Microbial Genomics最新文献

Identifying genetic variants associated with bacterial phenotypes, such as virulence, host preference and antimicrobial resistance, has great potential for a better understanding of the mechanisms involved in these traits. The availability of large collections of bacterial genomes has made genome-wide association studies (GWAS) a common approach for this purpose. The need to employ multiple software tools for data pre- and postprocessing limits the application of these methods by experienced bioinformaticians. To address this issue, we have developed a pipeline to perform bacterial GWAS from a set of assemblies and annotations, with multiple phenotypes as targets. The associations are run using five sets of genetic variants: unitigs, gene presence/absence, rare variants (i.e. gene burden test), gene-cluster-specific k-mers and all unitigs jointly. All variants passing the association threshold are further annotated to identify overrepresented biological processes and pathways. The results can be further augmented by generating a phylogenetic tree and predicting the presence of antimicrobial resistance and virulence-associated genes. We tested the microGWAS pipeline on a previously reported dataset on Escherichia coli virulence, successfully identifying the causal variants and providing further interpretation of the association results. The microGWAS pipeline integrates state-of-the-art tools to perform bacterial GWAS into a single, user-friendly and reproducible pipeline, allowing for the democratization of these analyses. The pipeline, together with its documentation, can be accessed at https://github.com/microbial-pangenomes-lab/microGWAS.

Whole-genome analysis has provided insights into the evolution of Chlamydia trachomatis and, recently, into circulating strains that cause lymphogranuloma venereum (LGV). A large LGV outbreak of a new ompA-genotype, L2b, was first reported in Europe in the early 2000s, primarily affecting men who have sex with men (MSM), and then expanded globally. More recent work shows that this outbreak is diversifying into variants of described ompA-genotypes, with the same L2b genomic backbone. This study extends the investigation of LGV cases to Argentina and Finland. In 2017, an LGV outbreak was described in Argentina characterized by distinct genomic features shown by both ompA-genotyping and Multi-Locus Sequence Typing (MLST) analysis. We have obtained whole-genome sequences from cultured isolates and clinical samples via SureSelect (Agilent) target enrichment. Based on ompA and phylogenetic analyses, we describe further diversity within the ompA-genotype L2b clade, illustrating the transmission dynamics in Argentina and Finland. A key finding is that of a novel clade of Argentinian samples, characterized by a proposed new ompA-genotype L4. Additionally, we present the genome sequence of a non-LGV strain associated with anorectal proctitis. These findings contribute to the investigation of LGV evolution, particularly with the presence of the novel L4 lineage, and provide insights into genomic diversity and transmission dynamics of C. trachomatis.

Acinetobacter baumannii is a nosocomial pathogen associated with various infections, including urinary tract infections (UTIs). In the course of an infection, A. baumannii is known to rapidly become resistant to antibiotic therapy, but much less is known about possible adaptation without antibiotic pressure. Through a retrospective study, we investigated within-host genetic diversity during a subclinical 5-year UTI in an animal-patient after withdrawal of colistin treatment. We conducted whole-genome sequencing and phenotypic assays on 17 clonally related isolates from the Sequence Type 25 lineage. Phylogenomic analysis revealed their proximity with animal and human strains from the same country suggesting zoonotic transmission (France). In this case study, the clonally related strains presented variations in genome sizes and nucleotide sequences. Over the course of the infection, A. baumannii underwent genome reduction through insertion sequence (IS) recombination, phage excision or plasmid curing. Alongside this global genome reduction, we observed an expansion of IS17, initially located on the endogenous large plasmid. Genetic variations were mainly located in biofilm formation and metabolism genes. We observed repeated variations affecting three biofilm genes and two adhesion operons associated with weak biofilm-forming capacity. Conversely, only two metabolic genes were recurrently affected, and phenotypic assays indicated a rather stable metabolism profile between the isolates suggesting minor adaptations to its host. Lastly, an overall decreased antibiotic resistance - expected in the absence of antibiotic treatment - contrasted with a conserved colistin resistance due to a pmrB mutation among the isolates.

