Temporal Temperature Gradient Gel Electrophoresis Research Articles

Consider Non-Syndromic Mitochondrial Disorders as Part of the Pediatric Phenotypic Spectrum

With interest we read the article by Ardissone et al. about a retrospective, singlecenter study of 150 pediatric patients with a genetically confirmed mitochondrial disorder (MID) due to a pathogenic mtDNA variant (mtDNA point mutation or single mtDNA deletion) [1]. The study is excellent but has limitations that are cause of concerns and should be discussed. A limitation of the study is that only syndromic MIDs were included [1]. Since MIDs often do not fit into any of the syndromic phenotypes, we should know whether these patients were deliberately excluded or did not appear at the study center at all. We should know whether the entire group of 902 patients also included patients with nonsyndromic MIDs. Another limitation is that heteroplasmy rates were only determined by estimating the densitometric ratio between wild-type and mutant mtDNA [1]. Heteroplasmy is usually determined by a number of quantitative methods, such as Sanger sequencing, high performance liquid chromatography, pyrosequencing, Snapshot, high resolution melt profiling (HRM), temporal temperature gradient gel electrophoresis (TTGE), invader assay, amplification refractory mutation system (ARMS), endonuclease method using Surveyor nuclease, and next generation sequencing (NGS) [2]. These techniques usually provide more accurate results than semi-quantitative estimation. There is a discrepancy between the statement in the method section ("only few data were not available from all patients”) and the results section (“CSF data were available from only 17 %, follow-up data only from 47%, and biochemical data only from 58 % of patients”) [1]. This discrepancy should be clarified. Another limitation is that the total observation time up to the last follow-up was not given [1]. Knowing this latency is important to assess the speed of disease progression and estimate the outcome. There is also a discrepancy between the statement in the result section that “outcome data were evaluated from 70 patients” and the detailed description in the results section, which included 71 patients. Which number is correct? Surprisingly, the biochemical examination was normal in almost a quarter of the patients with Leigh syndrome. We should know if these results are from studies of just muscle or fibroblasts, or if other tissues have been studied as well If only muscle or fibroblasts were examined, we should know whether or not these tissues were clinically affected in the one quarter of the Leigh patients. Surprisingly, only two thirds of the MELAS patients presented with stroke-like episodes (SLEs), which are pathognomonic for MELAS. We should know if this was due to the short observation or follow-up time in these patients. According to what criteria were these patients without SLE classified as MELAS? Were the Hirano or Japanese criteria applied? We should also know how “MELAS-like” was defined. There is no mention of the underlying mutations in the PEO patients. It would be interesting to know if these patients all carried a single mtDNA deletion, a nuclear DNA-located variant, or an mtDNA point mutation. Overall, the interesting study has limitations that call the results and their interpretation into question. With a prospective design and extensive and long-term follow-up, the study goals could be better achieved.

Effect of daily co-exposure to inulin and chlorpyrifos on selected microbiota endpoints in the SHIME® model

The intestinal microbiota has a key role in human health via the interaction with the somatic and immune cells in the digestive tract environment. Food, through matrix effect, nutrient and non-nutrient molecules, is a key regulator of microbiota diversity. As a food contaminant, the pesticide chlorpyrifos (CPF) has an effect on the composition of the intestinal microbiota and induces perturbation of microbiota. Prebiotics (and notably inulin) are known for their ability to promote an equilibrium of the microbiota that favours saccharolytic bacteria. The SHIME® dynamic in vitro model of the human intestine was exposed to CPF and inulin concomitantly for 30 days, in order to assess variations in both the bacterial populations and their metabolites. Various analyses of the microbiota (notably temporal temperature gradient gel electrophoresis) revealed a protective effect of the prebiotic through inhibition of the enterobacterial (E. coli) population. Bifidobacteria were only temporarily inhibited at D15 and recovered at D30. Although other potentially beneficial populations (lactobacilli) were not greatly modified, their activity and that of the saccharolytic bacteria in general were highlighted by an increase in levels of short-chain fatty acids and more specifically butyrate. Given the known role of host-microbiota communication, CPF's impact on the body's homeostasis remains to be determined.

