L. Jesús Garcia-Gil

Abnormal microbiota composition in the ileocolonic mucosa of Crohn's disease patients as revealed by polymerase chain reaction-denaturing gradient gel electrophoresis

Background Bacteria might play a role in the pathogenesis of Crohns disease (CD), and patients harbor a different type and density of gut microbiota compared with normal healthy subjects. Thus, the aim of this study was to compare the microbiota adhered to the mucosa of CD patients with that of healthy subjects. Methods Polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) of 16S rRNA gene fragments was used to identify the dominant bacterial species present in fresh biopsy samples obtained from the mucosa of 15 healthy and 19 CD subjects. Two patients suffering from ulcerative colitis and 1 suffering from ischemic colitis also were included. Results Individuals were clustered in 2 groups according to their molecular fingerprint, which differentiated the majority of CD specimens (88.2%) from the majority of healthy/ulcerative colitis/ischemic colitis specimens (82.3%). In addition, the patient‐to‐patient variability in microbiota was greater within the CD cluster than in the healthy/ulcerative colitis cluster (P = 0.000). One hundred forty‐one sequences were obtained from the PCR‐DGGE bands that were grouped into 58 different phylotypes, 8 of which were novel. BLAST analysis revealed that 74.5% of the sequences were similar to those of bacteria that have never been cultivated. In CD samples, prevalence values for Clostridium spp Ruminococcus torques and Escherichia coli were significantly higher, whereas Faecalibacterium was more frequently found in healthy specimens. Opportunistic pathogenic &ggr;‐proteobacteria were found occasionally, only in CD mucosal microbiota. Conclusions Microbiota attached to the ileocolonic mucosa of CD patients is distinguishable from that of healthy subjects. We postulate that individuals who are predisposed to CD are less able to regulate the microbial makeup of their intestines, which leads to an unstable microbial population.

Cultured Representatives of Two Major Phylogroups of Human Colonic Faecalibacterium prausnitzii Can Utilize Pectin, Uronic Acids, and Host-Derived Substrates for Growth

ABSTRACT Faecalibacterium prausnitzii is one of the most abundant commensal bacteria in the healthy human large intestine, but information on genetic diversity and substrate utilization is limited. Here, we examine the phylogeny, phenotypic characteristics, and influence of gut environmental factors on growth of F. prausnitzii strains isolated from healthy subjects. Phylogenetic analysis based on the 16S rRNA sequences indicated that the cultured strains were representative of F. prausnitzii sequences detected by direct analysis of fecal DNA and separated the available isolates into two phylogroups. Most F. prausnitzii strains tested grew well under anaerobic conditions on apple pectin. Furthermore, F. prausnitzii strains competed successfully in coculture with two other abundant pectin-utilizing species, Bacteroides thetaiotaomicron and Eubacterium eligens, with apple pectin as substrate, suggesting that this species makes a contribution to pectin fermentation in the colon. Many F. prausnitzii isolates were able to utilize uronic acids for growth, an ability previously thought to be confined to Bacteroides spp. among human colonic anaerobes. Most strains grew on N-acetylglucosamine, demonstrating an ability to utilize host-derived substrates. All strains tested were bile sensitive, showing at least 80% growth inhibition in the presence of 0.5 μg/ml bile salts, while inhibition at mildly acidic pH was strain dependent. These attributes help to explain the abundance of F. prausnitzii in the colonic community but also suggest factors in the gut environment that may limit its distribution.

Determination of the topography and biometry of chlorosomes by atomic force microscopy

