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PubMed2025

Systematic evaluation of metatranscriptomic differential gene expression in silico, in vitro, and in vivo enables elucidation of inter-species cross-feeding.

Metatranscriptomic (MTX) sequencing quantifies gene expression from the collective genomes of microbial communities (microbiomes), enabling assessment of functional activity rather than functional potential. While differential expression testing is instrumental to RNA-sequencing analysis, current metatranscriptomic approaches have been benchmarked only on simulated data and not under real operating conditions, resulting in a lack of standard practices. Here, we evaluate the performance of statistical differential expression methods on both simulated datasets and data collected from real bacterial 'mock communities' designed for this purpose. We assess the robustness of individual methods to organisms' low relative abundance, differential abundance, low prevalence, and transcription rate changes, showing that no existing methods perform adequately across all confounding conditions. We then apply the same approaches to metatranscriptomic datasets generated from gnotobiotic mice colonized with defined consortia of human bacterial strains and show that the method nominated by our mock community comparisons successfully inferred cross-feeding dynamics which were validated in vitro. We conclude that MTX method benchmarking on real, not simulated, datasets can and should optimize model implementation, enabling inference and validation of cross-feeding and other inter-species and host-microbe dynamics from in vivo studies.

Journal Article↗
PubMed1999

Statistics of DNA sequences: a low-frequency analysis.

We study statistical properties of DNA chains of thirteen microbial complete genomes. We find that the power spectrum of several of the sequences studied flattens off in the low frequency limit. This implies the correlation length in those sequences is much smaller than the entire DNA chain. Consequently, in contradiction with previous studies, we show that the fractal behavior of DNA chains does not always prevail through the entire DNA molecule.

Biometry↗
PubMed2001

Microbial genes in the human genome: lateral transfer or gene loss?

The human genome was analyzed for evidence that genes had been laterally transferred into the genome from prokaryotic organisms. Protein sequence comparisons of the proteomes of human, fruit fly, nematode worm, yeast, mustard weed, eukaryotic parasites, and all completed prokaryote genomes were performed, and all genes shared between human and each of the other groups of organisms were collected. About 40 genes were found to be exclusively shared by humans and bacteria and are candidate examples of horizontal transfer from bacteria to vertebrates. Gene loss combined with sample size effects and evolutionary rate variation provide an alternative, more biologically plausible explanation.

Animals↗
PubMed2005

Molecular characterization of vanB elements in naturally occurring gut anaerobes.

Previously, we reported the isolation of 10 vancomycin-resistant gram-positive anaerobic bacilli carrying the vanB ligase gene from nine hemodialysis patients (S. A. Ballard et al., Antimicrob. Agents Chemother. 49:77-81, 2005; T. P. Stinear et al., Lancet 357:855-856, 2001). In the present study, the molecular and evolutionary relationship of the vanB resistance element within these 10 anaerobes and two vancomycin-resistant Enterococcus faecium strains were examined. PCR analysis and nucleotide sequencing demonstrated that all 12 isolates carried the vanB operon associated with an element identical to Tn1549 and Tn5382 of Enterococcus. Restriction fragment length polymorphism analysis of the vanB operon in these isolates revealed two distinct patterns, and sequencing showed that minor base differences existed. PCR amplification of the joint region of a circular intermediate was demonstrated in nine of these organisms, a finding indicative of an ability to excise and circularize, an intermediate step in transposition and conjugative transfer. Southern hybridization with a vanB-vanX(B) probe suggests that there is one insert of the transposon in all isolates. Sequence analysis of the integration site revealed distinct sequences: the Tn1549/5382 element within E. faecium was inserted within the host chromosome, whereas nucleotide sequences surrounding the Tn1549/5382 element in the 10 anaerobes showed no significant homology to sequences in the GenBank database. We demonstrate considerable similarity between the Tn1549/5382 element identified in 10 anaerobe isolates with that found in enterococci. The homology and potential to transpose suggest a recent horizontal transfer event may have occurred. However, the original direction of transposition and the mechanism involved remains unknown.

