Fate of Bacillus cereus within phagocytic cells

In this study we assessed the interaction of different strains of Bacillus cereus with murine peritoneal macrophages and cultured phagocytic cells (Raw 264.7 cells). Association, internalization, intracellular survival, routing of bacteria to different compartments and expression of MHCII were assessed in cells infected with different strains of B. cereus in vegetative form. Association values (adhering + internalized bacteria) and phagocytosis were higher for strain B10502 than those for strains 2 and M2. However, after 90 min interaction, intracellular survival was higher for strain 2 than for strains M2 and B10502. Acquisition of lysosomal markers by B. cereus containing vacuoles (BcCV), assessed by LAMP1 and Lysotracker labelling occurred shortly after internalization. The highest ratio of LAMP1(+)-BcCV was found for strain M2. This strain was able to survive longer than strain B10502 which routes to LAMP1 containing vacuoles to a lesser extent. In addition, strain M2 stimulated expression of MHCII by infected cells. Confocal analyses 60 or 90 min post-infection showed different percentages of co-localization of bacteria with Lysotracker. Results suggest strain-dependent interaction and intracellular killing of B. cereus by phagocytic cells. These findings could be relevant for the pathogenic potential of Bacillus cereus strains.Keywords: Bacillus cereus · LAMP1 · phagocytosis · virulence · endocytic pathway · intracellular traffickin

Safety Characterization and Antimicrobial Properties of Kefir-Isolated <i>Lactobacillus kefiri</i>

Lactobacilli are generally regarded as safe; however, certain strains have been associated with cases of infection. Our workgroup has already assessed many functional properties of Lactobacillus kefiri, but parameters regarding safety must be studied before calling them probiotics. In this work, safety aspects and antimicrobial activity of L. kefiri strains were studied. None of the L. kefiri strains tested caused - or -hemolysis. All the strains were susceptible to tetracycline, clindamycin, streptomycin, ampicillin, erythromycin, kanamycin, and gentamicin; meanwhile, two strains were resistant to chloramphenicol. On the other hand, all L. kefiri strains were able to inhibit both Gram(+) and Gram(−) pathogens. Regarding the in vitro results, L. kefiri CIDCA 8348 was selected to perform in vivo studies. Mice treated daily with an oral dose of 108 CFU during 21 days showed no signs of pain, lethargy, dehydration, or diarrhea, and the histological studies were consistent with those findings. Moreover, no differences in proinflammatory cytokines secretion were observed between treated and control mice. No translocation of microorganisms to blood, spleen, or liver was observed. Regarding these findings, L. kefiri CIDCA 8348 is a microorganism isolated from a dairy product with a great potential as probiotic for human or animal use.Facultad de Ciencias Exacta

Safety Characterization and Antimicrobial Properties of Kefir-Isolated <i>Lactobacillus kefiri</i>

Lactobacilli are generally regarded as safe; however, certain strains have been associated with cases of infection. Our workgroup has already assessed many functional properties of Lactobacillus kefiri, but parameters regarding safety must be studied before calling them probiotics. In this work, safety aspects and antimicrobial activity of L. kefiri strains were studied. None of the L. kefiri strains tested caused - or -hemolysis. All the strains were susceptible to tetracycline, clindamycin, streptomycin, ampicillin, erythromycin, kanamycin, and gentamicin; meanwhile, two strains were resistant to chloramphenicol. On the other hand, all L. kefiri strains were able to inhibit both Gram(+) and Gram(−) pathogens. Regarding the in vitro results, L. kefiri CIDCA 8348 was selected to perform in vivo studies. Mice treated daily with an oral dose of 108 CFU during 21 days showed no signs of pain, lethargy, dehydration, or diarrhea, and the histological studies were consistent with those findings. Moreover, no differences in proinflammatory cytokines secretion were observed between treated and control mice. No translocation of microorganisms to blood, spleen, or liver was observed. Regarding these findings, L. kefiri CIDCA 8348 is a microorganism isolated from a dairy product with a great potential as probiotic for human or animal use.Facultad de Ciencias Exacta

Safety Characterization and Antimicrobial Properties of Kefir-Isolated <i>Lactobacillus kefiri</i>

