- Volume 8, Issue 10, 2022
Volume 8, Issue 10, 2022
- Research Articles
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- Genomic Methodologies
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An update to the database for Acinetobacter baumannii capsular polysaccharide locus typing extends the extensive and diverse repertoire of genes found at and outside the K locus
More LessSeveral novel non-antibiotic therapeutics for the critical priority bacterial pathogen, Acinetobacter baumannii , rely on specificity to the cell-surface capsular polysaccharide (CPS). Hence, prediction of CPS type deduced from genes in whole genome sequence data underpins the development and application of these therapies. In this study, we provide a comprehensive update to the A. baumannii K locus reference sequence database for CPS typing (available in Kaptive v. 2.0.1) to include 145 new KL, providing a total of 237 KL reference sequences. The database was also reconfigured for compatibility with the updated Kaptive v. 2.0.0 code that enables prediction of ‘K type’ from special logic parameters defined by detected combinations of KL and additional genes outside the K locus. Validation of the database against 8994 publicly available A. baumannii genome assemblies from NCBI databases identified the specific KL in 73.45 % of genomes with perfect, very high or high confidence. Poor sequence quality or the presence of insertion sequences were the main reasons for lower confidence levels. Overall, 17 KL were overrepresented in available genomes, with KL2 the most common followed by the related KL3 and KL22. Substantial variation in gene content of the central portion of the K locus, that usually includes genes specific to the CPS type, included 34 distinct groups of genes for synthesis of various complex sugars and >400 genes for forming linkages between sugars or adding non-sugar substituents. A repertoire of 681 gene types were found across the 237 KL, with 88.4 % found in <5 % of KL.
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Benchmarking taxonomic classifiers with Illumina and Nanopore sequence data for clinical metagenomic diagnostic applications
More LessCulture-independent metagenomic detection of microbial species has the potential to provide rapid and precise real-time diagnostic results. However, it is potentially limited by sequencing and taxonomic classification errors. We use simulated and real-world data to benchmark rates of species misclassification using 100 reference genomes for each of the ten common bloodstream pathogens and six frequent blood-culture contaminants (n=1568, only 68 genomes were available for Micrococcus luteus ). Simulating both with and without sequencing error for both the Illumina and Oxford Nanopore platforms, we evaluated commonly used classification tools including Kraken2, Bracken and Centrifuge, utilizing mini (8 GB) and standard (30–50 GB) databases. Bracken with the standard database performed best, the median percentage of reads across both sequencing platforms identified correctly to the species level was 97.8% (IQR 92.7:99.0) [range 5:100]. For Kraken2 with a mini database, a commonly used combination, median species-level identification was 86.4% (IQR 50.5:93.7) [range 4.3:100]. Classification performance varied by species, with Escherichia coli being more challenging to classify correctly (probability of reads being assigned to the correct species: 56.1–96.0%, varying by tool used). Human read misclassification was negligible. By filtering out shorter Nanopore reads we found performance similar or superior to Illumina sequencing, despite higher sequencing error rates. Misclassification was more common when the misclassified species had a higher average nucleotide identity to the true species. Our findings highlight taxonomic misclassification of sequencing data occurs and varies by sequencing and analysis workflow. To account for ‘bioinformatic contamination’ we present a contamination catalogue that can be used in metagenomic pipelines to ensure accurate results that can support clinical decision making.
