Identification and characterization of starter lactic acid bacteria and probiotics from Columbian dairy products

AIMS: Considering the significant rise in the probiotic market in Columbia, and given the lack of reports concerning the microbial population and strain performance in products from different producers, this study aims at determining the number of viable starter bacteria and probiotics in bio-yoghurts available at the Columbian market, identifying the species and analysing the performance of the isolated strains in bile acid resistance, antagonistic activity against pathogens, and adherence capacity to human intestinal epithelial cells. METHODS AND RESULTS: Seven bio-yoghurts were analysed for the bacterial species present. Species identification was carried out using 16S rRNA gene targeted PCR. The cultured bacteria were tested for bile acid resistance, adherence to a human intestinal epithelial cell line, and antagonism against the pathogen Salmonella enterica serovar Typhimurium. A total of 17 different strains were identified. Based on plate counting, all bio-yoghurts have at least total viable cells of approximately 10(7) CFU ml(-1). Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus were the most frequently isolated bacteria. Viable Bifidobacterium was only recovered from one product. However, after PCR analysis, DNA of this genus was confirmed in five out of seven products. Major differences were found for S. typhimurium antagonism. The adherence capacity to Caco-2 cells was observed in 10 of the isolated strains. In general, low survival to simulated gastric juice was observed. CONCLUSIONS: Some of the isolated strains have probiotic potential, although not all of them were present in the advised amount to exert beneficial health effects. However, the full correct scientific name of the isolated bacteria and their viable counts were not included on the product label. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the identification and functionality of starter bacteria and probiotics present in dairy products on the Columbian market.     

Stimulation by caveolin-1 of the hypotonicity-induced release of taurine and ATP at basolateral, but not apical, membrane of Caco-2 cells

Regulatory volume decrease (RVD) is a protective mechanism that allows mammalian cells to restore their volume when exposed to a hypotonic environment. A key component of RVD is the release of K⁺, Cl^–, and organic osmolytes, such as taurine, which then drives osmotic water efflux. Previous experiments have indicated that caveolin-1, a coat protein of caveolae microdomains in the plasma membrane, promotes the swelling-induced Cl^– current (I_Cl,swell) through volume-regulated anion channels. However, it is not known whether the stimulation by caveolin-1 is restricted to the release of Cl^– or whether it also affects the swelling-induced release of other components, such as organic osmolytes. To address this problem, we have studied I_Cl,swell and the hypotonicity-induced release of taurine and ATP in wild-type Caco-2 cells that are caveolin-1 deficient and in stably transfected Caco-2 cells that express caveolin-1. Electrophysiological characterization of wild-type and stably transfected Caco-2 showed that caveolin-1 promoted I_Cl,swell, but not cystic fibrosis transmembrane conductance regulator currents. Furthermore, caveolin-1 expression stimulated the hypotonicity-induced release of taurine and ATP in stably transfected Caco-2 cells grown as a monolayer. Interestingly, the effect of caveolin-1 was polarized because only the release at the basolateral membrane, but not at the apical membrane, was increased. It is therefore concluded that caveolin-1 facilitates the hypotonicity-induced release of Cl^–, taurine, and ATP, and that in polarized epithelial cells, the effect of caveolin-1 is compartmentalized to the basolateral membrane. caveolae; osmolyte; epithelial cell; chloride channel     

Copper tolerance in the cuprophyte Haumaniastrum katangense (S. Moore) P.A. Duvign. & Plancke

Cu tolerance and accumulation have been studied in Haumaniastrum katangense, a cuprophyte from Katanga (DR Congo), previously described as a copper hyperaccumulator. Nicotiana plumbaginifolia, a well-known non-tolerant and non-accumulator species, was used as a control. The germination rate of H. katangense was enhanced by copper and fungicide addition, suggesting that fungal pathogens, which restrain germination in normal conditions, are limiting. In hydroponic culture in the Hoagland medium, H. katangense did not grow well, in contrast to N. plumbaginifolia. Better growth was achieved by adding fungicide or higher copper concentrations. The maximal non-effective concentration (NEC) was 12 µM CuSO₄ for H. katangense grown in hydroponics, i.e. 24 times greater than Cu concentration in the Hoagland medium. By comparison, copper concentrations greater than 0.5 µM had a negative effect on the growth of N. plumbaginifolia. EC₅₀ (50% effective concentration) in hydroponics was 40 µM CuSO₄ for H. katangense and 6 µM CuSO₄ for N. plumbaginifolia. EC₁₀₀ (100% effective concentration) was 100 µM CuSO₄ for H. katangense and 15 µM CuSO₄ for N. plumbaginifolia. In soil, growth was also stimulated by Cu addition up to 300 mg kg^-1 CuSO₄. Surplus copper was also required for cultivating H. katangense in sterile conditions, suggesting that Cu excess may be necessary for needs other than pathogen defence. Cu accumulation in the shoot has been measured for N. plumbaginifolia and H. katangense at their respective NEC. Cu allocation in the two species showed a similar response to increasing Cu concentrations, i.e. root/shoot concentration ratio well above 1. In conclusion, H. katangense is highly tolerant to copper and has elevated copper requirement even in the absence of biotic interactions. Its accumulation pattern is typical of an excluder species.     

