- Volume 10, Issue 1, 2024

Generating complete, high-quality genome assemblies is key for any downstream analysis, such as comparative genomics. For bacterial genome assembly, various algorithms and fully automated pipelines exist, which are free-of-charge and easily accessible. However, these assembly tools often cannot unambiguously resolve a bacterial genome, for example due to the presence of sequence repeat structures on the chromosome or on plasmids. Then, a more sophisticated approach and/or manual curation is needed. Such modifications can be challenging, especially for non-bioinformaticians, because they are generally not considered as a straightforward process. In this study, we propose a standardized approach for manual genome completion focusing on the popular hybrid assembly pipeline Unicycler. The provided Galaxy workflow addresses two weaknesses in Unicycler’s hybrid assemblies: (i) collapse of inter-plasmidic repeats and (ii) false loss of single-copy sequences. To demonstrate and validate how to detect and resolve these assembly errors, we use two genomes from the Bacillus cereus group. By applying the proposed pipeline following an automated assembly, the genome sequence quality can be significantly improved.

The risk to human health from mosquito-borne viruses such as dengue, chikungunya and yellow fever is increasing due to increased human expansion, deforestation and climate change. To anticipate and predict the spread and transmission of mosquito-borne viruses, a better understanding of the transmission cycle in mosquito populations is needed. We present a pathogen-agnostic combined sequencing protocol for identifying vectors, viral pathogens and their hosts or reservoirs using portable Oxford Nanopore sequencing. Using mosquitoes collected in São Paulo, Brazil, we extracted RNA for virus identification and DNA for blood meal and mosquito identification. Mosquitoes and blood meals were identified by comparing cytochrome c oxidase I (COI) sequences against a curated Barcode of Life Data System (BOLD). Viruses were identified using the SMART-9N protocol, which allows amplified DNA to be prepared with native barcoding for nanopore sequencing. Kraken 2 was employed to detect viral pathogens and Minimap2 and BOLD identified the contents of the blood meal. Due to the high similarity of some species, mosquito identification was conducted using blast after generation of consensus COI sequences using RACON polishing. This protocol can simultaneously uncover viral diversity, mosquito species and mosquito feeding habits. It also has the potential to increase understanding of mosquito genetic diversity and transmission dynamics of zoonotic mosquito-borne viruses.

Amphibian skin microbiomes can play a critical role in host survival against emerging diseases by protecting their host against pathogens. While a plethora of biotic and abiotic factors have been shown to influence the taxonomic diversity of amphibian skin microbiomes it remains unclear whether functional genomic diversity varies in response to temporal and environmental factors. Here we applied a metagenomic approach to evaluate whether seasonality, distinct elevations/sites, and pathogen presence influenced the functional genomic diversity of the A. altamirani skin microbiome. We obtained a gene catalogue of 92 107 nonredundant annotated genes and a set of 50 unique metagenome assembled genomes (MAGs). Our analysis showed that genes linked to general and potential antifungal traits significantly differed across seasons and sampling locations at different elevations. Moreover, we found that the functional genomic diversity of A. altamirani skin microbiome differed between B. dendrobatidis infected and not infected axolotls only during winter, suggesting an interaction between seasonality and pathogen infection. In addition, we identified the presence of genes and biosynthetic gene clusters (BGCs) linked to potential antifungal functions such as biofilm formation, quorum sensing, secretion systems, secondary metabolite biosynthesis, and chitin degradation. Interestingly genes linked to these potential antifungal traits were mainly identified in Burkholderiales and Chitinophagales MAGs. Overall, our results identified functional traits linked to potential antifungal functions in the A. altamirani skin microbiome regardless of variation in the functional diversity across seasons, elevations/sites, and pathogen presence. Our findings suggest that potential antifungal traits found in Burkholderiales and Chitinophagales taxa could be related to the capacity of A. altamirani to survive in the presence of Bd, although further experimental analyses are required to test this hypothesis.

