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PubMed2025

Whole-Genome Sequencing Uncovers Chromosomal and Plasmid-Borne Multidrug Resistance and Virulence Genes in Poultry-Associated Escherichia coli from Nigeria.

BACKGROUND: Broad and unregulated antibiotic use in livestock production, particularly poultry farming, has increased the development and persistence of multidrug-resistant (MDR) bacterial strains in animals. These resistant pathogens and their antibiotic resistance genes (ARGs) can spread to humans through environmental exposure and the food chain, posing serious public health risks. Whole-genome sequencing (WGS), alongside phenotypic antimicrobial susceptibility testing (AST), enables a comprehensive understanding of resistance mechanisms and informs antimicrobial stewardship strategies, particularly in resource-limited settings. AIM: This study aimed to characterize the phenotypic and genotypic antimicrobial resistance profiles, plasmid content, and virulence factors of an MDR E. coli strain (S3) isolated from a poultry farm in Enugu State, Nigeria, to elucidate potential risks to public health and the role of poultry as a reservoir for resistance determinants. METHODS: E. coli strain S3 was isolated from chicken droppings using standard microbiological methods and confirmed by MALDI-TOF mass spectrometry. AST was assessed using disc diffusion and broth microdilution to determine minimum inhibitory concentrations (MICs) for ten antibiotics across multiple classes. WGS was performed with a hybrid approach combining Illumina and Nanopore platforms, followed by genome assembly and annotation. ARGs, plasmid replicons, and virulence factors were identified in silico using AMRFinderPlus, starAMR, RGI/CARD, PlasmidFinder, MOB-suite, and the Virulence Factor Database (VFDB). RESULTS: Phenotypic testing revealed extensive resistance, with complete resistance to six of seven tested antibiotics (cefotaxime, ampicillin, erythromycin, gentamicin, ciprofloxacin, and doxycycline). MICs exceeded clinical breakpoints for multiple classes, confirming an MDR phenotype. Genome analysis indicated a 5.33 Mb genome distributed across five contigs, including one chromosome and four plasmid-associated contigs. The strain harboured numerous ARGs, including bla CTX-M-15, bla OXA-1, bla TEM-1, aac(6')-Ib-cr, aadA5, aph(3")-Ib, sul1/sul2, tet(A), dfrA17, and mph(A), co-localized on plasmids indicative of horizontal gene transfer (HGT) potential. Plasmid types included Col156, IncF, and two rep clusters. Virulence profiling revealed genes associated with adhesion (pap cluster, ECP), iron acquisition (enterobactin, yersiniabactin, aerobactin, heme uptake), and toxins (sat, senB), highlighting the isolate's potential for urinary tract and intestinal infections. CONCLUSION: This study highlights the significant role of poultry-associated bacteria as reservoirs of AMR genes, particularly those harboured on mobile plasmids with potential for HGT. E. coli strain S3 exhibits extensive multidrug resistance and carries a complex plasmid repertoire facilitating horizontal transfer of ARGs. Coupled with a rich virulence gene profile, this strain underscores the public health risk posed by poultry-associated E. coli in Nigeria. These findings demonstrate the urgent need for stringent antimicrobial stewardship, regulatory oversight, and genomic surveillance in poultry production milieus to mitigate the dissemination of MDR pathogens.

Escherichia coli↗
PubMed2004

The genome-sequenced variant of Campylobacter jejuni NCTC 11168 and the original clonal clinical isolate differ markedly in colonization, gene expression, and virulence-associated phenotypes.