Isolated chlorosomes of several species of filamentous anoxygenic phototrophic bacteria (FAPB) and green sulfur bacteria (GSB) were examined by atomic force microscopy (AFM) to characterize their topography and biometry. Chlorosomes of Chloroflexusaurantiacus, Chloronema sp., and Chlorobium (Chl.) tepidum exhibited a smooth surface, whereas those of Chl. phaeobacteroides and Chl. vibrioforme showed a rough one. The potential artifactual nature of the two types of surfaces, which may have arisen because of sample manipulation or AFM processing, was ruled out when AFM images and transmission electron micrographs were compared. The difference in surface texture might be associated with the specific lipid and polypeptide composition of the chlorosomal envelope. The study of three-dimensional AFM images also provides information about the size and shape of individual chlorosomes. Chlorosomal volumes ranged from ca. 35 000 nm3 to 247 000 nm3 for Chl. vibrioforme and Chl. phaeobacteroides, respectively. The mean height was about 25 nm for all the species studied, except Chl. vibrioforme, which showed a height of only 14 nm, suggesting that GSB have 1–2 layers of bacteriochlorophyll (BChl) rods and GFB have ∼4. Moreover, the average number of BChl molecules per chlorosome was estimated according to models of BChl rod organisation. These calculations yielded upper limits ranging from 34 000 BChl molecules in Chl. vibrioforme to 240 000 in Chl. phaeobacteroides, values that greatly surpass those conventionally accepted.

Biofilm formation as a novel phenotypic feature of adherent-invasive Escherichia coli (AIEC)

BackgroundCrohns disease (CD) is a high morbidity chronic inflammatory disorder of unknown aetiology. Adherent-invasive Escherichia coli (AIEC) has been recently implicated in the origin and perpetuation of CD. Because bacterial biofilms in the gut mucosa are suspected to play a role in CD and biofilm formation is a feature of certain pathogenic E. coli strains, we compared the biofilm formation capacity of 27 AIEC and 38 non-AIEC strains isolated from the intestinal mucosa. Biofilm formation capacity was then contrasted with the AIEC phenotype, the serotype, the phylotype, and the presence of virulence genes.ResultsSpecific biofilm formation (SBF) indices were higher amongst AIEC than non-AIEC strains (P = 0.012). In addition, 65.4% of moderate to strong biofilms producers were AIEC, whereas 74.4% of weak biofilm producers were non-AIEC (P = 0.002). These data indicate that AIEC strains were more efficient biofilm producers than non-AIEC strains. Moreover, adhesion (P = 0.009) and invasion (P = 0.003) indices correlated positively with higher SBF indices. Additionally, motility (100%, P < 0.001), H1 type flagellin (53.8%, P < 0.001), serogroups O83 (19.2%, P = 0.008) and O22 (26.9%, P = 0.001), the presence of virulence genes such as sfa/focDE (38.5%, P = 0.003) and ibeA (26.9%, P = 0.017), and B2 phylotype (80.8%, P < 0.001) were frequent characteristics amongst biofilm producers.ConclusionThe principal contribution of the present work is the finding that biofilm formation capacity is a novel, complementary pathogenic feature of the recently described AIEC pathovar. Characterization of AIEC specific genetic determinants, and the regulatory pathways, involved in biofilm formation will likely bring new insights into AIEC pathogenesis.

THE MOLAR EXTINCTION COEFFICIENT OF BACTERIOCHLOROPHYLL E AND THE PIGMENT STOICHIOMETRY IN CHLOROBIUM PHAEOBACTEROIDES

We have determined the molar extinction coefficient of bacteriochlorophyll (BChl) e, the main light-harvesting pigment from brown-coloured photosynthetic sulfur bacteria. The extinction coefficient was determined using pure [Pr,E]BChl eF isolated by reversed-phase HPLC from crude pigment extracts of Chlorobium (Chl.) phaeobacteroides strain CL1401. The extinction coefficients at the Soret and Qy bands were determined in four organic solvents. The extinction coefficient of BChl e differs from those of other related Chlorobium chlorophylls (BChl c and BChl d) but is similar to that of chlorophyll b. The determined extinction coefficient was used to calculate the stoichiometric BChl e to BChl a and BChl e to carotenoids ratios in whole cells and isolated chlorosomes from Chl. phaeobacteroides strain CL1401 using the spectrum-reconstruction method (SRCM) described by Naqvi et al. (1997) (Spectrochim Acta A Mol Biomol Spectrosc 53: 2229–2234) . In isolated chlorosomes, BChl a content was ca. 1% of the total BChl content and the stoichiometric ratio of BChl e to carotenoids was 6. In whole cells, however, BChl a content was 3–4%, owing to the presence of BChl a-containing elements, i.e. FMO protein and reaction centre. An average of 5 BChl e molecules per carotenoid was determined in whole cells.

