Nuria Jiménez-Hernández

Metatranscriptomic Approach to Analyze the Functional Human Gut Microbiota

The human gut is the natural habitat for a large and dynamic bacterial community that has a great relevance for health. Metagenomics is increasing our knowledge of gene content as well as of functional and genetic variability in this microbiome. However, little is known about the active bacteria and their function(s) in the gastrointestinal tract. We performed a metatranscriptomic study on ten healthy volunteers to elucidate the active members of the gut microbiome and their functionality under conditions of health. First, the microbial cDNAs obtained from each sample were sequenced using 454 technology. The analysis of 16S transcripts showed the phylogenetic structure of the active microbial community. Lachnospiraceae, Ruminococcaceae, Bacteroidaceae, Prevotellaceae, and Rickenellaceae were the predominant families detected in the active microbiota. The characterization of mRNAs revealed a uniform functional pattern in healthy individuals. The main functional roles of the gut microbiota were carbohydrate metabolism, energy production and synthesis of cellular components. In contrast, housekeeping activities such as amino acid and lipid metabolism were underrepresented in the metatranscriptome. Our results provide new insights into the functionality of the complex gut microbiota in healthy individuals. In this RNA-based survey, we also detected small RNAs, which are important regulatory elements in prokaryotic physiology and pathogenicity.

Assessing Gut Microbial Diversity from Feces and Rectal Mucosa

Gut microbiota is the most complex bacterial community in the human body and its study may give important clues to the etiology of different intestinal diseases. Most studies carried out so far have used fecal samples, assuming that these samples have a similar distribution to the communities present throughout the colon. The present study was designed to test this assumption by comparing samples from the rectal mucosa and feces of nine healthy volunteers by sequencing libraries of 16S rRNA genes. At the family taxonomic level, where rarefaction curves indicate that the observed number of taxa is close to the expected one, we observe under different statistical analyses that fecal and mucosal samples cluster separately. The same is found at the level of species considering phylogenetic information. Consequently, it cannot be stated that both samples from a given individual are of similar composition. We believe that the evidence in support of this statement is strong and that it would not change by increasing the number of individuals and/or performing massive sequencing. We do not expect clinicians to stop using feces for research, but we think it is important to caution them on their potential lack of representativeness with respect to the bacterial biofilm on the rectal mucosa.

Evidence of recombination in intrapatient populations of hepatitis C virus.

Hepatitis C virus (HCV) is a major cause of liver disease worldwide and a potential cause of substantial morbidity and mortality in the future. HCV is characterized by a high level of genetic heterogeneity. Although homologous recombination has been demonstrated in many members of the family Flaviviridae, to which HCV belongs, there are only a few studies reporting recombination on natural populations of HCV, suggesting that these events are rare in vivo. Furthermore, these few studies have focused on recombination between different HCV genotypes/subtypes but there are no reports on the extent of intra-genotype or intra-subtype recombination between viral strains infecting the same patient. Given the important implications of recombination for RNA virus evolution, our aim in this study has been to assess the existence and eventually the frequency of intragenic recombination on HCV. For this, we retrospectively have analyzed two regions of the HCV genome (NS5A and E1-E2) in samples from two different groups: (i) patients infected only with HCV (either treated with interferon plus ribavirin or treatment naïve), and (ii) HCV-HIV co-infected patients (with and without treatment against HIV). The complete data set comprised 17712 sequences from 136 serum samples derived from 111 patients. Recombination analyses were performed using 6 different methods implemented in the program RDP3. Recombination events were considered when detected by at least 3 of the 6 methods used and were identified in 10.7% of the amplified samples, distributed throughout all the groups described and the two genomic regions studied. The resulting recombination events were further verified by detailed phylogenetic analyses. The complete experimental procedure was applied to an artificial mixture of relatively closely viral populations and the ensuing analyses failed to reveal artifactual recombination. From these results we conclude that recombination should be considered as a potentially relevant mechanism generating genetic variation in HCV and with important implications for the treatment of this infection.

Structural alterations of faecal and mucosa-associated bacterial communities in irritable bowel syndrome.

Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder in western countries. Previous studies on IBS, mostly based on faecal samples, suggest alterations in the intestinal microbiota. However, no consensus has been reached regarding the association between specific bacteria and IBS. We explore the alterations of intestinal bacterial communities in IBS using massive sequencing of amplified 16S rRNA genes. Mucosal biopsies of the ascending and descending colon and faeces from 16 IBS patients and 9 healthy controls were analysed. Strong inter-individual variation was observed in the composition of the bacterial communities in both patients and controls. These communities showed less diversity in IBS cases. There were larger differences in the microbiota composition between biopsies and faeces than between patients and controls. We found a few over-represented and under-represented taxa in IBS cases with respect to controls. The detected alterations varied by site, with no changes being consistent across sample types.