The genome sequence of the enteric bacterial pathogen Campylobacter jejuni NCTC 11168 (11168-GS) was published in 2000, providing a valuable resource for the identification of C. jejuni-specific colonization and virulence factors. Surprisingly, the 11168-GS clone was subsequently found to colonize 1-day-old chicks following oral challenge very poorly compared to other strains. In contrast, we have found that the original clinical isolate from which 11168-GS was derived, 11168-O, is an excellent colonizer of chicks. Other marked phenotypic differences were also identified: 11168-O invaded and translocated through tissue culture cells far more efficiently and rapidly than 11168-GS, was significantly more motile, and displayed a different morphology. Serotyping, multiple high-resolution molecular genotyping procedures, and subtractive hybridization did not yield observable genetic differences between the variants, suggesting that they are clonal. However, microarray transcriptional profiling of these strains under microaerobic and severely oxygen-limited conditions revealed dramatic expression differences for several gene families. Many of the differences were in respiration and metabolism genes and operons, suggesting that adaptation to different oxygen tensions may influence colonization potential. This correlates biologically with our observation that anaerobically priming 11168-GS or aerobically passaging 11168-O caused an increase or decrease, respectively, in colonization compared to the parent strain. Expression differences were also observed for several flagellar genes and other less well-characterized genes that may participate in motility. Targeted sequencing of the sigma factors revealed specific DNA differences undetected by the other genomic methods [corrected].

Animals↗
PubMed2002

Among 46 near full length HIV type 1 genome sequences from Rakai District, Uganda, subtype D and AD recombinants predominate.

The impact of HIV-1 genetic diversity on candidate vaccines is uncertain. To minimize genetic diversity in the evaluation of HIV-1 vaccines, vaccine products must be matched to the predominant subtype in a vaccine cohort. To that end, full genome sequencing was used to detect and characterize HIV-1 subtypes and recombinant strains from individuals in Rakai District, Uganda. DNA extracted from peripheral blood mononuclear cells (PMBC) was PCR amplified using primers in the long terminal repeats (LTRs) to generate nearly full length genomes. Amplicons were directly sequenced with dye terminators and automated sequencers. Sequences were phylogenetically analyzed and recombinants were detected and mapped with distance scan and bootscan. Among 46 sequences, 54% were subtype D, 15% were subtype A, and 30% were recombinant. All recombinants were individually unique, and most combined subtypes A and D. Subtype D comprised more than 70% of all the HIV-1 genomes in Rakai when both pure subtypes and recombinants were considered. Candidate vaccines based on HIV-1 subtype D would be appropriate for evaluation in Rakai District, Uganda.

Adolescent↗
PubMed2003

Distribution of hepatitis B virus (HBV) genotypes among HBV carriers in the Cote d'Ivoire: complete genome sequence and phylogenetic relatedness of HBV genotype E.

The characteristics of hepatitis B virus (HBV) genotype E are not well known because only a few studies have been carried out by complete genome analysis. The aim of this study was to elucidate the distribution of HBV genotypes in Cote d'Ivoire, and to clarify the genotype-related characteristics of genotype E. The distribution of HBV genotypes among 48 HBV carriers in Cote d'Ivoire was determined using serological and genetic methods. The characteristics of genotype E were evaluated by complete genome sequences, and further investigations of small S gene, basic core promoter (BCP) mutation, and precore mutation were undertaken. HBV genotype distribution among the 48 carriers was 6.3% for genotype A, 6.3% for genotype D, and 87.4% for genotype E. Complete genomes of two genotype E strains were sequenced, and found to have 98.2% to 99.2% homology at the nucleotide level when compared with genotype E strains reported previously. In 24 genotype E carriers, the precore mutation was detected in 75% of the patients without HBeAg, in contrast to only 25% of the patients with HBeAg (P < 0.05). All 24 strains have T at nucleotide 1858 in the precore region. In contrast, BCP double mutation was detected in 17% of the patients with HBeAg, and 33% of the patients without HBeAg. These results indicated as the following: (1) genotypes A, D, and E of HBV exist in Cote d'Ivoire and genotype E is the most prevalent; (2) genotype E spread with low genetic diversity over the complete genome in West Africa; (3) HBV precore and/or BCP double variants were common among the patients with genotype E infections.

Amino Acid Sequence↗
PubMed2026

Whole-genome sequencing and analysis of the endophytic fungus Alternaria alternata Y-2 from Leymus chinensis.