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- Functional Genomics and Microbe–Niche Interactions
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Trichoderma atroviride hyphal regeneration and conidiation depend on cell-signaling processes regulated by a microRNA-like RNA
The ability to respond to injury is essential for the survival of an organism and involves analogous mechanisms in animals and plants. Such mechanisms integrate coordinated genetic and metabolic reprogramming events requiring regulation by small RNAs for adequate healing of the wounded area. We have previously reported that the response to injury of the filamentous fungus Trichoderma atroviride involves molecular mechanisms closely resembling those of plants and animals that lead to the formation of new hyphae (regeneration) and the development of asexual reproduction structures (conidiophores). However, the involvement of microRNAs in this process has not been investigated in fungi. In this work, we explore the participation of microRNA-like RNAs (milRNAs) molecules by sequencing messenger and small RNAs during the injury response of the WT strain and RNAi mutants. We found that Dcr2 appears to play an important role in hyphal regeneration and is required to produce the majority of sRNAs in T. atroviride. We also determined that the three main milRNAs produced via Dcr2 are induced during the damage-triggered developmental process. Importantly, elimination of a single milRNA phenocopied the main defects observed in the dcr2 mutant. Our results demonstrate the essential role of milRNAs in hyphal regeneration and asexual development by post-transcriptionally regulating cellular signalling processes involving phosphorylation events. These observations allow us to conclude that fungi, like plants and animals, in response to damage activate fine-tuning regulatory mechanisms.
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In silico investigation of the genus Campylobacter type VI secretion system reveals genetic diversity in organization and putative effectors
Bacterial type VI secretion systems (T6SSs) are contractile nanomachines that deliver proteinic substrates into target prokaryotic or eukaryotic cells and the surrounding milieu. The genus Campylobacter encompasses 39 recognized species and 13 subspecies, with many belonging to a group known as ‘emerging Campylobacter pathogens’. Within Campylobacter , seven species have been identified to harbour a complete T6SS cluster but have yet to be comparatively assessed. In this study, using systematic bioinformatics approaches and the T6SS-positive Campylobacter jejuni 488 strain as a reference, we explored the genus-wide prevalence, similarity and make-up of the T6SS amongst 372 publicly available ‘complete’ Campylobacter genomes. Our analyses predict that approximately one-third of Campylobacter species possess a T6SS. We also putatively report the first identification of a T6SS in four species: Campylobacter cuniculorum, Campylobacter helveticus, Campylobacter armoricus and Campylobacter ornithocola . The Campylobacter T6SSs cluster into three distinct organizations (I–III), of which two break down into further variants. Thirty T6SS-containing genomes were found to harbour more than one vgrG gene, with Campylobacter lari strain NCTC 11845 possessing five. Analysis of the C. jejuni Pathogenicity Island-1 confirmed its conservation amongst T6SS-positive C. jejuni strains, as well as highlighting its diverse genetic composition, including additional putative effector–immunity pairs (e.g. PoNe and DUF1911 domains). Effector–immunity pairs were also observed neighbouring vgrGs in several other Campylobacter species, in addition to putative genes encoding nucleases, lysozymes, ATPases and a ferric ATP-binding cassette uptake system. These observations highlight the diverse genetic make-up of the T6SS within Campylobacter and provide further evidence of its role in pathogenesis.
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- Microbial Communities
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Luminal and mucosa-associated caecal microbiota of chickens after experimental Campylobacter jejuni infection in the absence of Campylobacter-specific phages of group II and III
Campylobacteriosis is still the most commonly reported zoonosis in the European Union causing gastrointestinal disease in humans. One of the most common sources for these food-borne infections is broiler meat. Interactions between Campylobacter (C.) jejuni and the intestinal microbiota might influence Campylobacter colonization in chickens. The aim of the present study was to gain further knowledge about exclusive interactions of the host microbiota with C. jejuni in Campylobacter -specific phage-free chickens under standardized conditions and special biosafety precautions.
Therefore, 12 artificially infected ( C. jejuni inoculum with a challenge dose of 7.64 log10 c.f.u.) and 12 control chickens of the breed Ross 308 were kept under special biosafety measures in an animal facility. At day 42 of life, microbiota studies were performed on samples of caecal digesta and mucus. No Campylobacter -specific phages were detected by real-time PCR analysis of caecal digesta of control or artificially infected chickens. Amplification of the 16S rRNA gene was performed within the hypervariable region V4 and subsequently sequenced with Illumina MiSeq platform. R (version 4.0.2) was used to compare the microbiota between C. jejuni -negative and C. jejuni -positive chickens. The factor chickens’ infection status contributed significantly to the differences in microbial composition of mucosal samples, explaining 10.6 % of the microbiota variation (P=0.007) and in digesta samples, explaining 9.69 % of the microbiota variation (P=0.015). The strongest difference between C. jejuni -non-infected and C. jejuni -infected birds was observed for the family Peptococcaceae whose presence in C. jejuni -infected birds could not be demonstrated. Further, several genera of the family Ruminococcaceae appeared to be depressed in its abundance due to Campylobacter infection. A negative correlation was found between Christensenellaceae R-7 group and Campylobacter in C. jejuni -colonised chickens, both genera potentially competing for substrate. This makes Christensenellaceae R-7 group highly interesting for further studies that aim to find control options for Campylobacter infections and assess the relevance of this finding for chicken health and Campylobacter colonization.