Genetic variability in hepatitis C virus and its role in antiviral treatment response

Summary.  Hepatitis C virus (HCV) is a major health problem worldwide, infecting an estimated 170 million people. The high genetic variability of HCV contributes to the chronicity of hepatitis C. Here, we report results from a large‐scale sequence analysis of 67 patients infected with HCV genotype 1, 23 with subtype 1a and 44 with subtype 1b. Two regions of the HCV genome were analysed in samples prior to combined therapy with alpha interferon plus ribavirin, one compressing the hypervariable regions (HVR1, HVR2 and HVR3) of the E2 glycoprotein and another one including the interferon‐sensitive determining region (ISDR) and the V3 domain of the NS5A protein. Genetic diversity measures showed a clear tendency to higher genetic variability levels in nonresponder patients to antiviral treatment than in responder patients, although highly disperse values were present within each response group for both subtypes. A more detailed analysis of amino acid composition revealed the presence of several subtype‐specific variants in a few positions, but no discriminating positions between responder and nonresponder patients were detected. Our results also revealed that most amino acid positions were highly conserved, especially for subtype 1a. We conclude that the outcome of the antiviral treatment might depend not only on the nature of one or a few independent positions, but more likely on the combination of several positions along the HCV genome. Moreover, the own host’s ability to generate an appropriate systemic response, in combination with the action of antivirals, is also likely to be essential for treatment outcome.

Daily follow-up of bacterial communities in the human gut reveals stable composition and host-specific patterns of interaction

In the last decade, an extensive effort has been made to characterize the human intestinal microbiota by means of SSU rRNA gene sequence and metagenomic analysis. Relatively few studies have followed intestinal bacterial communities over time to assess their stability in the absence of perturbation. In this study, we have monitored the faecal bacteria of three healthy subjects during 15 consecutive days. The global community structure was analysed through SSU rRNA gene sequencing. In agreement with previous studies, we found that the between-subject variation in community structure was larger than within. The composition was fairly stable throughout, although daily fluctuations were detected for all genera and phylotypes at 97% of sequence identity. While the core shared between subjects was very small, each subject harboured a stable high-abundance core composed of a small number of bacterial groups (9% of the phylotypes accounted for between 74% and 93% of the sequences). This may suggest that studies aimed at linking the microbiota composition with disease risk should be limited to the numerically most dominant phylotypes, as the rest appears transient. Networks of potential interactions between co-occurring genera were also subject-specific, even for the same bacterial genus, which might be reflecting host-specific selective pressures and historic events.

Genetic Variability of Hepatitis C Virus before and after Combined Therapy of Interferon plus Ribavirin

We present an analysis of the selective forces acting on two hepatitis C virus genome regions previously postulated to be involved in the viral response to combined antiviral therapy. One includes the three hypervariable regions in the envelope E2 glycoprotein, and the other encompasses the PKR binding domain and the V3 domain in the NS5A region. We used a cohort of 22 non-responder patients to combined therapy (interferon alpha-2a plus ribavirin) for which samples were obtained before initiation of therapy and after 6 or/and 12 months of treatment. A range of 25–100 clones per patient, genome region and time sample were sequenced. These were used to detect general patterns of adaptation, to identify particular adaptation mechanisms and to analyze the patterns of evolutionary change in both genome regions. These analyses failed to detect a common adaptive mechanism for the lack of response to antiviral treatment in these patients. On the contrary, a wide range of situations were observed, from patients showing no positively selected sites to others with many, and with completely different topologies in the reconstructed phylogenetic trees. Altogether, these results suggest that viral strategies to evade selection pressure from the immune system and antiviral therapies do not result from a single mechanism and they are likely based on a range of different alternatives, in which several different changes, or their combination, along the HCV genome confer viruses the ability to overcome strong selective pressures.

Refined analysis of genetic variability parameters in hepatitis C virus and the ability to predict antiviral treatment response.