To explore the genetic basis and functional potential of beneficial symbiosis between the endophytic fungus Alternaria alternata Y-2 and its host Leymus chinensis, we performed Illumina-based draft whole-genome sequencing and systematic bioinformatic analysis. Although this assembly does not reach telomere-to-telomere completeness, it provides high-quality gene-level information for gene prediction, functional annotation, carbohydrate-active enzyme (CAZyme) identification, and secondary metabolite biosynthetic gene cluster analysis. The final genome size of A. alternata Y-2 was 34,383,676&#xa0;bp with a GC content of 51.0%, containing 12,724 predicted protein-coding genes, 90 tRNAs, and 12 rRNAs. BUSCO assessment showed 98.9% completeness, supporting the high quality of this draft genome. A total of 12,627 genes were successfully annotated in the NCBI NR database, and 17,183 genes were functionally categorized using GO terms. In total, 448 CAZyme genes and 21 secondary metabolite biosynthetic gene clusters were identified, which are potentially involved in lignocellulose degradation, cellular redox homeostasis and biosynthesis of bioactive metabolites. Based on ITS sequence alignment, NR annotation, and phylogenetic analysis of single-copy orthologous genes, the strain was confidently identified as A. alternata. This study firstly reports the draft genome of an endophytic A. alternata strain derived from L. chinensis and provides valuable genetic resources for exploring the endophytic lifestyle, stress tolerance, and bioactive metabolite potential of this fungus.

Alternaria↗
PubMed1993

Methylation analysis by genomic sequencing of 5' region of mouse Pgk-1 gene and a cautionary note concerning the method.

We have used genomic sequencing aided by ligation-mediated PCR (LMPCR) to assay for 5-methylcytosine in the CpG-rich promoter region of the mouse X-linked phosphoglycerate kinase gene (Pgk-1). Earlier studies showed that there was very heavy methylation of CpG dinucleotides in the CpG-rich promoter of the human PGK1 gene on the inactive X chromosome (the Xi), but that these same sites were completely unmethylated on the active X chromosome (the Xa). For mouse Pgk-1, previous restriction enzyme analysis had shown apparently complete methylation of only one cytosine in the promoter region on the Xi, at HpaII site H7, which is located in the untranslated region, 28 nucleotides upstream of the translation start site. We analyzed this potentially critical region by combining the use of HpaII with LMPCR, and find that the CpG dinucleotides near H7 are either unmethylated or only partially methylated on the Xi. LMPCR analysis of male and female DNA over a 490-bp sequence including the promoter and enhancer extend the finding of relative hypomethylation on the mouse Xi to include all CpG dinucleotides in this region. These results are relevant to the role of DNA methylation in stabilizing the inactive state of chromatin. In addition, we find that caution must be exercised in using LMPCR for methylation analysis of some sequences. A DNA concentration-dependent band-suppression artifact can incorrectly suggest methylation of both CpG and nonCpG dinucleotides.

5-Methylcytosine↗
PubMed1989

Genome sequence of tick-borne encephalitis virus (Western subtype) and comparative analysis of nonstructural proteins with other flaviviruses.

The genome sequence of tick-borne encephalitis (TBE) virus (Western subtype vaccine strain Neudoerfl) was determined. This extends the previously published sequence of the structural proteins to the nonstructural protein region and noncoding sequences at the 5'- and 3'-termini. The amino-termini of the individual proteins were assigned by comparison with other flavivirus sequences. Amino acid homology calculations between TBE virus and mosquito-borne flaviviruses were performed for all nonstructural proteins. An evolutionary tree based on protein NS1 is presented that reveals the molecular basis of relationships among flaviviruses. Tick-borne and mosquito-borne flaviviruses share a common hydrophilicity profile and also other features of their primary sequences, such as the presumably functional Gly-Asp-Asp sequence element within protein NS5. Other characteristics, such as the potential N-glycosylation sites of protein NS1 and a potential proteolytic cleavage site within protein NS4B, are conserved within the mosquito-borne group, but differ in the TBE virus sequence.

Amino Acid Sequence↗
PubMed2026

Draft genome sequences of 12 fungi occurring in cyanobacteria-dominated biotic soil crusts in the Sonoran Desert.

