG levels. In addition, spleen cells of mice immunized with Venus-Acglu-BLPs027 secreted TNF-α, IFN-γ and IL-4 when stimulated ex vivo in a dose-dependent manner. We constructed a Gateway compatible destination vector to easily fuse the selected LysM domain to proteins of interest for antigen display to develop mucosal subunit vaccines.