High bacterial activity in nutrient rich saltwater: Evidence from the uncoupling between salinity and nutrients in the Patos Lagoon estuary

In estuaries, a common pattern found is of higher bacterial activity in freshwater than in saltwater, an indication of the existence of an activity gradient. It is not clear, however, whether this arises from a natural property of saltwater bacterial communities or from metabolic constraints imposed from the natural co-variation between salinity and nutrients in estuarine systems. In this study, we managed to detect in situ higher activity levels of free-living bacteria in nutrient-rich saltwater entering the Patos Lagoon estuary. The bacterial community composition (BCC - Temporal Temperature Gradient Gel Electrophoresis) and activity (community-level physiological profiles, CLPP- Ecoplate™ - Biolog) were evaluated during two days in a transect along the estuary until the adjacent coastal region. The unexpected higher nutrient content and bacterial activity in saltwater found suggest that the bacterial activity is a primary response to nutrient availability, whereas salinity is the main factor structuring BCC in this estuary. Our results reject the idea of a natural low activity of saltwater bacteria by showing that their activity can increase in situ given that the proper conditions are met, especially in estuaries that display circulation patterns that promote the occurrence of nutrient-rich saltwater.

Bacterial diversity of artisanal cheese from the Amazonian region of Brazil during the dry and rainy seasons

The microbiota from artisanal cheeses produced in the Amazonian region is evaluated. Samples of artisanal cheeses were obtained from markets in Conceição do Araguaia and Redenção (Pará, Brazil) over rainy and dry seasons, and their biodiversity was assessed by culture-dependent and culture-independent methods. Mean counts of lactic acid bacteria (LAB) in cheeses ranged from 7.32 to 8.84 log CFU/g, for both seasons. Members of genera Lactobacillus, Lactococcus, Weissella, Enterococcus, Pediococcus, and Leuconostoc were predominant. The amplification of the 16S rRNA V6-V9 region, followed by a temporal temperature-gradient gel electrophoresis (TTGE) and sequencing of the TTGE bands revealed important differences in the microbial composition variability between samples from the two seasons and among cheese samples analyzed. TTGE showed the presence of microorganisms that are frequently found in cheese, such as L. lactis subsp. lactis, as well as other non-usual species, such as Macrococcus caseolyticus and Corynebacterium variabile. Moreover, TTGE analysis revealed the presence of microorganisms that have been isolated from other types of foods (Paralactobacillus selangorenses) along with some not usually found in foods, such as Exiguobacterium acetylicum, plus the presence of pathogenic microorganisms (Granulicatella elegans and Aerococcus sanguinicola). The present molecular approaches combined with culture-dependent methods provided a more detailed description of the microbial ecology of traditional cheeses from the Amazonian region in northern Brazil.

First Report of Swim Bladder-Associated Microbiota in Rainbow Trout (Oncorhynchus mykiss).

The aim of the present study was to identify major bacteria associated with the swim bladder in the rainbow trout, Oncorhynchus mykiss. We extracted DNA from the swim bladder and gut contents in order to perform a temporal temperature gradient gel electrophoresis (TTGE) analysis of 16S rRNA amplicons for bacterial identification to further compare both profiles. Arthrobacter and Cellulosimicrobium were the major genera observed in the swim bladder in fish, but were not present in fish gut contents; Mycoplasma were instead observed in these samples. Further research to investigate the possible symbiotic roles of the swim bladder-associated microbiota in salmonids is needed.