Effect of Carotenoid Biosynthesis Inhibition on the Chlorosome Organization in Chlorobium phaeobacteroides Strain CL1401

We have studied the effect of the absence of carotenoids on the organization of bacteriochlorophylls (BChls) in chlorosomes of Chlorobium (Chl.) phaeobacteroides strain CL1401. Carotenoid‐depleted chlorosomes were obtained by means of 2‐hydroxybiphenyl–supplemented cultures. In the presence of the inhibitor, isorenieratene (Isr) and β‐Isr biosynthesis were inhibited to more than 95%, leading to an accumulation of the colorless precursor phytoene inside the chlorosomes. In addition, there was a 30–40% decrease in the baseplate BChl a content. The absorption spectrum of the carotenoid‐depleted chlorosomes showed a 10 nm blue shift in the BChl e Qy absorption peak. Under reducing conditions, a decrease in the BChl a/BChl e fluorescence emission ratio was observed in carotenoid‐depleted chlorosomes relative to that in control chlorosomes, caused mainly by the decrease in the BChl a content. The steady‐state fluorescence emission anisotropy in the BChl e region dropped from ∼0.24 for native chlorosomes to ∼0.14 for carotenoid‐depleted ones, indicating reorganization of BChl e. The circular dichroism (CD) signal of the carotenoid‐depleted chlorosomes was increased two times in the BChl e Qy region. A simple model based on the structure proposed was used to explain the observed effects. Carotenoids might affect the angle between the direction of the BChl e Qy transition and the axis of the rod. The orientation of BChl a in the baseplate remains unchanged in carotenoid‐depleted chlorosomes, although there is a partial loss of BChl a as a consequence of a decrease in the baseplate size. The carotenoids are most likely rather close to the BChls and appear to be important for the aggregate structure in Chl. phaeobacteroides.

Excitation energy transfer in chlorosomes of Chlorobium phaeobacteroides strain CL1401: the role of carotenoids

The role of carotenoids in chlorosomes of the green sulfur bacterium Chlorobium phaeobacteroides, containing bacteriochlorophyll (BChl) e and the carotenoid (Car) isorenieratene as main pigments, was studied by steady-state fluorescence excitation, picosecond single-photon timing and femtosecond transient absorption (TA) spectroscopy. In order to obtain information about energy transfer from Cars in this photosynthetic light-harvesting antenna with high spectral overlap between Cars and BChls, Car-depleted chlorosomes, obtained by inhibition of Car biosynthesis by 2-hydroxybiphenyl, were employed in a comparative study with control chlorosomes. Excitation spectra measured at room temperature give an efficiency of 60–70% for the excitation energy transfer from Cars to BChls in control chlorosomes. Femtosecond TA measurements enabled an identification of the excited state absorption band of Cars and the lifetime of their S1 state was determined to be ∼10 ps. Based on this lifetime, we concluded that the involvement of this state in energy transfer is unlikely. Furthermore, evidence was obtained for the presence of an ultrafast (>100 fs) energy transfer process from the S2 state of Cars to BChls in control chlorosomes. Using two time-resolved techniques, we further found that the absence of Cars leads to overall slower decay kinetics probed within the Qy band of BChl e aggregates, and that two time constants are generally required to describe energy transfer from aggregated BChl e to baseplate BChl a.

Mucosa-associated Faecalibacterium prausnitzii phylotype richness is reduced in patients with inflammatory bowel disease.