We present draft genome sequences for 12 fungal isolates, including two isolates each of Trichoderma, Exophiala, and Cladosporium, and one isolate each of Fusarium, Cladorrhinum, Epicoccum, Chaetomiaceae, Didymellaceae, and Pleosporales, from cyanobacteria-dominated biological soil crusts in the Sonoran Desert (Arizona, USA), sequenced on a NovaSeq 6000 at &#x2265;40&#xd7; coverage.

Ascomycota↗
PubMed2005

Genomic sequences encoding two types of medaka hemopexin-like protein Wap65, and their gene expression profiles in embryos.

Medaka genomic BAC clones, which contained two types of medaka hemopexin-like protein gene (Wap65), mWap65-1 and mWap65-2, were screened and their genomic sequences were determined by the shotgun strategy. The exon-intron organizations were highly conserved between both mWap65s and human hemopexin genes. The 5'-flanking regions of mWap65-1 and mWap65-2 contained various putative transcription factor binding sites including elements for developmental regulation. The expression patterns of mWap65s during embryonic development were examined by quantitative real-time PCR, demonstrating that both mWap65 transcripts were observed in early embryonic stages, but their expression patterns were different. Interestingly, in situ hybridization revealed that mWap65-2 transcripts were restricted to liver, whereas mWap65-1 transcripts were detected along the edge of pectoral fin buds and the median fin fold of tail buds in embryos at stage 32. Furthermore, we generated transgenic medaka expressing GFP driven by mWap65-1 and mWap65-2 promoters and observed GFP expression patterns during ontogeny. Although localizations of GFP varied among individuals, embryos uniformly expressed GFP 1 day after injection of mWap65-1-hrGFP and mWap65-2-hrGFP constructs, suggesting that mWap65-1 and mWap65-2 promoters were activated in very early stages. The differences between mWap65-1 and mWap65-2 in their expression profiles indicate their distinct roles during ontogeny.

Animals↗
PubMed2005

The genome sequence of Clostridium botulinum type C neurotoxin-converting phage and the molecular mechanisms of unstable lysogeny.

Botulinum neurotoxins (BoNTXs) produced by Clostridium botulinum are among the most poisonous substances known. Of the seven types of BoNTXs, genes for type C1 and D toxins (BoNTX/C1 and D) are carried by bacteriophages. The gene for exoenzyme C3 also resides on these phages. Here, we present the complete genome sequence of c-st, a representative of BoNTX/C1-converting phages. The genome is a linear double-stranded DNA of 185,682 bp with 404-bp terminal direct repeats, the largest known temperate phage genome. We identified 198 potential protein-coding regions, including the genes for production of BoNTX/C1 and exoenzyme C3. Very exceptionally, as a viable bacteriophage, a number of insertion sequences were found on the c-st genome. By analyzing the molecular structure of the c-st genome in lysogens, we also found that it exists as a circular plasmid prophage. These features account for the unstable lysogeny of BoNTX phages, which has historically been called "pseudolysogeny." The PCR scanning analysis of other BoNTX/C1 and D phages based on the c-st sequence further revealed that BoNTX phages comprise a divergent phage family, probably generated by exchanging genomic segments among BoNTX phages and their relatives.

Bacteriophages↗
PubMed2000

Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing.

Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.

Amino Acid Sequence↗
PubMed2004

Genome sequence of the deep-sea gamma-proteobacterium Idiomarina loihiensis reveals amino acid fermentation as a source of carbon and energy.