Establishment and development of the intestinal microbiota of preterm infants in a Lebanese tertiary hospital

The establishment and development of the intestinal microbiota is known to be associated with profound short- and long-term effects on the health of full-term infants (FTI), but studies are just starting for preterm infants (PTI). The data also mostly come from western countries and little information is available for the Middle East. Here, we determined the composition and dynamics of the intestinal microbiota during the first month of life for PTI (n = 66) and FTI (n = 17) in Lebanon. Fecal samples were collected weekly and analyzed by quantitative PCR (q-PCR) and temporal temperature gradient gel electrophoresis (TTGE). We observed differences in the establishment and composition of the intestinal microbiota between the two groups. q-PCR showed that PTI were more highly colonized by Staphylococcus than FTI in the first three weeks of life; whereas FTI were more highly colonized by Clostridium clusters I and XI. At one month of life, PTI were mainly colonized by facultative anaerobes and a few strict anaerobes, such as Clostridium cluster I and Bifidobacterium. The type of feeding and antibiotic treatments significantly affected intestinal colonization. TTGE revealed low species diversity in both groups and high inter-individual variability in PTI. Our findings show that PTI had altered intestinal colonization with a higher occurrence of potential pathogens (Enterobacter, Clostridium sp) than FTI. This suggests the need for intervention strategies for PTI to modulate their intestinal microbiota and promote their health.

Gut microbiota related to Giardia duodenalis, Entamoeba spp. and Blastocystis hominis infections in humans from Côte d'Ivoire.

Literature data provide little information about protozoa infections and gut microbiota compositional shifts in humans. This preliminary study aimed to describe the fecal bacterial community composition of people from Côte d'Ivoire harboring Giardia duodenalis, Entamoeba spp., and Blastocystis hominis, in trying to discover possible alterations in their fecal microbiota structure related to the presence of such parasites. Twenty fecal samples were collected from people inhabiting three different localities of Côte d'Ivoire for copromicroscopic analysis and molecular identification of G. duodenalis, Entamoeba spp., and B. hominis. Temporal temperature gradient gel electrophoresis (TTGE) was used to obtain a fingerprint of the overall bacterial community; quantitative polymerase chain reaction (qPCR) was used to define the relative abundances of selected bacterial species/group, and multivariate statistical analyses were employed to correlate all data. Cluster analysis revealed a significant separation of TTGE profiles into four clusters (p < 0.0001), with a marked difference for G. duodenalis-positive samples in relation to the others (p = 5.4×10-6). Interestingly, qPCR data showed how G. duodenalis-positive samples were related to a dysbiotic condition that favors potentially harmful species (such as Escherichia coli), while Entamoeba spp./B. hominis-positive subjects were linked to a eubiotic condition, as shown by a significantly higher Faecalibacterium prausnitzii-Escherichia coli ratio. This preliminary investigation demonstrates a differential fecal microbiota structure in subjects infected with G. duodenalis or Entamoeba spp./B. hominis, paving the way for using further next-generation DNA technologies to better understand host-parasite-bacteria interactions, aimed at identifying potential indicators of microbiota changes.

Structural differences in gut bacteria communities in developmental stages of natural populations of Lutzomyia evansi from Colombia's Caribbean coast.