The global nutritional regulator CodY is an essential protein in the human pathogen Streptococcus pneumoniae

CodY is a global regulator highly conserved in low-G+C Gram-positive bacteria. It plays a key role in the adaptation of Bacillus subtilis to nutritional limitation through repression of a large gene set during exponential growth and relief of repression upon starvation. In several pathogenic bacteria, CodY regulates major virulence genes. Our interest in Streptococcus pneumoniae CodY originates from our observations that the oligopeptide permease Ami was involved in repression of competence for genetic transformation. We hypothesized that peptide uptake through Ami feeds amino acid pools, which are sensed by CodY to repress competence. As our initial attempts at inactivating codY failed, we launched an in-depth analysis into the question of the essentiality of codY. We report that codY cannot be inactivated unless a complementing ectopic copy is present. We obtained genetic evidence that a recently published D39 codY knock-out contains additional mutations allowing survival of codY mutant cells. Whole genome sequencing revealed mutations in fatC, which encodes a ferric iron permease, and amiC. This combination of mutations was confirmed to allow tolerance of codY inactivation. The amiC mutation is in itself sufficient to account for the strong derepression of competence development observed in D39 codY cells.     

Carbonic Anhydrase Is Essential for Streptococcus pneumoniae Growth in Environmental Ambient Air