Summary.  Hepatitis C virus (HCV) infects approximately 3% of the world population. The chronicity of hepatitis C seems to depend on the level of genetic variability. We have recently (Torres‐Puente et al., J Viral Hepat, 2008; 15: 188) reported genetic variability estimates from a large‐scale sequence analysis of 67 patients infected with HCV subtypes 1a (23 patients) and 1b (44 patients) and related them to response, or lack of, to alpha‐interferon plus ribavirin treatment.. Two HCV genome regions were analysed in samples prior to antiviral therapy, one compressing the three hypervariable regions of the E2 glycoprotein and another one including the interferon sensitive determining region and the V3 domain of the NS5A protein. Haplotype and nucleotide diversity measures showed a clear tendency to higher genetic variability levels in nonresponder than in responder patients. Here, we have refined the analysis of genetic variability (haplotype and nucleotide diversity, number of haplotypes and mutations) by considering their distribution in each of the biologically meaningful subregions mentioned above, as well as in their surrounding and intervening regions. Variability levels are very heterogeneous among the different subregions, being higher for nonresponder patients. Interestingly, significant differences were detected in the biologically relevant regions, but also in the surrounding regions, suggesting that the level of variability of the whole HCV genome, rather than exclusively that from the hypervariable regions, is the main indicator of the treatment response. Finally, the number of haplotypes and mutations seem to be better discriminators than haplotype and nucleotide diversity, especially in the NS5A region.

The role of positive selection in hepatitis C virus

Hepatitis C virus (HCV) is a major health problem worldwide, infecting an estimated 170 million people. In this study, we have employed a large data set of sequences (14,654 sequences from between 25 and 100 clone sequences per analyzed region and per patient) from 67 patients infected with HCV genotype 1 (23 subtype 1a and 44 subtype 1b). For all patients, a sample prior to combined therapy with alpha interferon plus ribavirin was available, whereas for some patients additional samples after 6 or 12 months of treatment were also available. Twenty-seven patients responded to treatment (12 subtype 1a and 15 subtype 1b) and forty patients did not respond to treatment (11 subtype 1a vs. 29 subtype 1b). Two regions of the HCV genome were analyzed, one compressing the hypervariable regions (HVR1, HVR2 and HVR3) of the envelope 2 glycoprotein and another one including the interferon sensitive determining region (ISDR) and the V3 domain of the NS5A protein. Previously (Cuevas, J.M., Torres-Puente, M., Jiménez-Hernández, N., Bracho, M.A., García-Robles, I., Wrobel, B., Carnicer, F., del Olmo, J., Ortega, E., Moya, A., González-Candelas, F., 2008b. Genetic variability of hepatitis C virus before and after combined therapy of interferon plus ribavirin. Plos One 3 (8), e3058), several amino acid positions in both regions analyzed were detected to be under positive selection. Here, we have compared the amino acid composition of each positively selected position between responder and non-responder patients for both subtypes. If we exclude some non-conclusive cases, no clear differences were detected in any case. In conclusion, identifying specific positions as completely discriminatory of treatment response seems to be a difficult task. Our results, in concordance with previous studies, suggest that HCV evasion strategies are more likely based on a global increased variability, which would yield combinations of mutations with an increased resistance, than on the fixation of specific amino acids conferring resistance to antiviral treatment or immune response. In this sense, the particular systemic response from each patient could play an essential role in determining the outcome of the antiviral treatment.

Combined therapy of interferon plus ribavirin promotes multiple adaptive solutions in hepatitis C virus.

Hepatitis C virus (HCV) presents several regions involved potentially in evading antiviral treatment and host immune system. Two regions, known as PKR‐BD and V3 domains, have been proposed to be involved in resistance to interferon. Additionally, hypervariable regions in the envelope E2 glycoprotein are also good candidates to participate in evasion from the immune system. In this study, we have used a cohort of 22 non‐responder patients to combined therapy (interferon alpha‐2a plus ribavirin) for which samples obtained just before initiation of therapy and after 6 or/and 12 months of treatment were available. A range of 25–100 clones per patient, genome region and time sample were obtained. The predominant amino acid sequences for each time sample and patient were determined. Next, the sequences of the PKR‐BD and V3 domains and the hypervariable regions from different time samples were compared for each patient. The highest levels of variability were detected at the three hypervariable regions of the E2 protein and, to a lower extent, at the V3 domain of the NS5A protein. However, no clear patterns of adaptation to the host immune system or to antiviral treatment were detected. In summary, although high levels of variability are correlated to viral adaptive response, antiviral treatment does not seem to promote convergent adaptive changes. Consequently, other regions must be involved in evasion strategies likely based on a combination of multiple mechanisms, in which pools of changes along the HCV genome could confer viruses the ability to overcome strong selective pressures. J. Med. Virol. 81:650–656, 2009