In this study, two novel bacterial strains were isolated from coastal sediment of Weihai, China. The two strains were Gram-stain-negative and facultatively aerobic, designated 3-1745T and A346T. Based on phenotypic, genetic and phylogenetic properties, strains 3-1745T and A346T represent two novel species of the genus Marinobacterium . The results of genome analysis revealed many central carbohydrate metabolism pathways such as gluconeogenesis, pyruvate oxidation, tricyclic acid cycle, pentose phosphate pathway and PRPP biosynthesis in the genus Marinobacterium . The ability of strains 3-1745T and A346T to utilize volatile fatty acids was experimentally confirmed. Polyhydroxyalkanoate synthases (PhaA, PhaB and PhaC) for the synthesis of polyhydroxyalkanoates were prevalent in the genus Marinobacterium . Multiple BGCs (biosynthetic gene clusters) including betalactone, ectoine, ranthipeptide, redox-cofactor, RiPPs (ribosomally synthesized post-translationally modified peptides) and T3PKS (polyketide synthases) in the genome of the genus Marinobacterium were found. Additional genome analyses suggested that the genus Marinobacterium contained diverse potential mechanisms of salt tolerance and mainly utilized oligosaccharides. This is the first report on broad genomic analyses of the genus Marinobacterium with the description of two novel species and potential ecological and biotechnological implications.

African swine fever (ASF) is a severe haemorrhagic disease caused by the African swine fever virus (ASFV), transmitted by ticks, resulting in high mortality among domestic pigs and wild boars. The global spread of ASFV poses significant economic threats to the swine industry. This study employs diverse analytical methods to explore ASFV’s evolution and host adaptation, focusing on codon usage patterns and associated factors. Utilizing phylogenetic analysis methods including neighbour-joining and maximum-likelihood, 64 ASFV strains were categorized into four clades. Codon usage bias (CUB) is modest in ASFV coding sequences. This research identifies multiple factors – such as nucleotide composition, mutational pressures, natural selection and geographical diversity – contributing to the formation of CUB in ASFV. Analysis of relative synonymous codon usage reveals CUB variations within clades and among ASFVs and their hosts. Both Codon Adaptation Index and Similarity Index analyses confirm that ASFV strains are highly adapted to soft ticks (Ornithodoros moubata) but less so to domestic pigs, which could be a result of the long-term co-evolution of ASFV with ticks. This study sheds light on the factors influencing ASFV’s codon usage and fitness dynamics, enriching our understanding of its evolution, adaptation and host interactions.

Phages integrated into a bacterial genome – called prophages – continuously monitor the vigour of the host bacteria to determine when to escape the genome and to protect their host from other phage infections, and they may provide genes that promote bacterial growth. Prophages are essential to almost all microbiomes, including the human microbiome. However, most human microbiome studies have focused on bacteria, ignoring free and integrated phages, so we know little about how these prophages affect the human microbiome. To address this gap in our knowledge, we compared the prophages identified in 14 987 bacterial genomes isolated from human body sites to characterize prophage DNA in the human microbiome. Here, we show that prophage DNA is ubiquitous, comprising on average 1–5 % of each bacterial genome. The prophage content per genome varies with the isolation site on the human body, the health of the human and whether the disease was symptomatic. The presence of prophages promotes bacterial growth and sculpts the microbiome. However, the disparities caused by prophages vary throughout the body.

The extent of intraspecific genomic variation is key to understanding species evolutionary history, including recent adaptive shifts. Intraspecific genomic variation remains poorly explored in eukaryotic micro-organisms, especially in the nuclear dimorphic ciliates, despite their fundamental role as laboratory model systems and their ecological importance in many ecosystems. We sequenced the macronuclear genome of 22 laboratory strains of the oligohymenophoran Tetrahymena thermophila, a model species in both cellular biology and evolutionary ecology. We explored polymorphisms at the junctions of programmed eliminated sequences, and reveal their utility to barcode very closely related cells. As for other species of the genus Tetrahymena, we confirm micronuclear centromeres as gene diversification centres in T. thermophila, but also reveal a two-speed evolution in these regions. In the rest of the genome, we highlight recent diversification of genes coding for extracellular proteins and cell adhesion. We discuss all these findings in relation to this ciliate’s ecology and cellular characteristics.