Lactobacilli are generally regarded as safe; however, certain strains have been associated with cases of infection. Our workgroup has already assessed many functional properties of Lactobacillus kefiri, but parameters regarding safety must be studied before calling them probiotics. In this work, safety aspects and antimicrobial activity of L. kefiri strains were studied. None of the L. kefiri strains tested caused - or -hemolysis. All the strains were susceptible to tetracycline, clindamycin, streptomycin, ampicillin, erythromycin, kanamycin, and gentamicin; meanwhile, two strains were resistant to chloramphenicol. On the other hand, all L. kefiri strains were able to inhibit both Gram(+) and Gram(−) pathogens. Regarding the in vitro results, L. kefiri CIDCA 8348 was selected to perform in vivo studies. Mice treated daily with an oral dose of 108 CFU during 21 days showed no signs of pain, lethargy, dehydration, or diarrhea, and the histological studies were consistent with those findings. Moreover, no differences in proinflammatory cytokines secretion were observed between treated and control mice. No translocation of microorganisms to blood, spleen, or liver was observed. Regarding these findings, L. kefiri CIDCA 8348 is a microorganism isolated from a dairy product with a great potential as probiotic for human or animal use.Facultad de Ciencias Exacta

Genetic Drivers of Kidney Defects in the DiGeorge Syndrome

Background The DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown. Methods We conducted a genomewide search for structural variants in two cohorts: 2080 patients with congenital kidney and urinary tract anomalies and 22,094 controls. We performed exome and targeted resequencing in samples obtained from 586 additional patients with congenital kidney anomalies. We also carried out functional studies using zebrafish and mice. Results We identified heterozygous deletions of 22q11.2 in 1.1% of the patients with congenital kidney anomalies and in 0.01% of population controls (odds ratio, 81.5; P=4.5×10(-14)). We localized the main drivers of renal disease in the DiGeorge syndrome to a 370-kb region containing nine genes. In zebrafish embryos, an induced loss of function in snap29, aifm3, and crkl resulted in renal defects; the loss of crkl alone was sufficient to induce defects. Five of 586 patients with congenital urinary anomalies had newly identified, heterozygous protein-altering variants, including a premature termination codon, in CRKL. The inactivation of Crkl in the mouse model induced developmental defects similar to those observed in patients with congenital urinary anomalies. Conclusions We identified a recurrent 370-kb deletion at the 22q11.2 locus as a driver of kidney defects in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Of the nine genes at this locus, SNAP29, AIFM3, and CRKL appear to be critical to the phenotype, with haploinsufficiency of CRKL emerging as the main genetic driver. (Funded by the National Institutes of Health and others.)

Lactobacillus delbrueckii subsp lactis CIDCA 133 modulates response of human epithelial and dendritic cells infected with Bacillus cereus

It is known that probiotic microorganisms are able to modulate pathogen virulence. This ability is strain dependent and involves multiple interactions between microorganisms and relevant host's cell populations. In the present work we focus on the effect of a potentially probiotic lactobacillus strain (Lactobacillus delbrueckii subsp. lactis CIDCA 133) in an in vitro model of Bacillus cereus infection. Our results showed that infection of intestinal epithelial HT-29 cells by B. cereus induces nuclear factor kappa B (NF-κB) pathway. Noteworthy, the presence of strain L. delbrueckii subsp. lactis CIDCA 133 increases stimulation. However, B. cereus-induced interleukin (IL)-8 production by epithelial cells is partially abrogated by L. delbrueckii subsp. lactis CIDCA 133. These findings suggest that signalling pathways other than that of NF-κB are involved. In a co-culture system (HT-29 and monocyte-derived dendritic cells), B. cereus was able to translocate from the epithelial (upper) to the dendritic cell compartment (lower). This translocation was partially abrogated by the presence of lactobacilli in the upper compartment. In addition, infection of epithelial cells in the co-culture model, led to an increase in the expression of CD86 by dendritic cells. This effect could not be modified in the presence of lactobacilli. Interestingly, infection of enterocytes with B. cereus triggers production of proinflammatory cytokines by dendritic cells (IL-8, IL-6 and tumour necrosis factor alpha (TNF-α)). The production of TNF-α (a protective cytokine in B. cereus infections) by dendritic cells was increased in the presence of lactobacilli. The present work demonstrates for the first time the effect of L. delbrueckii subsp. lactis CIDCA 133, a potentially probiotic strain, in an in vitro model of B. cereus infection. The presence of the probiotic strain modulates cell response both in infected epithelial and dendritic cells thus suggesting a possible beneficial effect of selected lactobacilli strains on the course of B. cereus infection.Fil: Rolny, Ivanna Sabrina. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Tiscornia, I.. Instituto Pasteur de Montevideo; Uruguay. Universidad ORT Uruguay; UruguayFil: Racedo, Silvia María. Medical University of Graz; Austria. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Perez, Pablo Fernando. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: Bollati Fogolin, M.. Instituto Pasteur de Montevideo; Urugua