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Dissemination of metaldehyde catabolic pathways is driven by mobile genetic elements in Proteobacteria
Bioremediation of metaldehyde from drinking water using metaldehyde-degrading strains has recently emerged as a promising alternative. Whole-genome sequencing was used to obtain full genomes for metaldehyde degraders Acinetobacter calcoaceticus E1 and Sphingobium CMET-H. For the former, the genetic context of the metaldehyde-degrading genes had not been explored, while for the latter, none of the degrading genes themselves had been identified. In A. calcoaceticus E1, IS91 and IS6-family insertion sequences (ISs) were found surrounding the metaldehyde-degrading gene cluster located in plasmid pAME76. This cluster was located in closely-related plasmids and associated to identical ISs in most metaldehyde-degrading β- and γ-Proteobacteria, indicating horizontal gene transfer (HGT). For Sphingobium CMET-H, sequence analysis suggested a phytanoyl-CoA family oxygenase as a metaldehyde-degrading gene candidate due to its close homology to a previously identified metaldehyde-degrading gene known as mahX. Heterologous gene expression in Escherichia coli alongside degradation tests verified its functional significance and the degrading gene homolog was henceforth called mahS. It was found that mahS is hosted within the conjugative plasmid pSM1 and its genetic context suggested a crossover between the metaldehyde and acetoin degradation pathways. Here, specific replicons and ISs responsible for maintaining and dispersing metaldehyde-degrading genes in α, β and γ-Proteobacteria through HGT were identified and described. In addition, a homologous gene implicated in the first step of metaldehyde utilisation in an α-Proteobacteria was uncovered. Insights into specific steps of this possible degradation pathway are provided.
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- Pathogens and Epidemiology
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Endemic erythromycin resistant Corynebacterium diphtheriae in Vietnam in the 1990s
Diphtheria is a potentially fatal respiratory disease caused by toxigenic forms of the Gram-positive bacterium Corynebacterium diphtheriae . Despite the availability of treatments (antitoxin and antimicrobials) and effective vaccines, the disease still occurs sporadically in low-income countries and in higher income where use of diphtheria vaccine is inconsistent. Diphtheria was highly endemic in Vietnam in the 1990s; here, we aimed to provide some historical context to the circulation of erythromycin resistant organisms in Vietnam during this period. After recovering 54 C . diphtheriae isolated from clinical cases of diphtheria in Ho Chi Minh City between 1992 and 1998 we conducted whole genome sequencing and analysis. Our data outlined substantial genetic diversity among the isolates, illustrated by seven distinct Sequence Types (STs), but punctuated by the sustained circulation of ST67 and ST209. With the exception of one isolate, all sequences contained the tox gene, which was classically located on a corynebacteriophage. All erythromycin resistant isolates, accounting for 13 % of organisms in this study, harboured a novel 18 kb erm(X)-carrying plasmid, which exhibited limited sequence homology to previously described resistance plasmids in C. diphtheriae . Our study provides historic context for the circulation of antimicrobial resistant C. diphtheriae in Vietnam; these data provide a framework for the current trajectory in global antimicrobial resistance trends.