Epidemiological data have indicated that invasive infections caused by the Gram-positive cocci Streptococcus pyogenes (group A streptococcus, GAS) have increased in many Australian states over the past two decades. In July 2022, invasive GAS (iGAS) infections became nationally notifiable in Australia via public-health agencies. Surveillance for S. pyogenes infections has been sporadic within the state of New South Wales (NSW). This has led to a lack of genetic data on GAS strains in circulation, particularly for non-invasive infections, which are the leading cause of GAS’s burden on the Australian healthcare system. To address this gap, we used whole-genome sequencing to analyse the genomes of 318  S . pyogenes isolates collected within two geographical regions of NSW. Invasive isolates were collected in 2007–2017, whilst non-invasive isolates were collected in 2019–2021. We found that at least 66 different emm-types were associated with clinical disease within NSW. There was no evidence of any Australian-specific clones in circulation. The M1UK variant of the emm1 global pandemic clone (M1global) has been detected in our isolates from 2013 onwards. We detected antimicrobial-resistance genes (mainly tetM, ermA or ermB genes) in less than 10 % of our 318 isolates, which were more commonly associated with non-invasive infections. Superantigen virulence gene carriage was reasonably proportionate between non-invasive and invasive infection isolates. Our study adds rich data on the genetic makeup of historical S. pyogenes infections within Australia. Ongoing surveillance of invasive and non-invasive GAS infections within NSW by whole-genome sequencing is warranted to inform on outbreaks, antimicrobial resistance and vaccine coverage.

Listeria monocytogenes is a food-borne pathogen, typically affecting the elderly, immunocompromised patients and pregnant women. The aim of this study was to determine the population structure of L. monocytogenes clonal complex 1 (CC1) in the UK and describe the genomic epidemiology of this clinically significant CC. We interrogated a working dataset of 4073 sequences of L. monocytogenes isolated between January 2015 and December 2020 from human clinical specimens, food and/or food-production environments. A minimum spanning tree was reconstructed to determine the population structure of L. monocytogenes in the UK. Subsequent analysis focused on L. monocytogenes CC1, as the cause of the highest proportion of invasive listeriosis in humans. Sequencing data was integrated with metadata on food and environmental isolates, and information from patient questionnaires, including age, sex and clinical outcomes. All isolates either belonged to lineage I (n=1299/4073, 32%) or lineage II (n=2774/4073, 68%), with clinical isolates from human cases more likely to belong to lineage I (n=546/928, 59%) and food isolates more likely to belong to lineage II (n=2352/3067, 77%). Of the four largest CCs, CC1 (n=237) had the highest proportion of isolates from human cases of disease (CC1 n=160/237, 67.5 %; CC121 n=13/843, 2 %; CC9 n=53/360, 15 %; CC2 n=69/339, 20%). Within CC1, most cases were female (n=95/160, 59%, P=0.01771) and the highest proportion of cases were in people >60 years old (39/95, 41%, P=1.314×10−6) with a high number of them aged 20–39 years old (n=35/95, 37%) most linked to pregnancy-related listeriosis (n=29/35, 83%). Most of the male cases were in men aged over 60 years old (40/65, 62%), and most of the fatal cases in both males and females were identified in this age group (42/55, 76%). Phylogenetic analysis revealed 23 5 SNP single linkage clusters comprising 80/237 (34 %) isolates with cluster sizes ranging from 2 to 19. Five 5 SNP clusters comprised isolates from human cases and an implicated food item. Expanding the analysis to 25 SNP single linkage clusters resolved an additional two clusters linking human cases to a potential food vehicle. Analysis of demographic and clinical outcome data identified CC1 as a clinically significant cause of invasive listeriosis in the elderly population and in women of child-bearing age. Phylogenetic analysis revealed the population structure of CC1 in the UK comprised small, sparsely populated genomic clusters. Only clusters containing isolates from an implicated food vehicle, or food processing or farming environments, were resolved, emphasizing the need for clinical, food and animal-health agencies to share sequencing data in real time, and the importance of a One Health approach to public-health surveillance of listeriosis.