The respiratory tract pathogen Streptococcus pneumoniae needs to adapt to the different levels of carbon dioxide (CO₂) it encounters during transmission, colonization, and infection. Since CO₂ is important for various cellular processes, factors that allow optimal CO₂ sequestering are likely to be important for pneumococcal growth and survival. In this study, we showed that the putative pneumococcal carbonic anhydrase (PCA) is essential for in vitro growth of S. pneumoniae under the CO₂-poor conditions found in environmental ambient air. Enzymatic analysis showed that PCA catalyzes the reversible hydration of CO₂ to bicarbonate (HCO₃⁻), an essential step to prevent the cellular release of CO₂. The addition of unsaturated fatty acids (UFAs) reversed the CO₂-dependent in vitro growth inhibition of S. pneumoniae strains lacking the pca gene (Δpca), indicating that PCA-mediated CO₂ fixation is at least associated with HCO₃⁻-dependent de novo biosynthesis of UFAs. Besides being necessary for growth in environmental ambient conditions, PCA-mediated CO₂ fixation pathways appear to be required for intracellular survival in host cells. This effect was especially pronounced during invasion of human brain microvascular endothelial cells (HBMEC) and uptake by murine J774 macrophage cells but not during interaction of S. pneumoniae with Detroit 562 pharyngeal epithelial cells. Finally, the highly conserved pca gene was found to be invariably present in both CO₂-independent and naturally circulating CO₂-dependent strains, suggesting a conserved essential role for PCA and PCA-mediated CO₂ fixation pathways for pneumococcal growth and survival.The Gram-positive bacterium Streptococcus pneumoniae, or pneumococcus, is a human respiratory tract pathogen that contributes significantly to global mortality and morbidity. In addition, it is an important asymptomatic colonizer of the human nasopharynx, with carriage rates around 10% in adults and over 40% in children (6). Pneumococcal colonization and infection are closely linked, but knowledge of the factors that contribute to transmission, carriage, disease, and transition from carriage to disease is still limited. Research on components that physically contribute to host-pathogen interactions, such as capsular polysaccharides, adhesins, and toxins, has provided valuable insights into the process of pneumococcal pathogenesis (20). In contrast, the influence of environmental factors on pneumococcal growth and survival remains fairly unexplored.S. pneumoniae needs to adapt to various aerobic and anaerobic conditions, reflecting the different niches it occupies during transmission, colonization, and invasive disease. During niche transition, oxygen (O₂) levels change considerably. Levels of O₂ are 21% in ambient air, decrease to 10 to 15% in the alveoli of the lungs, and are about 5% in resting cells. In O₂-rich conditions, S. pneumoniae expresses pyruvate oxidase (SpxB), which generates acetyl-phosphate as a source of ATP and hydrogen peroxide (H₂O₂) for interspecies competition at the mucosal surfaces of the nasopharynx (41). The presence of O₂ is also a prerequisite for the pneumococcal X state (4, 14), which is a physiological condition that allows for genetic transformation and an adequate response to environmental stress (32). Recently, it was shown that the fatty acid (FA) content of the pneumococcal cell membrane (31) and the expression of 69 genes (8) change in response to the availability of O₂. Finally, changes in O₂ levels can also affect production of the polysaccharide capsule (48), which is the major pneumococcal virulence determinant.Similar to those of O₂, the levels of carbon dioxide (CO₂) vary considerably among the different pneumococcal niches inside and outside the host. Ambient levels of CO₂ in the environment are 0.038%, while CO₂ levels inside the human body, in particular in the lower respiratory tract, can reach 5% or more. The importance of this gaseous compound for S. pneumoniae is illustrated by the observation that the depletion of CO₂ from ambient air completely inhibits pneumococcal growth (21). Moreover, about 8% of all clinical isolates require a CO₂-enriched environment for growth in laboratory conditions (3). This intrinsic CO₂ dependence of S. pneumoniae and many other (micro)organisms is most likely related to an anabolic need for CO₂ or bicarbonate (HCO_3⁻) during biosynthesis of nucleic acids, amino acids, and FAs (1). Pathogens can often sequester CO₂ directly from host tissues, but in the absence of sufficient levels of extracellular CO₂, endogenous CO₂ needs to be enzymatically fixated. Carbonic anhydrases (CAs; EC 4.2.1.1) are enzymes that catalyze the reversible reaction CO₂ + H₂O ↔ HCO₃⁻ + H⁺. Because HCO₃⁻ cannot passively diffuse across biological membranes, its formation significantly delays the release of intracellular CO₂. At least five different classes of CAs have been described, and most eukaryotic, prokaryotic, and archaeal species express at least one CA class (39, 40).Genome analysis (39) has revealed that S. pneumoniae has one putative CA, a β-class CA that is highly conserved in all available pneumococcal genome sequences. Pneumococcal CA (PCA) is highly homologous to CAs in other streptococcal species, such as Streptococcus pyogenes. The closest nonstreptococcal PCA homologs are found in Mycobacterium species, while PCA homologs in other respiratory tract pathogens such as Neisseria meningitidis and Haemophilus influenzae are more divergent (40). The aim of this study was to investigate the functional characteristics of the pca gene and the encoded PCA enzyme in S. pneumoniae and to establish the relevance of PCA for pneumococcal growth and survival under CO₂-poor conditions in vitro. Further, we examined the importance of PCA during host-pathogen interaction.     

Quantitative proteome profiling of respiratory virus-infected lung epithelial cells

Respiratory virus infections are among the primary causes of morbidity and mortality in humans. Influenza virus, respiratory syncytial virus (RSV), parainfluenza (PIV) and human metapneumovirus (hMPV) are major causes of respiratory illness in humans. Especially young children and the elderly are susceptible to infections with these viruses. In this study we aim to gain detailed insight into the molecular pathogenesis of respiratory virus infections by studying the protein expression profiles of infected lung epithelial cells.A549 cells were exposed to a set of respiratory viruses [RSV, hMPV, PIV and Measles virus (MV)] using both live and UV-inactivated virus preparations. Cells were harvested at different time points after infection and processed for proteomics analysis by 2-dimensional difference gel electrophoresis. Samples derived from infected cells were compared to mock-infected cells to identify proteins that are differentially expressed due to infection.We show that RSV, hMPV, PIV3, and MV induced similar core host responses and that mainly proteins involved in defense against ER stress and apoptosis were affected which points towards an induction of apoptosis upon infection. By 2-D DIGE analyses we have gathered information on the induction of apoptosis by respiratory viruses in A549 cells.