The combined application of furazolidone and vancomycin has previously been shown to be synergistic against Gram-negative pathogens, with great therapeutic promise. However, the emergence and mechanism of resistance to this antibiotic combination have not been characterized. To fill this gap, we here selected Escherichia coli progeny for growth on the furazolidone-vancomycin combination at the concentration where the parent was sensitive. We show that selected clones were associated with increased resistance to neither, only one drug, or both furazolidone and vancomycin, but in all cases were associated with a decrease in the growth inhibition synergy. Using whole-genome sequencing, we identified various gene mutations in the resistant mutants. We further investigated the mechanism behind the most frequently arising mutations, those in the riboflavin biosynthesis genes ribB and ribE, that represent novel mutations causing furazolidone resistance and diminished vancomycin-furazolidone synergy. It was found that these ribB/ribE mutations act predominantly by decreasing the activity of the NfsA and NfsB nitroreductases. The emergence of the ribB/ribE mutations imposes a significant fitness cost on bacterial growth. Surprisingly, supplementing the medium with riboflavin, which compensates for the affected riboflavin biosynthesis pathway, could restore the normal growth of the ribB/ribE mutants while having no effects on the furazolidone resistance phenotype. Searching the ribB/ribE mutations in the public sequencing database detects the presence of the furazolidone-resistance-conferring ribE mutations (TKAG^131-134 deletion or duplication) in clinical isolates from different countries. Hypotheses explaining why these ribE mutations were found in clinical isolates despite having poor fitness were further discussed.

Edwardsiella ictaluri continues to pose a significant risk to the health and production of striped catfish (Pangasianodon hypophthalmus) in Vietnam. Whilst recent advances in genomic sequencing provide an insight into the global genomic diversity of this important fish pathogen, genome-wide analysis of Vietnamese isolates recovered over time is lacking. In this study, we used a whole-genome sequencing approach to compare the genomes of 31 E. ictaluri isolates recovered over a 20-year period (2001-2021) and performed comparative genomic analysis to explore temporal changes in genome diversity, population structure and mechanisms driving pathogenesis and antimicrobial resistance. Our findings revealed an open pan-genome with 4148 genes and a core genome (3 060 genes) accounting for over two-thirds of the genome. Moreover, we found the genomes sequenced to classify into two distinct lineages and estimated the ancestral origin of these lineages within Vietnam to date back to the 1950s. Plasmids were highly prevalent in Vietnamese E. ictaluri, with isolates harbouring up to four plasmids within their genome. Further, a diverse mobilome was observed with nine different plasmid types detected across the genome collection. Exploration of putative plasmids revealed a diverse set of antimicrobial resistance genes (ARGs) against key antibiotics used in Vietnamese aquaculture and virulence genes associated with protein secretion systems. Correlation analysis revealed the total number of ARGs detected in genomes to increase with isolate recovery time. Whilst the number of virulence genes remained relatively stable, temporal variation was noted in several virulence factors related to motility and immune system modulation. Findings from this study highlight the need for continued genomic surveillance to monitor changes in antimicrobial resistance and pathogenesis, to help inform the development of disease control and management strategies.

The prediction of the plasmid host range is crucial for investigating the dissemination of plasmids and the transfer of resistance and virulence genes mediated by plasmids. Several machine learning-based tools have been developed to predict plasmid host ranges. These tools have been trained and tested based on the bacterial host records of plasmids in related databases. Typically, a plasmid genome in databases such as the National Center for Biotechnology Information is annotated with only one or a few bacterial hosts, which does not encompass all possible hosts. Consequently, existing methods may significantly underestimate the host ranges of mobile plasmids. In this work, we propose a novel method named HRPredict, which employs a word vector model to digitally represent the encoded proteins on plasmid genomes. Since it is difficult to confirm which host a particular plasmid definitely cannot enter, we developed a machine learning approach for predicting whether a plasmid can enter a specific bacterium as a no-negative samples learning task. Using multiple one-class support vector machine (SVM) models that do not require negative samples for training, HRPredict predicts the host range of plasmids across 45 families, 56 genera and 56 species. In the benchmark test set, we constructed reliable negative samples for each host taxonomic unit via two indirect methods, and we found that the area under the curve (AUC), F1-score, recall, precision and accuracy of most taxonomic unit prediction models exceeded 0.9. Among the 13 broad-host-range plasmid types, HRPredict demonstrated greater coverage than HOTSPOT and PlasmidHostFinder, thus successfully predicting the majority of hosts previously reported. Through feature importance calculation for each SVM model, we found that genes closely related to the plasmid host range are involved in functions such as bacterial adaptability, pathogenicity and survival. These findings provide significant insight into the mechanisms through which bacteria adjust to diverse environments through plasmids. The HRPredict algorithm is expected to facilitate in-depth research on the spread of broad-host-range plasmids and enable host-range predictions for novel plasmids reconstructed from microbiome sequencing data.