BackgroundLutzomyia evansi, a phlebotomine insect endemic to Colombia’s Caribbean coast, is considered to be the main vector of visceral and cutaneous leishmaniasis in the region. Although insects of this species can harbor pathogenic and non-pathogenic microorganisms in their intestinal microbiota, there is little information available about the diversity of gut bacteria present in Lutzomyia evansi. In this study, conventional microbiological methods and molecular tools were used to assess the composition of bacterial communities associated with Lutzomyia evansi guts in immature and adult stages of natural populations from the department of Sucre (Caribbean coast of Colombia).MethodsSand flies were collected from two locations (peri-urban and jungle biotype) in the Department of Sucre (Caribbean coast of Colombia). A total of 752 Lutzomyia evansi intestines were dissected. In this study, 125 bacterial strains were isolated from different culture media (LB Agar, MacConkey Agar). Different methods were used for bacterial identification, including ribosomal intergenic spacer analysis (RISA) and analysis of the 16S rRNA and gyrB gene sequences. The genetic profiles of the bacterial populations were generated and temporal temperature gradient gel electrophoresis (TTGE) was used to compare them with total gut DNA. We also used PCR and DNA sequence analysis to determine the presence of Wolbachia endosymbiont bacteria and Leishmania parasites.ResultsThe culture-dependent technique showed that the dominant intestinal bacteria isolated belong to Acinetobacter, Enterobacter, Pseudomonas, Ochrobactrum, Shinella and Paenibacillus in the larval stage; Lysobacter, Microbacterium, Streptomyces, Bacillus and Rummeliibacillus in the pupal stage; and Staphylococcus, Streptomyces, Brevibacterium, Acinetobacter, Enterobacter and Pantoea in the adult stage. Statistical analysis revealed significant differences between the fingerprint patterns of the PCR-TTGE bands in bacterial communities from immature and adult stages. Additionally, differences were found in bacterial community structure in fed females, unfed females, males and larvae. The intestinal bacteria detected by PCR-TTGE were Enterobacter cloacae and Bacillus thuringiensis, which were present in different life stages of Lu. evansi, and Burkholderia cenocepacia and Bacillus gibsonii, which were detected only in the larval stage. Wolbachia and Leishmania were not detected in gut samples of Lutzomyia evansi.ConclusionsThe analyses conducted using microbiological and molecular approaches indicated significant variations in the bacterial communities associated with the gut of Lu. evansi, depending on the developmental stage and food source. We propose that these elements affect microbial diversity in L. evansi guts and may in turn influence pathogen transmission to humans bitten by this insect.

Comparative Analysis of Denaturing Gradient Gel Electrophoresis and Temporal Temperature Gradient Gel Electrophoresis Profiles as a Tool for the Differentiation of Candida Species.

Background:Candida species are usually opportunistic organisms that cause acute to chronic infections when conditions in the host are favorable. Accurate identification of Candida species is an essential pre-requisite for improved therapeutic strategy. Identification of Candida species by conventional methods is time-consuming with low sensitivity, yet molecular approaches have provided an alternative way for early diagnosis of invasive candidiasis. Denaturing gradient gel electrophoresis (DGGE) and temporal temperature gradient gel electrophoresis (TTGE) are polymerase chain reaction (PCR)-based approaches that are used for studying the community structure of microorganisms. By using these methods, simultaneous identification of multiple yeast species will be possible and reliable results will be obtained quickly.Objectives:In this study, DGGE and TTGE methods were set up and evaluated for the detection of different Candida species, and their results were compared.Materials and Methods:Five different Candida species were cultured on potato dextrose agar medium for 24 hours. Next, total DNA was extracted by the phenol-chloroform method. Two sets of primers, ITS3-GC/ITS4 and NL1-GC/LS2 were applied to amplify the desired regions. The amplified fragments were then used to analyze DGGE and TTGE profiles.Results:The results showed that NL1-GC/LS2 primer set could yield species-specific amplicons, which were well distinguished and allowed better species discrimination than that generated by the ITS3-GC/ITS4 primer set, in both DGGE and TTGE profiles. All five Candida species were discriminated by DGGE and TTGE using the NL1-GC/LS2 primer set.Conclusions:Comparison of DGGE and TTGE profiles obtained from NL1-GC/LS2 amplicons exhibited the same patterns. Although both DGGE and TTGE techniques are capable of detecting Candida species, TTGE is recommended because of easier performance and lower costs.

Molecular mechanisms of lung cancer development at its different stages in nuclear industry workers