Pet turtles are a well-recognized source of human salmonellosis, posing a threat to human health, particularly children who commonly keep pet turtles. To date, the genomic characteristics of Salmonella among pet turtles and children has not been well described. We investigated the prevalence, antimicrobial resistance (AMR) and genomic characteristics of Salmonella from pet turtles in Beijing, China. In total, 9.6 % (46/480) of pet turtles were positive for Salmonella with S. Thompson being the dominant serovar (19/46) in 2019. Moreover, 80.4 % of Salmonella were multi-drug resistant (MDR) and 60.7 % were resistant to ampicillin, streptomycin, sulfonamides and tetracycline (ASSuT). We further compared the genomes of S. Thompson isolates from pet turtles (n=19) with those from children with diarrhoea (n=28) in the same region and year, most of which were sequence type (ST)26, with one novel ST7937 identified from a child-associated isolate. S. Thompson isolates from children with diarrhoea exhibited less resistance than isolates from pet turtles. Most MDR isolates possessed multiple AMR genes, including the AmpC β-lactamase-encoding genes bla DHA-15 and bla DHA-1 which co-occurred with the IncA/C and IncHI plasmid replicon types. To the best of our knowledge, this is the first time that the bla DHA-15 gene has been detected from Salmonella . Several pet turtle-associated S. Thompson isolates comprised phylogenetically close clusters with those from children with diarrhoea (<20 SNP differences). Bayesian analysis demonstrated that the Chinese ST26 S. Thompson strains had a recent evolutionary history and evolved into two major clades, with one clade acquiring various resistant plasmids. Our findings revealed the emergence of MDR Salmonella among pet turtles in China and provided evidence for the interspecies transmission of S. Thompson.

Campylobacter spp. are a common cause of bacterial gastroenteritis in Australia, primarily acquired from contaminated meat. We investigated the relationship between genomic virulence characteristics and the severity of campylobacteriosis, hospitalisation, and other host factors.

We recruited 571 campylobacteriosis cases from three Australian states and territories (2018–2019). We collected demographic, health status, risk factors, and self-reported disease data. We whole genome sequenced 422 C . jejuni and 84 C . coli case isolates along with 616 retail meat isolates. We classified case illness severity using a modified Vesikari scoring system, performed phylogenomic analysis, and explored risk factors for hospitalisation and illness severity.

On average, cases experienced a 7.5 day diarrhoeal illness with additional symptoms including stomach cramps (87.1 %), fever (75.6 %), and nausea (72.0 %). Cases aged ≥75 years had milder symptoms, lower Vesikari scores, and higher odds of hospitalisation compared to younger cases. Chronic gastrointestinal illnesses also increased odds of hospitalisation. We observed significant diversity among isolates, with 65 C . jejuni and 21 C . coli sequence types. Antimicrobial resistance genes were detected in 20.4 % of isolates, but multidrug resistance was rare (0.04 %). Key virulence genes such as cdtABC ( C. jejuni ) and cadF were prevalent (>90 % presence) but did not correlate with disease severity or hospitalisation. However, certain genes (e.g. fliK, Cj1136, and Cj1138) appeared to distinguish human C. jejuni cases from food source isolates.

