Genomic diversity of Escherichia coli isolates from backyard chickens and guinea fowl in the Gambia

Chickens and guinea fowl are commonly reared in Gambian homes as affordable sources of protein. Using standard microbiological techniques, we obtained 68 caecal isolates of Escherichia coli from 10 chickens and 9 guinea fowl in rural Gambia. After Illumina whole-genome sequencing, 28 sequence types were detected in the isolates (4 of them novel), of which ST155 was the most common (22/68, 32 %). These strains span four of the eight main phylogroups of E. coli, with phylogroups B1 and A being most prevalent. Nearly a third of the isolates harboured at least one antimicrobial resistance gene, while most of the ST155 isolates (14/22, 64 %) encoded resistance to ≥3 classes of clinically relevant antibiotics, as well as putative virulence factors, suggesting pathogenic potential in humans. Furthermore, hierarchical clustering revealed that several Gambian poultry strains were closely related to isolates from humans. Although the ST155 lineage is common in poultry from Africa and South America, the Gambian ST155 isolates belong to a unique cgMLST cluster comprising closely related (38-39 alleles differences) isolates from poultry and livestock from sub-Saharan Africa - suggesting that strains can be exchanged between poultry and livestock in this setting. Continued surveillance of E. coli and other potential pathogens in rural backyard poultry from sub-Saharan Africa is warranted

Acquisition of a large virulence plasmid (pINV) promoted temperature-dependent virulence and global dispersal of O96:H19 enteroinvasive Escherichia coli

Enteroinvasive Escherichia coli (EIEC) and Shigella are closely related agents of bacillary dysentery. It is widely viewed that EIEC and Shigella species evolved from E. coli via independent acquisitions of a large virulence plasmid (pINV) encoding a type 3 secretion system (T3SS). Sequence Type (ST)99 O96:H19 E. coli is a novel clone of EIEC responsible for recent outbreaks in Europe and South America. Here, we use 92 whole genome sequences to reconstruct a dated phylogeny of ST99 E. coli, revealing distinct phylogenomic clusters of pINV-positive and -negative isolates. To study the impact of pINV acquisition on the virulence of this clone, we developed an EIEC-zebrafish infection model showing that virulence of ST99 EIEC is thermoregulated. Strikingly, zebrafish infection using a T3SS-deficient ST99 EIEC strain and the oldest available pINV-negative isolate reveals a separate, temperature-independent mechanism of virulence, indicating that ST99 non-EIEC strains were virulent before pINV acquisition. Taken together, these results suggest that an already pathogenic E. coli acquired pINV and that virulence of ST99 isolates became thermoregulated once pINV was acquired

Remarkable genomic diversity among Escherichia isolates recovered from healthy chickens

The genus Escherichia has been extensively studied and it is known to encompass a range of commensal and pathogenic bacteria that primarily inhabit the gastrointestinal tracts of warm-blooded vertebrates. However, the presence of E. coli as a model organism and potential pathogen has diverted attention away from commensal strains and other species in the genus. To investigate the diversity of Escherichia in healthy chickens, we collected fecal samples from antibiotic-free Lohmann Brown layer hens and determined the genome sequences of 100 isolates, 81 of which were indistinguishable at the HC0 level of the Hierarchical Clustering of Core Genome Multi-Locus Sequence Typing scheme. Despite initial selection on CHROMagar Orientation medium, which is considered selective for E. coli, in silico phylotyping and core genome single nucleotide polymorphism analysis revealed the presence of at least one representative of all major clades of Escherichia, except for E. albertii, Shigella, and E. coli phylogroup B2 and cryptic clade I. The most frequent phylogenomic groups were E. coli phylogroups A and B1 and E. ruysiae (clades III and IV). We compiled a collection of reference strains isolated from avian sources (predominantly chicken), representing every Escherichia phylogroup and species, and used it to confirm the phylogeny and diversity of our isolates. Overall, the isolates carried low numbers of the virulence and antibiotic resistance genes typically seen in avian pathogenic E. coli. Notably, the clades not recovered are ones that have been most strongly associated with virulence by other studies

Within-host microevolution of Streptococcus pneumoniae is rapid and adaptive during natural colonisation

Genomic evolution, transmission and pathogenesis of Streptococcus pneumoniae, an opportunistic human-adapted pathogen, is driven principally by nasopharyngeal carriage. However, little is known about genomic changes during natural colonisation. Here, we use whole-genome sequencing to investigate within-host microevolution of naturally carried pneumococci in ninety-eight infants intensively sampled sequentially from birth until twelve months in a high-carriage African setting. We show that neutral evolution and nucleotide substitution rates up to forty-fold faster than observed over longer timescales in S. pneumoniae and other bacteria drives high within-host pneumococcal genetic diversity. Highly divergent co-existing strain variants emerge during colonisation episodes through real-time intra-host homologous recombination while the rest are co-transmitted or acquired independently during multiple colonisation episodes. Genic and intergenic parallel evolution occur particularly in antibiotic resistance, immune evasion and epithelial adhesion genes. Our findings suggest that within-host microevolution is rapid and adaptive during natural colonisation