to assess mutational events in exons 5, 7, and 8 of the p53 gene and to reveal mutant p53 protein in verified cases of morphologically altered (proliferative and precancerous changes, lung cancer) and histologically unaltered, lung tissues in workers exposed to occupational radiation. The investigation used formalin-fixed paraffin-embedded unaltered and altered lung tissue blocks (FFPBs) obtained from the human radiobiological tissue repository. The shelf-life of FFPBs was 5-31 years. An immunohistochemical technique using mouse antibodies against p53 protein (<<DAKO>>, Denmark), stained with diaminobenzidine (DAB) chromogen, was employed to determine p53 protein. DNA was isolated from lung tissue FFPBs with QIAmp DNA FFPE Tissue Kit, (<<QIAGEN>>, USA). Polymerase chain reaction (PCR) was performed to amplify the p53 gene exons 5, 7, and 8 selected for examination, by applying the sequences of genes and primers, the specificity of which was checked using the online resource (http://www.ncbi.nlm.nih.gov/blast). PCR products were detected by temporal temperature gradient gel-electrophoresis and the Sanger sequencing method. The obtained DNA fragments were analyzed on a sequencer ABI Prism 3100 Genetic Analizer (<<Applied Biosystems>>, USA). Computer-aided DNA analysis was made using the BLAST program. A package of applied Statistica 6.0 programs was employed for statistical data processing. Results. Immunohistochemical analysis showed that mutant p53 protein was absent in the cells of unaltered lung tissue and the number of cells with mutant p53 protein increased in all the patients with proliferative and precancerous changes and lung cancer, suggesting p53 protein dysfunction. The total number of p53 gene mutations in exons 5, 7, and 8, if there were proliferative and precancerous lung tissue changes and lung cancer, were 25, 20, and 40%, respectively. All the found mutations were transversions (the substitution of purine for pyrimidine or, conversely), indicating the action of exogenous mutagens. The results of this investigation have confirmed other investigators' data showing that p53 gene mutations in lung cancer are observed in 40-70% of cases. The differences in the number of cases of altered lung tissue with mutations in the p53 gene (not more than 40%) and in those of p53 protein expression were found in 100%, suggesting the regulation of p53 gene function in the cell at multiple levels.

Association of germ-free mice with a simplified human intestinal microbiota results in a shortened intestine

Genetic, nutritional, and gut microbiota-derived factors have been proposed to play a role in the development of the whole intestine that is around 40% longer in PRM/Alf mice compared with other mouse strains. The PRM/Alf genotype explains 60% of this length difference. The remaining 40% are due to a maternal effect that could depend on the gut microbiota transmitted by the mother to their pups. Germ-free PRM/Alf mice and C3H/He mice were associated with a simplified human microbiota (SIHUMI) to study its impact on gut length. The small intestines of the SIHUMI-associated mice were 16.4% (PRM/Alf) and 9.7% (C3H/He) shorter than those of the corresponding germ-free counterparts. Temporal temperature gradient gel electrophoresis and quantitative real-time PCR revealed differences in microbiota composition between both SIHUMI-associated mouse strains. Anaerostipes caccae was one log lower in PRM/Alf mice than in C3H/He mice. Since polyamines and short-chain fatty acids (SCFAs) are important intestinal growth factors, their concentrations were explored. Cecal concentrations of putrescine, spermine, spermidine, and N-acetylspermine were 1.5-fold, 3.7-fold, 2.2-fold, and 1.4-fold higher, respectively, in the SIHUMI-C3H/He mice compared with the SIHUMI-PRM/Alf mice. In addition, cecal acetate, propionate, and butyrate concentrations in SIHUMI-C3H/He mice were 1.4-fold, 1.1-fold, and 2.1-fold higher, respectively, than in SIHUMI-PRM/Alf mice. These results indicate that polyamines and SCFAs did not promote gut lengthening in any of the two mouse strains. This suggests that as yet unknown factors provided by the SIHUMI prevented gut lengthening in the SIHUMI-associated mice compared with the germfree mice.

Effects of non-chemical soil fumigant treatments on root colonisation with arbuscular mycorrhizal fungi and strawberry fruit production