Campylobacteriosis generally presents similarly across cases, though some are more severe. Genotypic virulence factors identified in the literature to-date do not predict disease severity but may differentiate human C. jejuni cases from food source isolates. Host factors like age and comorbidities have a greater influence on health outcomes than virulence factors.

Xanthomonas translucens, the causal agent of bacterial leaf streak disease (BLS) in cereals, is a re-emerging pathogen that is becoming increasingly destructive across the world. While BLS has caused yield losses in the past, there is anecdotal evidence that newer isolates may be more virulent. We observed that two X. translucens isolates collected from two sites in Colorado, USA, are more aggressive on current wheat and barley varieties compared to older isolates, and we hypothesize that genetic changes between recent and older isolates contribute to the differences in isolate aggressiveness. To test this, we phenotyped and genetically characterized two X. translucens isolates collected from Colorado in 2018, which we designated CO236 (from barley) and CO237 (from wheat). Using pathovar-specific phenotyping and PCR primers, we determined that CO236 belongs to pathovar translucens (Xtt) and CO237 belongs to pathovar undulosa (Xtu). We sequenced the full genomes of the isolates using Oxford Nanopore long-read sequencing, and compared their whole genomes against published X. translucens genomes. This analysis confirmed our pathovar designations for Xtt CO236 and Xtu CO237, and showed that, at the whole-genome level, there were no obvious genomic structural changes between Xtt CO236 and Xtu CO237 and other respective published pathovar genomes. Focusing on pathovar undulosa (Xtu CO237), we then compared putative type III effectors among all available Xtu isolate genomes and found that they were highly conserved. However, there were striking differences in the presence and sequence of various transcription activator-like effectors between Xtu CO237 and published undulosa genomes, which correlate with isolate virulence. Here, we explore the potential implications of the differences in these virulence factors, and provide possible explanations for the increased virulence of recently emerged isolates.

Candida glabrata is a commensal yeast of the gastrointestinal tract and skin of humans. However, it causes opportunistic infections in immunocompromised patients, and is the second most common Candida pathogen causing bloodstream infections. Although there are many studies on the epidemiology of C. glabrata infections, the fine- and large-scale geographical nature of C. glabrata remain incompletely understood. Here we investigated both the fine- and large-scale population structure of C. glabrata through genome sequencing of 80 clinical isolates obtained from six tertiary hospitals in Qatar and by comparing with global collections. Our fine-scale analyses revealed high genetic diversity within the Qatari population of C. glabrata and identified signatures of recombination, inbreeding and clonal expansion within and between hospitals, including evidence for nosocomial transmission among coronavirus disease 2019 (COVID-19) patients. In addition to signatures of recombination at the population level, both MATa and MATα alleles were detected in most hospitals, indicating the potential for sexual reproduction in clinical environments. Comparisons with global samples showed that the Qatari C. glabrata population was very similar to those from other parts of the world, consistent with the significant role of recent anthropogenic activities in shaping its population structure. Genome-wide association studies identified both known and novel genomic variants associated with reduced susceptibilities to fluconazole, 5-flucytosine and echinocandins. Together, our genomic analyses revealed the diversity, transmission patterns and antifungal drug resistance mechanisms of C. glabrata in Qatar as well as the relationships between Qatari isolates and those from other parts of the world.