Carriage Dynamics of Pneumococcal Serotypes in Naturally Colonized Infants in a Rural African Setting During the First Year of Life

Streptococcus pneumoniae (the pneumococcus) carriage precedes invasive disease and influences population-wide strain dynamics, but limited data exist on temporal carriage patterns of serotypes due to the prohibitive costs of longitudinal studies. Here, we report carriage prevalence, clearance and acquisition rates of pneumococcal serotypes sampled from newborn infants bi-weekly from weeks 1 to 27, and then bi-monthly from weeks 35 to 52 in the Gambia. We used sweep latex agglutination and whole genome sequencing to serotype the isolates. We show rapid pneumococcal acquisition with nearly 31% of the infants colonized by the end of first week after birth and quickly exceeding 95% after 2 months. Co-colonization with multiple serotypes was consistently observed in over 40% of the infants at each sampling point during the first year of life. Overall, the mean acquisition time and carriage duration regardless of serotype was 38 and 24 days, respectively, but varied considerably between serotypes comparable to observations from other regions. Our data will inform disease prevention and control measures including providing baseline data for parameterising infectious disease mathematical models including those assessing the impact of clinical interventions such as pneumococcal conjugate vaccines

Acquisition of a large virulence plasmid (pINV) promoted temperature-dependent virulence and global dispersal of O96:H19 enteroinvasive Escherichia coli

Enteroinvasive Escherichia coli (EIEC) and Shigella are closely related agents of bacillary dysentery. It is widely viewed that EIEC and Shigella species evolved from E. coli via independent acquisitions of a large virulence plasmid (pINV) encoding a type 3 secretion system (T3SS). Sequence Type (ST)99 O96:H19 E. coli is a novel clone of EIEC responsible for recent outbreaks in Europe and South America. Here, we use 92 whole genome sequences to reconstruct a dated phylogeny of ST99 E. coli, revealing distinct phylogenomic clusters of pINV-positive and -negative isolates. To study the impact of pINV acquisition on the virulence of this clone, we developed an EIEC-zebrafish infection model showing that virulence of ST99 EIEC is thermoregulated. Strikingly, zebrafish infection using a T3SS-deficient ST99 EIEC strain and the oldest available pINV-negative isolate reveals a separate, temperature-independent mechanism of virulence, indicating that ST99 non-EIEC strains were virulent before pINV acquisition. Taken together, these results suggest that an already pathogenic E. coli acquired pINV and that virulence of ST99 isolates became thermoregulated once pINV was acquired.IMPORTANCEEnteroinvasive Escherichia coli (EIEC) and Shigella are etiological agents of bacillary dysentery. Sequence Type (ST)99 is a clone of EIEC hypothesized to cause human disease by the recent acquisition of pINV, a large plasmid encoding a type 3 secretion system (T3SS) that confers the ability to invade human cells. Using Bayesian analysis and zebrafish larvae infection, we show that the virulence of ST99 EIEC isolates is highly dependent on temperature, while T3SS-deficient isolates encode a separate temperature-independent mechanism of virulence. These results indicate that ST99 non-EIEC isolates may have been virulent before pINV acquisition and highlight an important role of pINV acquisition in the dispersal of ST99 EIEC in humans, allowing wider dissemination across Europe and South America

The use of representative community samples to assess SARS-CoV-2 lineage competition: Alpha outcompetes Beta and wild-type in England from January to March 2021.

Genomic surveillance for SARS-CoV-2 lineages informs our understanding of possible future changes in transmissibility and vaccine efficacy and will be a high priority for public health for the foreseeable future. However, small changes in the frequency of one lineage over another are often difficult to interpret because surveillance samples are obtained using a variety of methods all of which are known to contain biases. As a case study, using an approach which is largely free of biases, we here describe lineage dynamics and phylogenetic relationships of the Alpha and Beta variant in England during the first 3 months of 2021 using sequences obtained from a random community sample who provided a throat and nose swab for rt-PCR as part of the REal-time Assessment of Community Transmission-1 (REACT-1) study. Overall, diversity decreased during the first quarter of 2021, with the Alpha variant (first identified in Kent) becoming predominant, driven by a reproduction number 0.3 higher than for the prior wild-type. During January, positive samples were more likely to be Alpha in those aged 18 to 54 years old. Although individuals infected with the Alpha variant were no more likely to report one or more classic COVID-19 symptoms compared to those infected with wild-type, they were more likely to be antibody-positive 6 weeks after infection. Further, viral load was higher in those infected with the Alpha variant as measured by cycle threshold (Ct) values. The presence of infections with non-imported Beta variant (first identified in South Africa) during January, but not during February or March, suggests initial establishment in the community followed by fade-out. However, this occurred during a period of stringent social distancing. These results highlight how sequence data from representative community surveys such as REACT-1 can augment routine genomic surveillance during periods of lineage diversity