Carbapenem-resistant Acinetobacter baumannii is recognized by the World Health Organization (WHO) as one of the top priority pathogens. Despite its public health importance, genomic data of clinical isolates from Malaysia remain scarce. In this study, whole-genome sequencing was performed on 126 A. baumannii isolates collected from the main tertiary hospital in the state of Terengganu, Malaysia, over a 10-year period (2011-2020). Antimicrobial susceptibilities determined for 20 antibiotics belonging to 8 classes showed that 77.0% (n=97/126) of the isolates were categorized as multidrug resistant (MDR), with all MDR isolates being carbapenem resistant. Multilocus sequence typing analysis categorized the Terengganu A. baumannii clinical isolates into 34 Pasteur and 44 Oxford sequence types (STs), with ST2_Pasteur of the Global Clone 2 lineage identified as the dominant ST (n=76/126; 60.3%). The ST2_Pasteur isolates could be subdivided into six Oxford STs with the majority being ST195_Oxford (n=35) and ST208_Oxford (n=17). Various antimicrobial resistance genes were identified with the bla _OXA-23-encoded carbapenemase being the predominant acquired carbapenemase gene (n=90/126; 71.4%). Plasmid-encoded rep genes were identified in nearly all (n=122/126; 96.8%) of the isolates with the majority being Rep_3 family (n=121). Various virulence factors were identified, highlighting the pathogenic nature of this bacterium. Only 14/126 (11.1%) of the isolates were positive for the carriage of CRISPR-Cas arrays with none of the prevalent ST2_Pasteur isolates harbouring them. This study provided a genomic snapshot of the A. baumannii isolates obtained from a single tertiary healthcare centre in Malaysia over a 10-year period and showed the predominance of a single closely related ST2_Pasteur lineage, indicating the entrenchment of this clone in the hospital.

A healthy early-life gut microbiota plays an important role in maintaining immediate and long-term health. Perturbations, particularly in low- to middle-income communities, are associated with increased infection risk. Thus, a promising avenue for restoring a healthy infant microbiota is to select key beneficial bacterial candidates from underexplored microbiomes for developing new probiotic-based therapies. This study aimed to recover bifidobacteria and lactic acid bacteria from the faeces of healthy Cameroonian infants and unravel the genetic basis of their beneficial properties. Faecal samples were collected from 26 infants aged 0-5 months recruited in Dschang (Cameroon). Recovered bacterial isolates were subjected to whole-genome sequencing and in silico analysis to assess their potential for carbohydrate utilization, their antimicrobial capacities, host-adaptation capabilities and their safety. From the range of infant-associated Bifidobacterium and Enterococcus strains identified, Bifidobacterium species were found to harbour putative gene clusters implicated in human milk oligosaccharide metabolism. Genes linked to the production of antimicrobial peptides such as class IV lanthipeptides were found in Bifidobacterium pseudocatenulatum, while those implicated in biosynthesis of cytolysins, enterolysins, enterocins and propeptins, among others, were identified in enterococci. Bifidobacterial isolates did not contain genes associated with virulence; however, we detected the presence of putative tetracycline resistance genes in several strains belonging to Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. longum. Among the enterococci, Enterococcus mundtii PM10 did not carry any genes associated with antimicrobial resistance or virulence. The latter, together with all the Bifidobacterium strains, also encoded several putative adaptive and stress-response-related genes, suggesting robust gastroinstestinal tract colonization potential. This work provides the first genomic characterization of Bifidobacterium and Enterococcus isolates from Cameroonian infants. Several strains showed the genomic potential to confer beneficial properties. Further phenotypic and clinical investigations are needed to confirm their suitability as customized probiotics.