Campylobacter spp. are a leading cause of bacterial foodborne zoonosis worldwide, with poultry meat and products recognised as a significant source of human infection. In Vietnam there are few data regarding the occurrence, antimicrobial resistance, and genomic diversity of Campylobacter in poultry and poultry meat. The aim of this study was to estimate the prevalence of Campylobacter in chicken meat at retail in Hanoi, determine antimicrobial sensitivities of the Campylobacter isolated, and assess their genetic diversity. A total of 120 chicken meat samples were collected from eight traditional retail markets (n=80) and four supermarkets (n=40). Campylobacter was isolated following ISO 10272-1 : 2017 and identification verified by PCR. The prevalence of Campylobacter was 38.3 % (46/120) and C. coli was the most prevalent species in both retail markets (74 %) and supermarkets (88 %). The minimum inhibitory concentrations for ciprofloxacin, erythromycin, gentamicin, nalidixic acid, streptomycin, and tetracycline were determined by broth microdilution for 32 isolates. All characterised Campylobacter were resistant to ciprofloxacin, nalidixic acid, and tetracycline, with corresponding resistance determinants detected in the sequenced genomes. Most C. coli were multidrug resistant (24/28) and two harboured the erythromycin resistance gene ermB on a multiple drug-resistance genomic island, a potential mechanism for dissemination of resistance. The 32 isolates belonged to clonal complexes associated with both poultry and people, such as CC828 for C. coli . These results contribute to the One Health approach for addressing Campylobacter in Vietnam by providing detailed new insights into a main source of human infection and can inform the design of future surveillance approaches.

Pakistan is amongst the four countries with the highest number of pneumococcal deaths. While the PCV10 vaccine was introduced in Pakistan in October 2012, data regarding the impact of the vaccine on the population dynamics of Streptococcus pneumoniae in Pakistan remain obscure. Using whole genome sequencing of 190 isolates (nasopharyngeal carriage=75, disease=113, unknown sites=2) collected between 2002 and 2020, this study presents characteristics of pneumococcal strains in Pakistan in the pre- and post-vaccine era. The isolates were characterized on the basis of serotype distribution, genetic lineages (or Global Pneumococcal Sequence Cluster, GPSC) and antibiotic resistance. A high level of diversity in serotype and genetic lineages of pneumococci was observed in Pakistan. Among 190 isolates, we identified 54 serotypes, 67 GPSCs and 116 sequence types (STs) including 23 new STs. The most prevalent GPSCs and their associated serotypes in nasopharyngeal carriage were GPSC54 (expressing serotype 9V), GPSC5 (15A and 7B, and serogroup 24), GPSC25 (15B/15C), GPSC67 (18C) and GPSC376 (6A and 6D). Similarly, among 113 disease-causing isolates, the most prevalent GPSC/serotype combinations were GPSC2 (serotype 1), GPSC10 (serotypes 14, 10A, 19A and 19F), GPSC43 (serotypes 13, 11A, 23B, 35A and 9V), GPSC67 (serotypes 18A and 18C) and GPSC642 (serotype 11A). Of the 190 isolates, the highest levels of resistance were observed against penicillin (58.9 %, n=122), erythromycin (29.5 %, n=56), clindamycin (13.2 %, n=25), co-trimoxazole (94.2 %, n=179) and tetracycline/doxycycline (53.2 %, n=101). A higher proportion of disease-causing isolates were multidrug resistant as compared to carriage isolates (54 % vs 25 %). Our data suggest limited coverage of PCV10 in nasopharyngeal (21.6 %, 16/74) as well as disease-causing (38.1 %, 16/42) isolates among children ≤5 years old; however, higher valent vaccine PCV13 would increase the coverage rates to 33.8 % in nasopharyngeal and 54.8 % in disease-causing isolates, whereas PCV24/25 would offer the highest coverage rates. Owing to the diversity of serotypes observed during the post-vaccine period, the suggested inclusion of serotype in future vaccine formulations will require investigations with larger data sets with an extended temporal window. This article contains data hosted by Microreact.