The effects of biofumigation and soil heating on arbuscular mycorrhizal fungi (AMF) colonisation, strawberry growth and strawberry yield in pot experiments compared with untreated soil and chemical fumigation with dazomet were tested. Three different Brassicaceae species (Brassica juncea, Eruca sativa, Sinapis alba) were used as biofumigant plant green manure and soil heating was applied to simulate soil solarisation. Half of the plants were inoculated with indigenous arbuscular mycorrhizal fungi inoculum. With one exception (E. sativa) among the uninoculated plants, the treatments significantly decreased the mycorrhizal colonisation parameters compared with the untreated control. Dazomet displayed the greatest inhibitory effects on AMF establishment. In addition, the intensity and number of bands corresponding to Glomus spp. obtained with temporal temperature-gradient gel electrophoresis were lower for strawberry plants from biofumigant treatments than from the control. For the inoculated plants, there were almost no significant differences among the mycorrhizal colonisation parameters. The mass of leaves for the uninoculated and inoculated plants was higher for almost all non-chemical soil fumigant treatments compared with the control, except for heating of the uninoculated treatments. The number of strawberry fruits for the uninoculated biofumigant treatments was the highest, being higher than the values observed for the heating treatments, the chemical disinfection treatments and the control. There were no significant differences among the inoculated treatments. Biofumigation with Brassicaceae species resulted in higher soil organic matter and mineral nutrients and had a relatively small effect on AMF colonisation (F% = 59.0, 80.3, 47.3 for Bj, Es and Sa, respectively) compared with uninoculated controls (F% = 84.3). Despite the reduced AMF colonisation, biofumigation resulted in a higher fruit number and mass of leaves. Therefore, it represents a non-chemical soil fumigation method that should be applied in sustainable strawberry production.

Biological and chemical analyses of a laboratory-scale biofilter for oxygenate bioremediation in simulated groundwater

Microbial remediation of methyl tert-butyl ether-contaminated aquifers has been widely studied since their cost/efficiency ratios are lower than other remediating techniques. Based on previous studies, simultaneous assessment of two inocula (Acinetobacter calcoaceticus M10 and a co-culture between strain M10 and Rhodococcus ruber E10) was performed for methyl tert-butyl ether bioremediation in a designed pilot-scale biofilter. A non-inoculated biofilter was included for comparison. Temporary bioremediation of methyl tert-butyl ether (up to 80 % removal) from the biofilter inoculated with the consortium was observed within 44 days. In addition, the taxonomic profile (temporal temperature gradient gel electrophoresis from RNA extracts) from the latter biofilter contained more active strains than in the other two systems. The presence of strains M10 and E10 in temporal temperature gradient gel electrophoresis analysis from RNA extracts and high presence of strain E10 in temporal temperature gradient gel electrophoresis analysis of DNA extracts, along with alkB amplification of both strains in the biofilter, suggest that the co-culture inoculum was responsible for the methyl tert-butyl ether removal.

An improved method compatible with metagenomic analyses to extract genomic DNA from soils in Tuber melanosporum orchards

The development of high-throughput methods such as pyrosequencing and microarrays has greatly improved our understanding of the microbial diversity in complex environments such as soils. Nevertheless, albeit advancements in such techniques, the first major step is to obtain high quantity and good quality genomic DNA (gDNA). The work presented here aims to present an inherent problem with 260:230nm ratio of extracted gDNA from calcareous soils of Tuber melanosporum orchards and a protocol to overcome this problem. Using two commercial gDNA extraction kits on spatially distant truffle orchards, we demonstrated that the 260:230nm ratio was very low, consequentially yielding gDNA incompatible with microarray analyses. In order to solve this problem, optimization steps were tested including several wash steps performed before and/or after lysis. These washes significantly improved the gDNA quality (ratio 260:230nm >1·7) without modification of the structure of the bacterial communities as stated by temporal temperature gradient gel electrophoresis analysis. A final re-extraction with phenol/chloroform was required for one of the soil samples. A combination of wash steps included into the extraction protocol followed by phenol: chloroform re-extraction is recommended to obtain high-quality gDNA from calcareous soils of T.melanosporum orchards. The method recommended here significantly improves gDNA quality obtained from T.melanosporum orchards to make it acceptable for highly sensitive methods such as microarray.

