The Mucus-Binding Factor Mediates Lacticaseibacillus rhamnosus CRL1505 Adhesion but Not Immunomodulation in the Respiratory Tract.

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.

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.

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.

Characterization of Weissella viridescens UCO-SMC3 as a Potential Probiotic for the Skin: Its Beneficial Role in the Pathogenesis of Acne Vulgaris.

Previously, we isolated lactic acid bacteria from the slime of the garden snail Helix aspersa Müller and selected Weissella viridescens UCO-SMC3 because of its ability to inhibit in vitro the growth of the skin-associated pathogen Cutibacterium acnes. The present study aimed to characterize the antimicrobial and immunomodulatory properties of W. viridescens UCO-SMC3 and to demonstrate its beneficial effect in the treatment of acne vulgaris. Our in vitro studies showed that the UCO-SMC3 strain resists adverse gastrointestinal conditions, inhibits the growth of clinical isolates of C. acnes, and reduces the adhesion of the pathogen to keratinocytes. Furthermore, in vivo studies in a mice model of C. acnes infection demonstrated that W. viridescens UCO-SMC3 beneficially modulates the immune response against the skin pathogen. Both the oral and topical administration of the UCO-SCM3 strain was capable of reducing the replication of C. acnes in skin lesions and beneficially modulating the inflammatory response. Of note, orally administered W. viridescens UCO-SMC3 induced more remarkable changes in the immune response to C. acnes than the topical treatment. However, the topical administration of W. viridescens UCO-SMC3 was more efficient than the oral treatment to reduce pathogen bacterial loads in the skin, and effects probably related to its ability to inhibit and antagonize the adhesion of C. acnes. Furthermore, a pilot study in acne volunteers demonstrated the capacity of a facial cream containing the UCO-SMC3 strain to reduce acne lesions. The results presented here encourage further mechanistic and clinical investigations to characterize W. viridescens UCO-SMC3 as a probiotic for acne vulgaris treatment.

Biodiversity and technological-functional potential of lactic acid bacteria isolated from spontaneously fermented chia sourdough.

Chia, is a gluten-free, rich in proteins, oilseed that is "on trend" as an alternative ingredient in food production, adding nutritional value. As a reservoir of natural biodiversity, lactic acid bacteria development, during spontaneous chia flour fermentation (sourdough) for 10 days, were investigated by culturing and high throughput sequencing (HTS). Culture-dependent analysis showed a rapid increase in total LAB numbers from the second day of sourdough refreshment. Taxonomical identification of LAB isolates by rep-PCR and further 16S rRNA sequencing was performed. Besides Among identified LAB by culture-dependent approach, species from genus Enterococcus were the most abundant; Lactococcus (Lc. lactis), Lactobacillus (L. rhamnosus) and Weissella (W. cibaria) species were also isolated. By HTS, twelve OTUs belonging to LAB genera were identified during chia sourdough fermentation with an increased Lactobacillus diversity. Enterococcus (E.) faecium, E. mundtii, W. cibaria and L. rhamnosus were detected as dominant species in the final propagation stages while Bacillus and Clostridium were mostly present during first fermentation stages. The investigation of biotechnological and safety traits (acidification ability, protein hydrolysis, exopolysaccharides production, antimicrobial activity and antibiotic resistance) of 15 representative LAB strains was performed. Strains characterization led to the selection of Lc. lactis CH179, L. rhamnosus CH34 and W. cibaria CH28 as candidates to be used as novel functional starter culture for gluten-free chia fermented products. As far as we know, this is the first study providing information on the molecular inventory of LAB population during spontaneous fermentation of chia sourdough.