Many peptidoglycan-deficient bacteria such as the Mycoplasmatales are known host-associated lineages, lacking the environmental resistance mechanisms and metabolic capabilities necessary for a free-living lifestyle. Several peptidoglycan-deficient and non-sporulating orders of interest are thought to be descended from Gram-positive sporulating Bacilli through reductive evolution. Here we annotate 2650 genomes belonging to the class Bacilli , according to the Genome Taxonomy Database, to predict the peptidoglycan and sporulation phenotypes of three novel orders, RFN20, RF39 and ML615J-28, known only through environmental sequence surveys. These lineages are interspersed between peptidoglycan-deficient non-sporulating orders including the Mycoplasmatales and Acholeplasmatales , and more typical Gram-positive orders such as the Erysipelotrichales and Staphylococcales. We use the extant genotypes to perform ancestral state reconstructions. The novel orders are predicted to have small genomes with minimal metabolic capabilities and to comprise a mix of peptidoglycan-deficient and/or non-sporulating species. In contrast to expectations based on cultured representatives, the order Erysipelotrichales lacks many of the genes involved in peptidoglycan and endospore formation. The reconstructed evolutionary history of these traits suggests multiple independent whole-genome reductions and loss of phenotype via intermediate transition states that continue into the present. We suggest that the evolutionary history of the reduced-genome lineages within the class Bacilli is one driven by multiple independent transitions to host-associated lifestyles, with the degree of reduction in environmental resistance and metabolic capabilities correlated with degree of host association.

In temperate marine climate zones, seasonal changes in water temperature contribute to distinct populations of warm- and cold-water vibrios. We report here the complete genome sequence (BUSCO score=94.8) of the novel strain Vibrio sp. VB16 isolated in late winter from the intertidal zone near Virginia Beach, Virginia, USA with the ability to form colonies at 4 °C. The 5.2 Mbp genome is composed of a large (3.6 Mbp) and small (1.6 Mbp) chromosome. Based on paired average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA–DNA hybridization (dDDH), V. sp. VB16 is the same species as V. sp. UBA2437 from a North Sea tidal flat and is closely related to V. sp. DW001 from Antarctic sea ice. Our phylogenomic and bioinformatic analyses placed VB16, UBA2437 and DW001 into a cold-tolerant subclade within the albus clade, along with two non-cold-tolerant subclades. Orthovenn analysis indicated that VB16 and its other albus clade members shared 1544 gene orthologue clusters, including clusters for biosynthesis of polar flagella and tight adhesion pili that predict multiple lifestyles, either free-living or as an opportunistic pathogen within a marine eukaryotic host. The cold-tolerant subclade shared 552 orthologue proteins, including genes known to promote survival in cold or freezing temperatures, such as the eicosapentaenoic acid biosynthetic gene cluster, syp exopolysaccharide gene cluster and novel giant proteins with ice-binding domains. This subclade represents a group of psychrotolerant or ‘moderate psychrophile’ winter season Vibrio species. The discovery of this subclade opens the door for experimental work on the physiological features, virulence potential and ecological importance of this subclade.

Recombination events between Delta and Omicron SARS-CoV-2 lineages highlight the need for co-infection research. Existing studies focus on late-phase co-infections, with few examining earlier pandemic stages. This new study aims to globally identify and characterize co-infections using a bioinformatic pipeline to analyse genomic data from diverse locations and pandemic phases. Among 26988 high-quality SARS-CoV-2 isolates from 11 diverse project databases, we identified 141 potential co-infection cases (0.52%), surpassing previous prevalence estimates. These co-infections were observed throughout the pandemic timeline, with an increase noted after the emergence of the Omicron variant. Co-infections involving the Omicron variant were the most prevalent, potentially influenced by the high level of diversity within this lineage and its impact on the viral landscape. Additionally, we found co-infections involving the pre-Alpha/Alpha lineages, which have been rarely described, raising possibilities of contributing to new lineage emergence through recombination events. The analysis revealed co-infection cases involving both different and the same lineages/sublineages. Our study showcases the potential of our pipeline to leverage valuable information stored in global sequence repositories, advancing our understanding of SARS-CoV-2 co-infections. The prevalence of co-infections highlights the importance of monitoring viral diversity and its potential implications on disease dynamics. Integrating clinical data with genomic findings can further shed light on the clinical implications and outcomes of co-infections.