Anti-Bacterial Agents↗
PubMed1994

Stability of DNA fingerprint pattern produced with IS6110 in strains of Mycobacterium tuberculosis.

To assess the stability of IS6110 restriction fragment length polymorphism patterns, DNA fingerprints of 6 Mycobacterium bovis isolates from 1 patient and of 41 Mycobacterium tuberculosis isolates from 18 patients were compared. The fingerprint pattern for a given patient remained identical or nearly identical despite recovery of the isolates during intervals which ranged from 8 months to 4.5 years. Changes in drug resistance profile did not alter a strain's fingerprint pattern.

DNA Fingerprinting↗
PubMed1999

Performance of the Affymetrix GeneChip HIV PRT 440 platform for antiretroviral drug resistance genotyping of human immunodeficiency virus type 1 clades and viral isolates with length polymorphisms.

The performance of a silica chip-based resequencing method, the Affymetrix HIV PRT 440 assay (hereafter referred to as the Affymetrix assay), was evaluated on a panel of well-characterized nonclade B viral isolates and on isolates exhibiting length polymorphisms. Sequencing of human immunodeficiency virus type 1 (HIV-1) pol cDNAs from clades A, C, D, E, and F resulted in clade-specific regions of base-calling ambiguities in regions not known to be associated with resistance polymorphisms, as well as a small number of spurious resistance polymorphisms. The Affymetrix assay failed to detect the presence of additional serine codons distal to reverse transcriptase (RT) codon 68 that are associated with multinucleoside RT inhibitor resistance. The increasing prevalence of non-clade B HIV-1 strains in the United States and Europe and the identification of clinically relevant pol gene length polymorphisms will impact the generalizability of the Affymetrix assay, emphasizing the need to accommodate this expanding pool of pol genotypes in future assay versions.

Acquired Immunodeficiency Syndrome↗
PubMed1998

3'-Azido-3'-deoxythymidine (AZT) mediates cross-resistance to nucleoside analogs in the case of AZT-resistant human immunodeficiency virus type 1 variants.

Difficulties in deciphering the mechanisms of 3'-azido-3'-deoxythymidine (AZT)-resistance by human immunodeficiency virus type 1 (HIV-1) variants are due in part to an inability to reconstitute resistance in vitro using AZT-resistant reverse transcriptases. We decided to characterize mechanisms of AZT resistance in tissue culture infections by studying the ability of drug-resistant viruses to synthesize viral DNA in the presence or absence of drug. Through use of PCR amplifications, we discovered an AZT-mediated stimulation of reverse transcription by AZT-resistant viruses carrying the M41L and T215Y mutations that can apparently override the inhibitory effects of AZT-5'-triphosphate. In addition, the presence of AZT also causes viruses containing the M41L and T215Y substitutions to have diminished sensitivity to other nucleoside analogs (i.e., ddC, ddI, and d4T). This AZT-mediated cross-resistance may help to explain the virological failure of treatment regimens that included ddI plus AZT or ddC plus AZT in situations in which the T215Y and/or M41L mutations were present (F. Brun-Vézinet, C. Boucher, C. Loveday, D. Descamps, V. Fauveau, J. Izopet, D. Jeffries, S. Kaye, C. Krzyanowski, A. Nunn, R. Schuurman, J. M. Seigneurin, C. Tamalet, R. Tedder, J. Weber, and G. J. Weverling, Lancet 350:983-990, 1997). Our results suggest that the use of AZT may be contraindicated in those patients for whom resistance to this compound (M41L and/or T215Y) has been demonstrated.

Anti-HIV Agents↗
PubMed2026

Metagenome-assembled genomes for N2-fixing cyanobacterium Nostoc sp. TISTR 8405 and co-occurring microorganisms from a long-term laboratory culture.