Secreted compounds of the probiotic Bacillus clausii O/C inhibit the cytotoxic effects induced by Clostridium difficile and Bacillus cereus toxins

Although the use of probiotics based on Bacillus strains to fight off intestinal pathogens and antibiotic-associated diarrhea is widespread, the mechanisms involved in producing their beneficial effects remain unclear. Here, we studied the ability of compounds secreted by the probiotic Bacillus clausii strain O/C to counteract the cytotoxic effects induced by toxins of two pathogens, Clostridium difficile and Bacillus cereus, by evaluating eukaryotic cell viability and expression of selected genes. Coincubation of C. difficile and B. cereus toxic culture supernatants with the B. clausii supernatant completely prevented the damage induced by toxins in Vero and Caco-2 cells. The hemolytic effect of B. cereus was also avoided by the probiotic supernatant. Moreover, in these cells, the expression of rhoB, encoding a Rho GTPase target for C. difficile toxins, was normalized when C. difficile supernatant was pretreated using the B. clausii supernatant. All of the beneficial effects observed with the probiotic were abolished by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Suspecting the involvement of a secreted protease in this protective effect, a protease was purified from the B. clausii supernatant and identified as a serine protease (M-protease; GenBank accession number Q99405). Experiments on Vero cells demonstrated the antitoxic activity of the purified protease against pathogen supernatants. This is the first report showing the capacity of a protease secreted by probiotic bacteria to inhibit the cytotoxic effects of toxinogenic C. difficile and B. cereus strains. This extracellular compound could be responsible, at least in part, for the protective effects observed for this human probiotic in antibiotic-associated diarrhea

Safety Characterization and Antimicrobial Properties of Kefir-Isolated Lactobacillus kefiri

Lactobacilli are generally regarded as safe; however, certain strains have been associated with cases of infection. Our workgroup has already assessed many functional properties of Lactobacillus kefiri, but parameters regarding safety must be studied before calling them probiotics. In this work, safety aspects and antimicrobial activity of L. kefiri strains were studied. None of the L. kefiri strains tested caused α- or β-hemolysis. All the strains were susceptible to tetracycline, clindamycin, streptomycin, ampicillin, erythromycin, kanamycin, and gentamicin; meanwhile, two strains were resistant to chloramphenicol. On the other hand, all L. kefiri strains were able to inhibit both Gram(+) and Gram(−) pathogens. Regarding the in vitro results, L. kefiri CIDCA 8348 was selected to perform in vivo studies. Mice treated daily with an oral dose of 108 CFU during 21 days showed no signs of pain, lethargy, dehydration, or diarrhea, and the histological studies were consistent with those findings. Moreover, no differences in proinflammatory cytokines secretion were observed between treated and control mice. No translocation of microorganisms to blood, spleen, or liver was observed. Regarding these findings, L. kefiri CIDCA 8348 is a microorganism isolated from a dairy product with a great potential as probiotic for human or animal use

Reduced dosage of <i>β-catenin</i> provides significant rescue of cardiac outflow tract anomalies in a <i>Tbx1</i> conditional null mouse model of 22q11.2 deletion syndrome

<div><p>The 22q11.2 deletion syndrome (22q11.2DS; velo-cardio-facial syndrome; DiGeorge syndrome) is a congenital anomaly disorder in which haploinsufficiency of <i>TBX1</i>, encoding a T-box transcription factor, is the major candidate for cardiac outflow tract (OFT) malformations. Inactivation of <i>Tbx1</i> in the anterior heart field (AHF) mesoderm in the mouse results in premature expression of pro-differentiation genes and a persistent truncus arteriosus (PTA) in which septation does not form between the aorta and pulmonary trunk. Canonical <i>Wnt/β-catenin</i> has major roles in cardiac OFT development that may act upstream of <i>Tbx1</i>. Consistent with an antagonistic relationship, we found the opposite gene expression changes occurred in the AHF in <i>β-catenin</i> loss of function embryos compared to <i>Tbx1</i> loss of function embryos, providing an opportunity to test for genetic rescue. When both alleles of <i>Tbx1</i> and one allele of <i>β-catenin</i> were inactivated in the <i>Mef2c-AHF-Cre</i> domain, 61% of them (n = 34) showed partial or complete rescue of the PTA defect. Upregulated genes that were oppositely changed in expression in individual mutant embryos were normalized in significantly rescued embryos. Further, <i>β-catenin</i> was increased in expression when <i>Tbx1</i> was inactivated, suggesting that there may be a negative feedback loop between canonical Wnt and <i>Tbx1</i> in the AHF to allow the formation of the OFT. We suggest that alteration of this balance may contribute to variable expressivity in 22q11.2DS.</p></div