ABSTRACT Faecalibacterium prausnitzii depletion in intestinal diseases has been extensively reported, but little is known about intraspecies variability. This work aims to determine if subjects with gastrointestinal disease host mucosa-associated F. prausnitzii populations different from those hosted by healthy individuals. A new species-specific PCR-denaturing gradient gel electrophoresis (PCR-DGGE) method targeting the 16S rRNA gene was developed to fingerprint F. prausnitzii populations in biopsy specimens from 31 healthy control (H) subjects and 36 Crohns disease (CD), 23 ulcerative colitis (UC), 6 irritable bowel syndrome (IBS), and 22 colorectal cancer (CRC) patients. The richness of F. prausnitzii subtypes was lower in inflammatory bowel disease (IBD) patients than in H subjects. The most prevalent operational taxonomic units (OTUs) consisted of four phylotypes (OTUs with a 99% 16S rRNA gene sequence similarity [OTU99]), which were shared by all groups of patients. Their distribution and the presence of some disease-specific F. prausnitzii phylotypes allowed us to differentiate the populations in IBD and CRC patients from that in H subjects. At the level of a minimum similarity of 97% (OTU97), two phylogroups accounted for 98% of the sequences. Phylogroup I was found in 87% of H subjects but in under 50% of IBD patients (P = 0.003). In contrast, phylogroup II was detected in >75% of IBD patients and in only 52% of H subjects (P = 0.005). This study reveals that even though the main members of the F. prausnitzii population are present in both H subjects and individuals with gut diseases, richness is reduced in the latter and an altered phylotype distribution exists between diseases. This approach may serve as a basis for addressing the suitability of F. prausnitzii phylotypes to be quantified as a putative biomarker of disease and depicting the importance of the loss of these subtypes in disease pathogenesis.

Nanosecond Laser Photolysis Studies of Chlorosomes and Artificial Aggregates Containing Bacteriochlorophyll e: Evidence for the Proximity of Carotenoids and Bacteriochlorophyll a in Chlorosomes from Chlorobium phaeobacteroides strain CL1401¶

Time‐resolved, laser‐induced changes in absorbance, ΔA(λ; t), have been recorded with a view to probing pigment–pigment interactions in chlorosomes (control as well as carotenoid‐depleted) and artificial aggregates of bacteriochlorophyll e (BChle). Control chlorosomes were isolated from Chlorobium phaeobacteroides strain CL1401, whose chromophores comprise BChle, bacteriochlorophyll a (BChla) and several carotenoid (Car) pigments; Car‐depleted chlorosomes, from cells grown in cultures containing 2‐hydroxybiphenyl. Artificial aggregates were prepared by dispersing BChle in aqueous phase in the presence of monogalactosyl diglyceride. In chlorosomes ΔA(λ; t) shows, besides a signal attributable to triplet Car (with a half‐life of about 4 μs), signals in the Qy regions of both BChl. The BChla signal decays at the same rate as the Car signal, which is explained by postulating that some Car are in intimate contact with some baseplate BChla pigments, and that when a ground‐state Car changes into a triplet Car, the absorption spectrum of its BChla neighbors undergoes a concomitant change (termed transient environment‐induced perturbation). The signal in the Qy‐region of BChle behaves differently: its amplitude falls, under reducing conditions, by more than a factor of two during the first 0.5 μs (a period during which the Car signal suffers negligible diminution), and is much smaller under nonreducing conditions. The BChle signal is also attributed to transient environment‐induced perturbation, but in this case the perturber is a BChle photoproduct (probably a triplet or a radical ion). The absence of long‐lived BChle triplets in all three systems, and of long‐lived BChla triplets in chlorosomes, indicates that BChle in densely packed assemblies is less vulnerable to photodamage than monomeric BChle and that, in chlorosome, BChla rather than BChle needs, and receives, photoprotection from an adjacent Car.

Faecalibacterium prausnitzii : from microbiology to diagnostics and prognostics

There is an increasing interest in Faecalibacterium prausnitzii, one of the most abundant bacterial species found in the gut, given its potentially important role in promoting gut health. Although some studies have phenotypically characterized strains of this species, it remains a challenge to determine which factors have a key role in maintaining the abundance of this bacterium in the gut. Besides, phylogenetic analysis has shown that at least two different F. prausnitzii phylogroups can be found within this species and their distribution is different between healthy subjects and patients with gut disorders. It also remains unknown whether or not there are other phylogroups within this species, and also if other Faecalibacterium species exist. Finally, many studies have shown that F. prausnitzii abundance is reduced in different intestinal disorders. It has been proposed that F. prausnitzii monitoring may therefore serve as biomarker to assist in gut diseases diagnostics. In this mini-review, we aim to serve as an overview of F. prausnitzii phylogeny, ecophysiology and diversity. In addition, strategies to modulate the abundance of F. prausnitzii in the gut as well as its application as a biomarker for diagnostics and prognostics of gut diseases are discussed. This species may be a useful potential biomarker to assist in ulcerative colitis and Crohn’s disease discrimination.