We report the complete genome sequence of the deep-sea gamma-proteobacterium, Idiomarina loihiensis, isolated recently from a hydrothermal vent at 1,300-m depth on the Loihi submarine volcano, Hawaii. The I. loihiensis genome comprises a single chromosome of 2,839,318 base pairs, encoding 2,640 proteins, four rRNA operons, and 56 tRNA genes. A comparison of I. loihiensis to the genomes of other gamma-proteobacteria reveals abundance of amino acid transport and degradation enzymes, but a loss of sugar transport systems and certain enzymes of sugar metabolism. This finding suggests that I. loihiensis relies primarily on amino acid catabolism, rather than on sugar fermentation, for carbon and energy. Enzymes for biosynthesis of purines, pyrimidines, the majority of amino acids, and coenzymes are encoded in the genome, but biosynthetic pathways for Leu, Ile, Val, Thr, and Met are incomplete. Auxotrophy for Val and Thr was confirmed by in vivo experiments. The I. loihiensis genome contains a cluster of 32 genes encoding enzymes for exopolysaccharide and capsular polysaccharide synthesis. It also encodes diverse peptidases, a variety of peptide and amino acid uptake systems, and versatile signal transduction machinery. We propose that the source of amino acids for I. loihiensis growth are the proteinaceous particles present in the deep sea hydrothermal vent waters. I. loihiensis would colonize these particles by using the secreted exopolysaccharide, digest these proteins, and metabolize the resulting peptides and amino acids. In summary, the I. loihiensis genome reveals an integrated mechanism of metabolic adaptation to the constantly changing deep-sea hydrothermal ecosystem.

Amino Acids↗
PubMed1998

Completion of the Tula hantavirus genome sequence: properties of the L segment and heterogeneity found in the 3' termini of S and L genome RNAs.

In this study the L segment and the 5' and 3' termini of the S, M and L segments of the prototype Tula hantavirus (TUL) were sequenced, thus completing the first determination of the genome sequence of a hantavirus that has not been linked to any human disease. The TUL L segment comprises 6541 nt with one ORF of 6459 nt in the antigenome sense. This ORF potentially encodes a 2153 aa protein with a predicted molecular mass of 247 kDa. The amino acid sequence includes all the motifs conserved in RNA-dependent RNA polymerases. The 5' termini of all three genome RNAs (vRNAs) had the expected sequences conserved in hantaviruses. The 3' termini of M vRNAs were also conserved. However, the 3' termini of S and L vRNAs were heterogeneous as most of the sequenced 3' termini had either deletions of 1 to 22 nt or an extra 1 to 3 nt. No increase in the level of heterogeneity was seen in vRNAs of virions collected 3, 6, 9 and 12 days post-infection, suggesting that the heterogeneity already exists at the early stages of infection. The S and L vRNAs from infected cells had more truncated 3' termini than vRNAs from pelleted virus. Heterogeneity of the 3' termini of genome RNAs could decrease the efficiency of antigenome and mRNA syntheses and contribute to the slow growth observed for TUL and other hantaviruses in cell culture.

Genome, Viral↗
PubMed2004

A Fugu-Human Genome Synteny Viewer: web software for graphical display and annotation reports of synteny between Fugu genomic sequence and human genes.

A web server has been developed to access annotation and graphical reports of synteny and gene order between the Fugu genome and human genes. In this system, the assembled Fugu genomic sequences (also known as scaffolds) are annotated. The annotations for each Fugu scaffold are computed, stored and made publicly available. The annotations describe matches to human homologous genes. For each significant human gene match on the Fugu scaffold, the corresponding human chromosome map and measures of the significance of each match are given. The web-based server provides public access to these annotations and graphical displays of the results. The user is provided with a selection of views including a chromosome-colour-coded image and a table containing the details of the matches. The Fugu-Human Genome Synteny Viewer has been tested by comparing results with examples from a paper that includes a study of transcription factors, Fos and Jun encoding regions. The Fugu-human genome synteny views are available for each Fugu scaffold through the clonesearch web page located at the Fugu Genomics website (http://fugu.rfcgr.mrc.ac.uk/).

Animals↗
PubMed2005

The complete genome sequence of Francisella tularensis, the causative agent of tularemia.

