Enteric coated spheres produced by extrusion/spheronization provide effective gastric protection and efficient release of live therapeutic bacteria

We present a novel but simple enteric coated sphere formulation containing probiotic bacteria (Lactobacillus casei). Oral delivery of live bacterial cells (LBC) requires live cells to survive firstly manufacturing processes and secondly GI microbicidal defenses including gastric acid. We incorporated live L. casei directly in the granulation liquid, followed by granulation, extrusion, spheronization, drying and spray coating to produce dried live probiotic spheres. A blend of MCC, calcium-crosslinked alginate, and lactose was developed that gave improved live cell survival during manufacturing, and gave excellent protection from gastric acid plus rapid release in intestinal conditions. No significant loss of viability was observed in all steps except drying, which resulted in approximately 1 log loss of viable cells. Eudragit coating was used to protect dried live cells from acid, and microcrystalline cellulose (MCC) was combined with sodium alginate to achieve efficient sphere disintegration leading to rapid and complete bacterial cell release in intestinal conditions. Viability and release of L. casei was evaluated in vitro in simulated GI conditions. Uncoated spheres gave partial acid protection, but enteric coated spheres effectively protected dried probiotic LBC from acid for 2 h, and subsequently released all viable cells within 1h of transfer into simulated intestinal fluid.

New insights into the impact of Lactobacillus population on host-bacteria metabolic interplay

We aimed at evaluating the association between intestinal Lactobacillus sp. composition and their metabolic activity with the host metabolism in adult and elderly individuals. Faecal and plasma metabolites were measured and correlated to the Lactobacillus species distribution in healthy Estonian cohorts of adult (n=16; <48 y) and elderly (n=33; >65 y). Total cholesterol, LDL, C-reactive protein and glycated hemoglobin were statistically higher in elderly, while platelets, white blood cells and urinary creatinine were higher in adults. Aging was associated with the presence of L. paracasei and L. plantarum and the absence of L. salivarius and L. helveticus. High levels of intestinal Lactobacillus sp. were positively associated with increased concentrations of faecal short chain fatty acids, lactate and essential amino acids. In adults, high red blood cell distribution width was positively associated with presence of L. helveticus and absence of L. ruminis. L. helveticus was correlated to lactate and butyrate in faecal waters. This indicates a strong relationship between the composition of the gut Lactobacillus sp. and host metabolism. Our results confirm that aging is associated with modulations of blood biomarkers and intestinal Lactobacillus species composition. We identified specific Lactobacillus contributions to gut metabolic environment and related those to blood biomarkers. Such associations may prove useful to decipher the biological mechanisms underlying host-gut microbial metabolic interactions in an ageing population.

Culex pipiens development is greatly influenced by native bacteria and exogenous yeast

Culex pipiens is the most cosmopolitan mosquito of the Pipiens Assemblage. By studying the nature of interactions between this species and microorganisms common to its breeding environment we can unravel important pitfalls encountered during development. We tested the survival rate of larval stages, pupae and adults of a Cx. pipiens colony exposed to a variety of microorganisms in laboratory conditions and assessed the transmission to offspring (F1) by those organisms that secured development up to adulthood. Three complementary experiments were designed to: 1) explore the nutritional value of yeasts and other microorganisms during Cx. pipiens development; 2) elucidate the transstadial transmission of yeast to the host offspring; and 3) to examine the relevance of all these microorganisms in female choice for oviposition-substratum. The yeast Saccharomyces cerevisiae proved to be the most nutritional diet, but despite showing the highest survival rates, vertical transmission to F1 was never confirmed. In addition, during the oviposition trials, none of the gravid females was attracted to the yeast substratum. Notably, the two native bacterial strains, Klebsiella sp. and Aeromonas sp., were the preferred oviposition media, the same two bacteria that managed to feed neonates until molting into 2nd instar larvae. Our results not only suggest that Klebsiella sp. or Aeromonas sp. serve as attractants for oviposition habitat selection, but also nurture the most fragile instar, L1, to assure molting into a more resilient stage, L2, while yeast proves to be the most supportive diet for completing development. These experiments unearthed survival traits that might be considered in the future development of strategies of Cx. pipiens control. These studies can be extended to other members of the Pipiens Assemblage

Microbial culture and checkerboard DNA-DNA hybridization assessment of bacteria in root canals of primary teeth pre- and post-endodontic therapy with a calcium hydroxide/chlorhexidine paste