We report here metagenome-assembled genomes from a long-term laboratory culture of the nitrogen-fixing cyanobacterium Nostoc sp. TISTR 8405, originally sourced from a Thai freshwater lake. The community consists of two additional co-occurring microorganisms, Erythrobacter sp. THAI-01 and Allorhizobium sp. THAI-01, and contains putative plasmids associated with Nostoc and Allorhizobium, respectively.

co-culture↗
PubMed2006

Detection and quantification of Legionella pneumophila in water samples using competitive PCR.

The presence of high levels of Legionella pneumophila in man-made aquatic systems correlates with the incidence of nosocomial Legionnaires' disease. This requires a rapid, reliable, and sensitive quantification of L. pneumophila concentrations in suspected water systems. In this research, a homologous competitor was developed and evaluated in a L. pneumophila competitive polymerase chain reaction (cPCR) to quantify this human pathogen in a quick, cost-effective, and reliable way. Accuracy of cPCR was evaluated by analyzing cooling tower and tap water samples spiked with known concentrations of L. pneumophila bacteria, in parallel with the standard culture method. Legionella pneumophila amounts detected and calculated from cPCR and culture correlated very well: r = 0.998, P = 0.002 for tap water and r = 0.990, P = 0.009 for cooling tower water. Nevertheless, for both kinds of water samples, mean numbers of L. pneumophila calculated from cPCR results were always higher than those obtained by culture. This study makes it clear that the rapid, sensitive, and cost-effective L. pneumophila cPCR is a promising alternative to the standard time-consuming culture method and expensive real-time PCR to enumerate L. pneumophila bacteria in environmental water samples.

Colony Count, Microbial↗
PubMed2006

Chaos game representation for comparison of whole genomes.

BACKGROUND: Chaos game representation of genome sequences has been used for visual representation of genome sequence patterns as well as alignment-free comparisons of sequences based on oligonucleotide frequencies. However the potential of this representation for making alignment-based comparisons of whole genome sequences has not been exploited. RESULTS: We present here a fast algorithm for identifying all local alignments between two long DNA sequences using the sequence information contained in CGR points. The local alignments can be depicted graphically in a dot-matrix plot or in text form, and the significant similarities and differences between the two sequences can be identified. We demonstrate the method through comparison of whole genomes of several microbial species. Given two closely related genomes we generate information on mismatches, insertions, deletions and shuffles that differentiate the two genomes. CONCLUSION: Addition of the possibility of large scale sequence alignment to the repertoire of alignment-free sequence analysis applications of chaos game representation, positions CGR as a powerful sequence analysis tool.

Algorithms↗
PubMed1999

Novel patterns of amino acid mutations in the hepatitis B virus polymerase in association with resistance to lamivudine therapy in japanese patients with chronic hepatitis B.

Lamivudine is effective in suppressing replication of hepatitis B virus (HBV). However, the emergence of HBV variants resistant to lamivudine is a concern. Lamivudine resistance has been attributed mainly to a substitution of isoleucine or valine for methionine at residue 550 (M550I or M550V) in the catalytic site of the virus polymerase. A substitution of methionine for leucine at residue 526 (L526M) has also been identified. To examine such virus genotypic mutations in Japanese patients, we studied five patients with chronic hepatitis B, who showed HBV breakthrough while on a 1-year lamivudine treatment. The entire nucleotide and deduced amino acid sequences of the proposed reverse transcriptase domain of the polymerase gene were determined on HBV DNA amplified by polymerase chain reaction from patient sera collected at the start and at the end of therapy. The HBV sequences from all five patients were of genotype C. In four patients, a substitution of valine or isoleucine for leucine at residue 426, which has not been reported previously, emerged in combination with M550I. One also harbored L526M. In the remaining patient, an alteration of leucine to methionine at residue 428 co-occurred with M550V. Longitudinal study of the mutations showed that the two or three mutations in each patient emerged almost simultaneously 4 weeks before or at the time of breakthrough and were replaced by wild-type virus after completing the therapy. Our results indicate that occurrence of HBV polymerase mutations at residue 426 in combination with M550I is frequent in Japanese or genotype C virus-in- fected patients who develop resistance to lamivudine.

Adult↗