Francisella tularensis is one of the most infectious human pathogens known. In the past, both the former Soviet Union and the US had programs to develop weapons containing the bacterium. We report the complete genome sequence of a highly virulent isolate of F. tularensis (1,892,819 bp). The sequence uncovers previously uncharacterized genes encoding type IV pili, a surface polysaccharide and iron-acquisition systems. Several virulence-associated genes were located in a putative pathogenicity island, which was duplicated in the genome. More than 10% of the putative coding sequences contained insertion-deletion or substitution mutations and seemed to be deteriorating. The genome is rich in IS elements, including IS630 Tc-1 mariner family transposons, which are not expected in a prokaryote. We used a computational method for predicting metabolic pathways and found an unexpectedly high proportion of disrupted pathways, explaining the fastidious nutritional requirements of the bacterium. The loss of biosynthetic pathways indicates that F. tularensis is an obligate host-dependent bacterium in its natural life cycle. Our results have implications for our understanding of how highly virulent human pathogens evolve and will expedite strategies to combat them.

Base Sequence↗
PubMed2005

Identification of endopeptidase genes from the genomic sequence of Lactobacillus helveticus CNRZ32 and the role of these genes in hydrolysis of model bitter peptides.

Genes encoding three putative endopeptidases were identified from a draft-quality genome sequence of Lactobacillus helveticus CNRZ32 and designated pepO3, pepF, and pepE2. The ability of cell extracts from Escherichia coli DH5alpha derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, beta-casein (beta-CN) (f193-209) and alpha(S1)-casein (alpha(S1)-CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10 degrees C) was examined. CNRZ32 PepO3 was determined to be a functional paralog of PepO2 and hydrolyzed both peptides, while PepE and PepF had unique specificities towards alpha(S1)-CN (f1-9) and beta-CN (f193-209), respectively. CNRZ32 PepE2 and PepO did not hydrolyze either peptide under these conditions. To demonstrate the utility of these peptidases in cheese, PepE, PepO2, and PepO3 were expressed in Lactococcus lactis, a common cheese starter, using a high-copy vector pTRKH2 and under the control of the pepO3 promoter. Cell extracts of L. lactis derivatives expressing these peptidases were used to hydrolyze beta-CN (f193-209) and alpha(S1)-CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides alpha(S1)-CN (f1-9), alpha(S1)-CN (f1-13), and alpha(S1)-CN (f1-16) were identified from Cheddar cheese serum and included in the defined peptide mix. Our results demonstrate that in all systems examined, PepO2 and PepO3 had the highest activity with beta-CN (f193-209) and alpha(S1)-CN (f1-9). Cheese-derived peptides were observed to affect the activity of some of the enzymes examined, underscoring the importance of incorporating such peptides in model systems. These data indicate that L. helveticus CNRZ32 endopeptidases PepO2 and PepO3 are likely to play a key role in this strain's ability to reduce bitterness in cheese.

Amino Acid Sequence↗
PubMed2005

Quantifying ascertainment bias and species-specific length differences in human and chimpanzee microsatellites using genome sequences.

Surveys of variability of homologous microsatellite loci among species reveal an ascertainment bias for microsatellite length where microsatellite loci isolated in one species tend to be longer than homologous loci in related species. Here, we take advantage of the availability of aligned human and chimpanzee genome sequences to compare length difference of homologous microsatellites for loci identified in humans to length difference for loci identified in chimpanzees. We are able to quantify ascertainment bias for a range of motifs and microsatellite lengths. Because ascertainment bias should not exist if a microsatellite selected in one species is as likely to be longer as it is to be shorter than its homologue, we propose that the nature of ascertainment bias can provide evidence for understanding how microsatellites evolve. We show that bias is greater for longer microsatellites but also that many long microsatellites have short homologues. These results are consistent with the notion that growth of long microsatellites is constrained by an upper length boundary that, when reached, sometimes results in large deletions. By evaluating ascertainment bias separately for interrupted and uninterrupted repeats we also show that long microsatellites tend to become interrupted, thereby contributing a second component of ascertainment bias. Having accounted for ascertainment bias, in agreement with results published elsewhere, we find that microsatellites in humans are longer on average than those in chimpanzees. This length difference is similar among repeat motifs but surprisingly comprises two roughly equal components, one associated with the repeats themselves and one with the flanking sequences. The differences we find can only be explained if microsatellites are both evolving directionally under a biased mutation process and are doing so at different rates in different closely related species.

Animals↗