Biodiversity and dynamics of the bacterial community of packaged king scallop (Pecten maximus) meat during cold storage

The microbial biodiversity and dynamics of king scallops meat and coral during cold storage (cold chain rupture: 1/3 storage time at 4 °C followed by 2/3 at 8 °C), was assessed by combining culture-dependant and -independent methods. Products were packaged as follows: aerobic, vacuum packed and 3 different CO2/N2 modified atmospheres and the impact of these conditions on the microbial communities was assessed. Results indicated that under air (current packaging condition), the dominant species corresponded to Brochothrix thermosphacta, Pseudomonas spp. and Shewanella spp. These species have regularly been associated in the literature with food (especially seafood), and product spoilage. Moellerella wisconsensis was the only species detected on VRBG medium, however, its impact on the food product is unclear. Packaging conditions influenced the ecosystem equilibrium and biodiversity. Except for day 8, the lowest counts for all studied flora were observed for modified atmosphere packaging (MAP) containing >80% CO2. Moreover, in these conditions, higher biodiversity by Temporal Temperature Gradient Gel Electrophoresis (TTGE) and the non-detection of specific flora (i.e. Pseudoalteromonas haloplanktis) were observed. At day 8, scallops packaged using these conditions were still acceptable from a sensorial point of view (odour), although the initial load of the king scallop was high (total psychrotrophic flora reached 5.5 log CFU/g).

Long-Term Changes in Human Colonic Bifidobacterium Populations Induced by a 5-Day Oral Amoxicillin-Clavulanic Acid Treatment

The objective of this study was to assess the possible modifications due to amoxicillin-clavulanic acid (AMC) treatment on total bacteria and on Bifidobacterium species balance in human colonic microbiota. Eighteen healthy volunteers (19 to 36 years old) were given a 875/125 mg dose of AMC twice a day for 5 days. Fecal samples were obtained before and after antibiotic exposure. After total DNA extraction, total bacteria and bifidobacteria were specifically quantified using real-time PCR. Dominant species were monitored over time using bacterial and bifidobacterial Temporal Temperature Gradient gel Electrophoresis (TTGE). At the end of AMC exposure, total bacterial concentrations as well as bifidobacteria concentrations were significantly reduced compared to before AMC exposure:10.7±0.1 log10 16S rRNA gene copies/g vs 11.1±0.1 log10 (p = 0.003) and 8.1±0.5 log10 16S rRNA gene copies/g vs 9.4±0.3 log10 (p = 0.003), respectively. At the same time, the mean similarity percentages of TTGE bacteria and TTGE bifidobacteria profiles were significantly reduced compared to before AMC exposure: 51.6%±3.5% vs 81.4%±2.1% and 55.8%±7.6% vs 84.5%±4.1%, respectively. Occurrence of B. adolescentis, B. bifidum and B. pseudocatenulatum/B. catenulatum species significantly decreased. Occurrence of B. longum remained stable. Moreover, the number of distinct Bifidobacterium species per sample significantly decreased (1.5±0.3 vs 2.3±0.3; p = 0.01). Two months after AMC exposure, the mean similarity percentage of TTGE profiles was 55.6% for bacteria and 62.3% for bifidobacteria. These results clearly demonstrated that a common antibiotic treatment may qualitatively alter the colonic microbiota. Such modifications may have potential long-term physiological consequences.

16S rRNA Gene-targeted TTGE in Determining Diversity of Gut Microbiota during Acute Diarrhoea and Convalescence

The human gut microbiota play a vital role in health and nutrition but are greatly modified during severe diarrhoea due to purging and pathogenic colonization. To understand the extent of loss during and after diarrhoea, faecal samples collected from children (n=21) suffering from acute diarrhoea and from their healthy siblings (n=9) were analyzed by 16S rRNA gene-targeted universal primer polymerase chain reaction (PCR), followed by temporal temperature gradient gel electrophoresis (TTGE). The gut microbiota decreased significantly as indicated by the number of TTGE bands at day 0 of acute diarrhoea [patients vs healthy siblings: 11±0.9 vs 21.8±1.1 (mean ± standard error), p<0.01]. The number of bands showed a steady increase from day 1 to day 7; however, it remained significantly less than that in healthy siblings (15±0.9, p<0.01). These results suggest that appropriate therapeutic and post-diarrhoeal nutritional intervention might be beneficial for the early microbial restoration and recovery.