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A global pangenome for the wheat fungal pathogen Pyrenophora tritici-repentis and prediction of effector protein structural homology
The adaptive potential of plant fungal pathogens is largely governed by the gene content of a species, consisting of core and accessory genes across the pathogen isolate repertoire. To approximate the complete gene repertoire of a globally significant crop fungal pathogen, a pan genomic analysis was undertaken for Pyrenophora tritici-repentis (Ptr), the causal agent of tan (or yellow) spot disease in wheat. In this study, 15 new Ptr genomes were sequenced, assembled and annotated, including isolates from three races not previously sequenced. Together with 11 previously published Ptr genomes, a pangenome for 26 Ptr isolates from Australia, Europe, North Africa and America, representing nearly all known races, revealed a conserved core-gene content of 57 % and presents a new Ptr resource for searching natural homologues (orthologues not acquired by horizontal transfer from another species) using remote protein structural homology. Here, we identify for the first time a non-synonymous mutation in the Ptr necrotrophic effector gene ToxB, multiple copies of the inactive toxb within an isolate, a distant natural Pyrenophora homologue of a known Parastagonopora nodorum necrotrophic effector (SnTox3), and clear genomic break points for the ToxA effector horizontal transfer region. This comprehensive genomic analysis of Ptr races includes nine isolates sequenced via long read technologies. Accordingly, these resources provide a more complete representation of the species, and serve as a resource to monitor variations potentially involved in pathogenicity.
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The rate and role of pseudogenes of the Mycobacterium tuberculosis complex
Whole-genome sequence analyses have significantly contributed to the understanding of virulence and evolution of the Mycobacterium tuberculosis complex (MTBC), the causative pathogens of tuberculosis. Most MTBC evolutionary studies are focused on single nucleotide polymorphisms and deletions, but rare studies have evaluated gene content, whereas none has comprehensively evaluated pseudogenes. Accordingly, we describe an extensive study focused on quantifying and predicting possible functions of MTBC and Mycobacterium canettii pseudogenes. Using NCBI’s PGAP-detected pseudogenes, we analysed 25 837 pseudogenes from 158 MTBC and M. canetii strains and combined transcriptomics and proteomics of M. tuberculosis H37Rv to gain insights about pseudogenes' expression. Our results indicate significant variability concerning rate and conservancy of in silico predicted pseudogenes among different ecotypes and lineages of tuberculous mycobacteria and pseudogenization of important virulence factors and genes of the metabolism and antimicrobial resistance/tolerance. We show that in silico predicted pseudogenes contribute considerably to MTBC genetic diversity at the population level. Moreover, the transcription machinery of M. tuberculosis can fully transcribe most pseudogenes, indicating intact promoters and recent pseudogene evolutionary emergence. Proteomics of M. tuberculosis and close evaluation of mutational lesions driving pseudogenization suggest that few in silico predicted pseudogenes are likely capable of neofunctionalization, nonsense mutation reversal, or phase variation, contradicting the classical definition of pseudogenes. Such findings indicate that genome annotation should be accompanied by proteomics and protein function assays to improve its accuracy. While indels and insertion sequences are the main drivers of the observed mutational lesions in these species, population bottlenecks and genetic drift are likely the evolutionary processes acting on pseudogenes' emergence over time. Our findings unveil a new perspective on MTBC’s evolution and genetic diversity.
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Epidemiological links and antimicrobial resistance of clinical Salmonella enterica ST198 isolates: a nationwide microbial population genomic study in Switzerland
More LessSalmonella is a leading cause of foodborne outbreaks and systemic infections worldwide. Emerging multi-drug resistant Salmonella lineages such as a ciprofloxacin-resistant subclade (CIPR) within Salmonella enterica serovar Kentucky ST198 threaten the effective prevention and treatment of infections. To understand the genomic diversity and antimicrobial resistance gene content associated with S. Kentucky in Switzerland, we whole-genome sequenced 70 human clinical isolates obtained between 2010 and 2020. Most isolates belonged to ST198-CIPR. High- and low-level ciprofloxacin resistance among CIPR isolates was associated with variable mutations in ramR and acrB in combination with stable mutations in quinolone-resistance determining regions (QRDRs). Analysis of isolates from patients with prolonged ST198 colonization indicated subclonal adaptions with the ramR locus as a mutational hotspot. SNP analyses identified multiple clusters of near-identical isolates, which were often associated with travel but included spatiotemporally linked isolates from Switzerland. The largest SNP cluster was associated with travellers returning from Indonesia, and investigation of global data linked >60 additional ST198 salmonellosis isolates to this cluster. Our results emphasize the urgent need for implementing whole-genome sequencing as a routine tool for Salmonella surveillance and outbreak detection.