Aim. To investigate the root canal microbiota of primary teeth with apical periodontitis and the in vivo antimicrobial effects of a calcium hydroxide/chlorhexidine paste used as root canal dressing. Design. Baseline samples were collected from 30 root canals of primary teeth with apical periodontitis. Then, the root canals were filled with a calcium hydroxide paste containing 1% chlorhexidine for 14 days and the second bacteriologic samples were taken prior to root canal filling. Samples were submitted to microbiologic culture procedure to detect root canal bacteria and processed for checkerboard DNA-DNA hybridization. Results. Baseline microbial culture revealed high prevalence and cfu number of anaerobic, black-pigmented bacteroides, Streptococcus, and aerobic microorganisms. Following root canal dressing, the overall number of cfu was dramatically diminished compared to initial contamination (P < 0.05), although prevalence did not change (P > 0.05). Of 35 probes used for checkerboard DNA-DNA hybridization, 31 (88.57%) were present at baseline, and following root canal dressing, the number of positive probes reduced to 13 (37.14%). Similarly, the number of bacterial cells diminished folowing application of calcium hydroxide/chlorhexidine root canal dressing (P = 0.006). Conclusion. Apical periodontitis is caused by a polymicrobial infection, and a calcium hydroxide/chlorhexidine paste is effective in reducing the number of bacteria inside root canals when applied as a root canal dressing.

A new type of Brazilian propolis: Prenylated benzophenones in propolis from Amazon and effects against cariogenic bacteria

Ethanol extracts of four propolis samples (E1-E4) from Manaus (Brazilian Amazon) were analysed by HPLC/DAD/ESI-MS/MS and GC/EIMS. The major constituents of E2 and E4 were analysed by NMR ((1)H and (13)C) and ESI/MS/MS. The main constituents of E2 and E4 are polyprenylated benzophenones: 7-epi-nemorosone, 7-epi-clusianone (major E4 constituents), xanthochymol and gambogenone (major E2 constituents), making up a chemical profile so far unreported for Brazilian propolis. Aristhophenone, methyl insigninone, 18-ethyloxy-17-hydroxy-17,18-dihydroscrobiculatone B, and derivatives of dimethyl weddellianone A and B, propolones, and a scrobiculatone derivative, were detected as minor constituents. Triterpenoids (beta-amyrins, beta-amyrenone, lupeol and lupenone) were ubiquitous and predominant in El and E3. The extracts E2 and E4 were highly active against the cariogenic bacteria Streptococcus mitis, Streptococcus mutans and Streptococcus salivarius. E2 was more active than E4, probably due to a higher content of 2-epi-nemorosone, while the latter was richer in di-hydroxylated compounds. (C) 2010 Elsevier Ltd. All rights reserved.

Vibrios dominate as culturable nitrogen-fixing bacteria of the Brazilian coral Mussismilia hispida

Taxonomic characterization was performed on the putative N-2-fixing microbiota associated with the coral species Mussismilia hispida, and with its sympatric species Palythoa caribaeorum, P. variabilis, and Zoanthus solanderi, off the coast of Sao Sebastiao (Sao Paulo State, Brazil). The 95 isolates belonged to the Gammaproteobacteria according to the 16S rDNA gene sequences. In order to identify the isolates unambiguously, pyrH gene sequencing was carried out. The majority of the isolates (n = 76) fell within the Vibrio core group, with the highest gene sequence similarity being towards Vibrio harveyi and Vibrio alginolyticus. Nineteen representative isolates belonging to V. harveyi (n = 7), V. alginolyticus (n = 8), V. campbellii (n = 3), and V parahaemolyticus (n = 1) were capable of growing six successive times in nitrogen-free medium and some of them showed strong nitrogenase activity by means of the acetylene reduction assay (ARA). It was concluded that nitrogen fixation is a common phenotypic trait among Vibrio species of the core group. The fact that different Vibrio species can fix N, might explain why they are so abundant in the mucus of different coral species. (C) 2008 Published by Elsevier GmbH.

Occurrence of herpes simplex virus 1 and three periodontal bacteria in patients with chronic periodontitis and necrotic pulp

Viral and bacterial associations appear to be implicated in the development of periodontal infections. Little information is available describing the periodontopathic agents in root canals with necrotic pulp. In this study, the occurrence and the combinations among herpes simplex virus type 1 (HSV-1) and Dialister pneumosintes, Tannerella forsythia.. and Treponema denticola in patients with chronic periodontitis and necrotic pulp were evaluated. Clinical samples from healthy subjects and patients with periodontal or pulp infections were analyzed using a nested polymerase chain reaction PCR to detect HSV and PCR to detect the 3 periodontal bacteria. The presence of Tannerella forsythia and Treponema denticola was observed in healthy, periodontitis, and necrotic pulp patients. HSV was observed in periodontitis and necrotic pulp patients, and no healthy subject harbored D. pneumosintes or HSV. The occurrence of Tannerella forsythia was not statistically significant in patients with necrotic pulp (P = 0.704). Periodontal bacteria were observed varying from 10.3% to 20.7% in periodontitis and necrotic pulp patients. The presence of Treponema denticola - HSV association was predominant in patients showing necrotic pulp (24.1%); however, HSV alone was observed in one patient with periodontitis and in another patient with necrotic pulp. The presence of double association among bacteria or bacteria - HSV could indicate a role in both periodontitis and necrotic pulp, and Tannerella forsythia - Treponenta denticola - HSV and Tannerella forsythia - D. pneumosintes - Treponema denticola - HSV associations might be important in periodontitis.