Phylogeography and resistome of pneumococcal meningitis in West Africa before and after vaccine introduction

Despite contributing to the large disease burden in West Africa, little is known about the genomic epidemiology of Streptococcus pneumoniae which cause meningitis among children under 5 years old in the region. We analysed whole-genome sequencing data from 185 S. pneumoniae isolates recovered from suspected paediatric meningitis cases as part of the World Health Organization (WHO) invasive bacterial diseases surveillance from 2010 to 2016. The phylogeny was reconstructed, accessory genome similarity was computed and antimicrobial-resistance patterns were inferred from the genome data and compared to phenotypic resistance from disc diffusion. We studied the changes in the distribution of serotypes pre- and post-pneumococcal conjugate vaccine (PCV) introduction in the Central and Western sub-regions separately. The overall distribution of non-vaccine, PCV7 (4, 6B, 9V, 14, 18C, 19F and 23F) and additional PCV13 serotypes (1, 3, 5, 6A, 19A and 7F) did not change significantly before and after PCV introduction in the Central region (Fisher's test P value 0.27) despite an increase in the proportion of non-vaccine serotypes to 40 % (n=6) in the post-PCV introduction period compared to 21.9 % (n=14). In the Western sub-region, PCV13 serotypes were more dominant among isolates from The Gambia following the introduction of PCV7, 81 % (n=17), compared to the pre-PCV period in neighbouring Senegal, 51 % (n=27). The phylogeny illustrated the diversity of strains associated with paediatric meningitis in West Africa and highlighted the existence of phylogeographical clustering, with isolates from the same sub-region clustering and sharing similar accessory genome content. Antibiotic-resistance genotypes known to confer resistance to penicillin, chloramphenicol, co-trimoxazole and tetracycline were detected across all sub-regions. However, there was no discernible trend linking the presence of resistance genotypes with the vaccine introduction period or whether the strain was a vaccine or non-vaccine serotype. Resistance genotypes appeared to be conserved within selected sub-clades of the phylogenetic tree, suggesting clonal inheritance. Our data underscore the need for continued surveillance on the emergence of non-vaccine serotypes as well as chloramphenicol and penicillin resistance, as these antibiotics are likely still being used for empirical treatment in low-resource settings. This article contains data hosted by Microreact

Corrigendum: 'Nanopore-only assemblies for genomic surveillance of the global priority drug-resistant pathogen, Klebsiella pneumoniae'.

In the published version of the article, there were errors in the read accessions provided in Table S1, which are now fixed. The updated file (TableS1_v2_13March2023.csv) can be accessed on Figshare using the DOI: 10.6084/m9.figshare.19745608. Furthermore, we would like to correct the following sentence in the Data Summary section of the published version: ‘Illumina reads have been deposited in the NCBI SRA, under the BioProject ID: PRJEB6891, and ONT reads (SUP basecalled) have been deposited under BioProject PRJNA646837.’ The sentence should read: ‘Illumina and ONT reads have been deposited in the NCBI SRA, under the BioProject IDs: PRJEB6891, PRJNA646837 and PRJNA351909.’

Extensive microbial diversity within the chicken gut microbiome revealed by metagenomics and culture

Background The chicken is the most abundant food animal in the world. However, despite its importance, the chicken gut microbiome remains largely undefined. Here, we exploit culture-independent and culture-dependent approaches to reveal extensive taxonomic diversity within this complex microbial community. Results We performed metagenomic sequencing of fifty chicken faecal samples from two breeds and analysed these, alongside all (n = 582) relevant publicly available chicken metagenomes, to cluster over 20 million non-redundant genes and to construct over 5,500 metagenome-assembled bacterial genomes. In addition, we recovered nearly 600 bacteriophage genomes. This represents the most comprehensive view of taxonomic diversity within the chicken gut microbiome to date, encompassing hundreds of novel candidate bacterial genera and species. To provide a stable, clear and memorable nomenclature for novel species, we devised a scalable combinatorial system for the creation of hundreds of well-formed Latin binomials. We cultured and genome-sequenced bacterial isolates from chicken faeces, documenting over forty novel species, together with three species from the genus Escherichia, including the newly named species Escherichia whittamii. Conclusions Our metagenomic and culture-based analyses provide new insights into the bacterial, archaeal and bacteriophage components of the chicken gut microbiome. The resulting datasets expand the known diversity of the chicken gut microbiome and provide a key resource for future high-resolution taxonomic and functional studies on the chicken gut microbiome