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One clone to rule them all: Culture-independent genomics of Chlamydia psittaci from equine and avian hosts in Australia
Chlamydia psittaci is an avian pathogen with zoonotic potential. In Australia, C. psittaci has been well reported as a cause of reproductive loss in mares which subsequently have been the source of infection and illness in some in-contact humans. To date, molecular typing studies describe the predominant and clonal C. psittaci sequence type (ST)24 strains in horse, psittacine, and human infections. We sought to assess the clonality between ST24 strains and the emergence of equine ST24 with a comprehensive genomics approach. We used culture-independent probe-based and metagenomic whole-genome sequencing to investigate 13 C . psittaci genomes from horses, psittacines, and a pigeon from Australia. Published genomes of 36 C . psittaci strains were also used to contextualise our Australian dataset and investigate lineage diversity. We utilised a single-nucleotide polymorphism (SNP) based clustering and multi-locus sequence typing (MLST) approach. C. psittaci has four major phylogenetic groups (PG1-4) based on core-genome SNP-based phylogeny. PG1 contained clonal global and Australian equine, psittacine, and human ST24 genomes, with a median pairwise SNP distance of 68 SNPs. PG2, PG3, and PG4 had greater genomic diversity, including diverse STs collected from birds, livestock, human, and horse hosts from Europe and North America and a racing pigeon from Australia. We show that the clustering of C. psittaci by MLST was congruent with SNP-based phylogeny. The monophyletic ST24 clade has four major sub-lineages. The genomes of 17 Australian human, equine, and psittacine strains collected between 2008 and 2021 formed the predominant ST24 sub-lineage 1 (emerged circa 1979). Despite a temporal distribution of 13 years, the genomes within sub-lineage 1 had a median pairwise SNP distance of 32 SNPs, suggesting a recent population expansion or potential cross-host transmission. However, two C. psittaci genomes collected in 2015 from Victorian parrots clustered into distinct ST24 sub-lineage 4 (emerged circa 1965) with ovine strain C19/98 from Germany. This work describes a comprehensive phylogenomic characterisation of ST24 and identifies a timeline of potential bird-to-equine spillover events.
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- Short Communications
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- Pathogens and Epidemiology
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Whole genome sequence analysis of ESBL-producing Escherichia coli recovered from New Zealand freshwater sites
More LessExtended-spectrum beta lactamase (ESBL)-producing Escherichia coli are often isolated from humans with urinary tract infections and may display a multidrug-resistant phenotype. These pathogens represent a target for a One Health surveillance approach to investigate transmission between humans, animals and the environment. This study examines the multidrug-resistant phenotype and whole genome sequence data of four ESBL-producing E . coli isolated from freshwater in New Zealand. All four isolates were obtained from a catchment with a mixed urban and pastoral farming land-use. Three isolates were sequence type (ST) 131 (CTX-M-27-positive) and the other ST69 (CTX-M-15-positive); a phylogenetic comparison with other locally isolated strains demonstrated a close relationship with New Zealand clinical isolates. Genes associated with resistance to antifolates, tetracyclines, aminoglycosides and macrolides were identified in all four isolates, together with fluoroquinolone resistance in two isolates. The ST69 isolate harboured the bla CTX-M-15 gene on a IncHI2A plasmid, and two of the three ST131 isolates harboured the bla CTX-M-27 genes on IncF plasmids. The last ST131 isolate harboured bla CTX-M-27 on the chromosome in a unique site between gspC and gspD. These data highlight a probable human origin of the isolates with subsequent transmission from urban centres through wastewater to the wider environment.
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