Investigation of the impact of feeding Lactobacillus plantarum CRL 1815 encapsulated in microbially derived polymers on the rat faecal microbiota

The aim of this study was to evaluate the impact of the administration of microencapsulated Lactobacillus plantarum CRL 1815 with two combinations of microbially derived polysaccharides, xanthan:gellan gum (1%:0·75%) and jamilan:gellan gum (1%:1%), on the rat faecal microbiota. A 10-day feeding study was performed for each polymer combination in groups of 16 rats fed either with placebo capsules, free or encapsulated Lact.plantarum or water. The composition of the faecal microbiota was analysed by fluorescence in situ hybridization and temporal temperature gradient gel electrophoresis. Degradation of placebo capsules was detected, with increased levels of polysaccharide-degrading bacteria. Xanthan:gellan gum capsules were shown to reduce the Bifidobacterium population and increase the Clostridium histolyticum group levels, but not jamilan:gellan gum capsules. Only after administration of jamilan:gellan gum-probiotic capsules was detected a significant increase in Lactobacillus-Enterococcus group levels compared to controls (capsules and probiotic) as well as two bands were identified as Lact.plantarum in two profiles of ileum samples. Exopolysaccharides constitute an interesting approach for colon-targeted delivery of probiotics, where jamilan:gellan gum capsules present better biocompatibility and promising results as a probiotic carrier. This study introduces and highlights the importance of biological compatibility in the encapsulating material election, as they can modulate the gut microbiota by themselves, and the use of bacterial exopolysaccharides as a powerful source of new targeted-delivery coating material.

Long-term in situ dynamics of the fungal communities in a multi-contaminated soil are mainly driven by plants

The fungal communities of a multi-contaminated soil polluted by polycyclic aromatic hydrocarbons and heavy metals (NM) were studied within a long-term in situ experiment of natural attenuation assisted by plants. Three treatments were monitored: bare soil (NM-BS), soil planted with alfalfa and inoculated with mycorrhizal fungi (NM-Msm), and soil with spontaneous vegetation (NM-SV). The same soil after thermal desorption (TD) was planted with alfalfa and inoculated with mycorrhizal fungi (TD-Msm). Twice a year for 5years, the fungal abundance and the community structure were evaluated by real-time PCR and temporal temperature gradient gel electrophoresis targeting 18S rRNA genes. The fungal abundance increased over time and was higher in planted than in bare NM soil and in TD than in NM soil. The Shannon diversity index (H') increased during the first 2years with the emergence of more than 30 ribotypes, but decreased after 3years with the selection of a few competitive species, mostly Ascomycetes. H' was higher under complex plant assemblage (NM-SV) than in the NM-BS plots but did not differ between NM and TD soils planted with alfalfa. These results indicated that even in a highly polluted soil, the plant cover was the main driver of the fungal community structure.

PCR-TTGE Analysis of 16S rRNA from Rainbow Trout (Oncorhynchus mykiss) Gut Microbiota Reveals Host-Specific Communities of Active Bacteria

This study assessed the relative contributions of host genetics and diet in shaping the gut microbiota of rainbow trout. Full sibling fish from four unrelated families, each consisting of individuals derived from the mating of one male and one female belonging to a breeding program, were fed diets containing either vegetable proteins or vegetable oils for two months in comparison to a control diet consisting of only fish protein and fish oil. Two parallel approaches were applied on the same samples: transcriptionally active bacterial populations were examined based on RNA analysis and were compared with bacterial populations obtained from DNA analysis. Comparison of temporal temperature gradient gel electrophoresis (TTGE) profiles from DNA and RNA showed important differences, indicating that active bacterial populations were better described by RNA analysis. Results showed that some bacterial groups were significantly (P<0.05) associated with specific families, indicating that microbiota composition may be influenced by the host. In addition, the effect of diet on microbiota composition was dependent on the trout family.