Screening for endophytic nitrogen-fixing bacteria in Brazilian sugar cane varieties used in organic farming and description of Stenotrophomonas pavanii sp. nov.

A Gram-negative, rod-shaped, non-spore-forming and nitrogen-fixing bacterium, designated ICB 89(T), was isolated from stems of a Brazilian sugar cane variety widely used in organic farming. 16S rRNA gene sequence analysis revealed that strain ICB 89(T) belonged to the genus Stenotrophomonas and was most closely related to Stenotrophomonas maltophilia LMG 958(T), Stenotrophomonas rhizophila LMG 22075(T), Stenotrophomonas nitritireducens L2(T), [Pseudomonas] geniculata ATCC 19374(T), [Pseudomonas] hibiscicola ATCC 19867(T) and [Pseudomonas] beteli ATCC 19861(T). DNA-DNA hybridization together with chemotaxonomic data and biochemical characteristics allowed the differentiation of strain ICB 89(T) from its nearest phylogenetic neighbours. Therefore, strain ICB 89(T) represents a novel species, for which the name Stenotrophomonas pavanii sp. nov. is proposed. The type strain is ICB 89(T) (=CBMAI 564(T) =LMG 25348(T)).

Presence of periodontopathic bacteria in coronary arteries from patients with chronic periodontitis

In this study the presence of periodontopathic pathogens in atheromatous plaques removed from coronary arteries of patients with chronic periodontitis and periodontally healthy subjects by PCR was detected. Our results indicate a significant association between the presence of Porphyromonas gingivalis and atheromas, and the periodontal bacteria in oral biofilm may find a way to reach arteries. (C) 2010 Elsevier Ltd. All rights reserved.

Influence of organic fertilization on the number of culturable diazotrophic endophytic bacteria isolated from sugarcane

The numbers of culturable diazotrophic endophytic bacteria (CDEB) from roots stems and leaves of sugarcane submitted to organic inorganic or no fertilization were compared In order to determine the size of the N(2) fixing populations the Most Probable Number technique (MPN) was used The quantification of diazotrophic bacteria by using the acetylene reduction assay (ARA) was more accurate than observing the bacterial growth in the vials to confirm N(2) fixing capability the detection of gene nifH was performed on a sample of 105 Isolated bacteria The production of extracellular enzymes involved in the penetration of the plants by the bacteria was also studied The results showed that organic fertilization enhances the number of CDEB when compared with conventional fertilization used throughout the growing season The maximum number of bacteria was detected in the roots Roots and stems presented the greatest number of CDEB in the middle of the cropping season and in leaves numbers varied according to the treatment Using two pairs of primers and two different methods the nifH gene was found in 104 of the 105 tested isolates Larger amounts of pectinase were released by isolates from sugarcane treated with conventional fertilizers (66%) whereas larger amounts of cellulase were released by strains isolated from sugarcane treated with organic fertilizers (80%) (C) 2010 Elsevier Masson SAS All rights reserved

Quantitative detection of periodontopathic bacteria in atherosclerotic plaques from coronary arteries

Oral pathogens, including periodontopathic bacteria, are thought to be aetiological factors in the development of cardiovascular disease. In this study, the presence of Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum-periodonticum-simiae group, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Tannerella forsythia in atheromatous plaques from coronary arteries was determined by real-time PCR. Forty-four patients displaying cardiovascular disease were submitted to periodontal examination and endarterectomy of coronary arteries. Approximately 60-100 mg atherosclerotic tissue was removed surgically and DNA was obtained. Quantitative detection of periodontopathic bacteria was performed using universal and species-specific TaqMan probe/primer sets. Total bacterial and periodontopathic bacterial DNA were found in 94.9 and 92.3 %, respectively, of the atheromatous plaques from periodontitis patients, and in 80.0 and 20.0%, respectively, of atherosclerotic tissues from periodontally healthy subjects. All periodontal bacteria except for the F. nucleatum-periodonticum-simiae group were detected, and their DNA represented 47.3 % of the total bacterial DNA obtained from periodontitis; patients. Porphyromonas gingivalis, A. actinomycetemcomitans and Prevotella intermedia were detected most often. The presence of two or more periodontal species could be observed in 64.1 % of the samples. In addition, even in samples in which a single periodontal species was detected, additional unidentified microbial DNA could be observed. The significant number of periodontopathic bacterial DNA species in atherosclerotic tissue samples from patients with periodontitis suggests that the presence of these micro-organisms in coronary lesions is not coincidental and that they may in fact contribute to the development of vascular diseases.

Screening of bacteria to produce polyhydroxyalkanoates from xylose

Although xylose is a major constituent of lignocellulosic feedstock and the second most abundant sugar in nature, only 22% of 3,152 screened bacterial isolates showed significant growth in xylose in 24 h. Of those 684, only 24% accumulated polyhydroxyalkanoates after 72 h. A mangrove isolate, identified as Bacillus sp. MA3.3, yielded the best results in literature thus far for Gram-positive strains in experiments with glucose and xylose as the sole carbon source. When glucose or xylose were supplied, poly-3-hydroxybutyrate (PHB) contents of cell dry weight were, respectively, 62 and 64%, PHB yield 0.25 and 0.24 g g(-1) and PHB productivity (P(PHB)) 0.10 and 0.06 g l(-1) h(-1). This 40% P(PHB) difference may be related to the theoretical ATP production per 3-hydroxybutyrate (3HB) monomer calculated as 3 mol mol(-1) for xylose, less than half of the ATP/3HB produced from glucose (7 mol mol(-1)). In PHB production using sugar mixtures, all parameters were strongly reduced due to carbon catabolite repression. PHB production using Gram-positive strains is particularly interesting for medical applications because these bacteria do not produce lipopolysaccharide endotoxins which can induce immunogenic reactions. Moreover, the combination of inexpensive substrates and products of more value may lead to the economical sustainability of industrial PHB production.

NAD Biosynthesis Evolution in Bacteria: Lateral Gene Transfer of Kynurenine Pathway in Xanthomonadales and Flavobacteriales

The biosynthesis of quinolinate, the de novo precursor of nicotinamide adenine dinucleotide (NAD), may be performed by two distinct pathways, namely, the bacterial aspartate (aspartate-to-quinolinate) and the eukaryotic kynurenine (tryptophan-to-quinolinate). Even though the separation into eukaryotic and bacterial routes is long established, recent genomic surveys have challenged this view, because certain bacterial species also carry the genes for the kynurenine pathway. In this work, both quinolinate biosynthetic pathways were investigated in the Bacteria clade and with special attention to Xanthomonadales and Bacteroidetes, from an evolutionary viewpoint. Genomic screening has revealed that a small number of bacterial species possess some of the genes for the kynurenine pathway, which is complete in the genus Xanthomonas and in the order Flavobacteriales, where the aspartate pathway is absent. The opposite pattern (presence of the aspartate pathway and absence of the kynurenine pathway) in close relatives (Xylella ssp. and the order Bacteroidales, respectively) points to the idea of a recent acquisition of the kynurenine pathway through lateral gene transfer in these bacterial groups. In fact, sequence similarity comparison and phylogenetic reconstruction both suggest that at least part of the genes of the kynurenine pathway in Xanthomonas and Flavobacteriales is shared by eukaryotes. These results reinforce the idea of the role that lateral gene transfer plays in the configuration of bacterial genomes, thereby providing alternative metabolic pathways, even with the replacement of primary and essential cell functions, as exemplified by NAD biosynthesis.

Growth, encystment and survival of Acanthamoeba castellanii grazing on different bacteria

Free-living amoebae of the genus Acanthamoeba are widely distributed in soil and water collections, where trophozoites (vegetative, multiplicative stages) feed mainly by phagocytosis and thus control bacterial populations in the environment. Here, we examined the growth, encystment and survival of Acanthamoeba castellanii receiving different bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Bacillus subtilis, Bacillus megaterium, Micrococcus luteus, and Staphylococcus aureus) in nonnutrient saline. All bacteria assayed induced a dose-dependent proliferative response, in most cases maximized with a bacterial dose of 1 x 10(9) mL(-1); except for M. luteus, trophozoites grew better with viable than with heat-killed bacteria. In addition, Acanthamoeba growth was improved by adding bacteria on alternate days. Single-dose experiments indicated a temporal association between the growth of trophozoite and bacterial consumption, and higher consumption of M. luteus, E. coli and P. aeruginosa, bacterial species that allowed the highest trophozoite yields. Long-term Acanthamoeba-bacteria incubation revealed that encystment was significantly delayed by almost all the bacteria assayed (including S. aureus, which elicited a poor growth response) and that the presence of bacteria markedly increased cyst yield; final cyst recovery clearly depended on both the dose and the type of the bacterium given, being much higher with E. coli, M. luteus and P. aeruginosa.