Pathogenomics for accurate diagnosis, treatment, prognosis of oncology: a cutting edge overview.

The capability to gather heterogeneous data, alongside the increasing power of artificial intelligence to examine it, leading a revolution in harnessing multimodal data in the life sciences. However, most approaches are limited to unimodal data, leaving integrated approaches across modalities relatively underdeveloped in computational pathology. Pathogenomics, as an invasive method to integrate advanced molecular diagnostics from genomic data, morphological information from histopathological imaging, and codified clinical data enable the discovery of new multimodal cancer biomarkers to propel the field of precision oncology in the coming decade. In this perspective, we offer our opinions on synthesizing complementary modalities of data with emerging multimodal artificial intelligence methods in pathogenomics. It includes correlation between the pathological and genomic profile of cancer, fusion of histology, and genomics profile of cancer. We also present challenges, opportunities, and avenues for future work.

Pathogenomics of Helicobacter pylori.

The human stomach bacterium Helicobacter pylori, the causative agent of gastritis, ulcers and adenocarcinoma, possesses very high genetic diversity. H. pylori has been associated with anatomically modern humans since their origins over 100,000 years ago and has co-evolved with its human host ever since. Predominantly intrafamilial and local transmission, along with genetic isolation, genetic drift, and selection have facilitated the development of distinct bacterial populations that are characteristic for large geographical areas. H. pylori utilizes a large arsenal of virulence and colonization factors to mediate the interaction with its host. Those include various adhesins, the vacuolating cytotoxin VacA, urease, serine protease HtrA, the cytotoxin-associated genes pathogenicity island (cagPAI)-encoded type-IV secretion system and its effector protein CagA, all of which contribute to disease development. While many pathogenicity-related factors are present in all strains, some belong to the auxiliary genome and are associated with specific phylogeographic populations. H. pylori is naturally competent for DNA uptake and recombination, and its genome evolution is driven by extraordinarily high recombination and mutation rates that are by far exceeding those in other bacteria. Comparative genome analyses revealed that adaptation of H. pylori to individual hosts is associated with strong selection for particular protein variants that facilitate immune evasion, especially in surface-exposed and in secreted virulence factors. Recent studies identified single-nucleotide polymorphisms (SNPs) in H. pylori that are associated with the development of severe gastric disease, including gastric cancer. Here, we review the current knowledge about the pathogenomics of H. pylori.

Uncovering the history of recombination and population structure in western Canadian stripe rust populations through mating type alleles.

BACKGROUND: The population structure of crop pathogens such as Puccinia striiformis f. sp. tritici (Pst), the cause of wheat stripe rust, is of interest to researchers looking to understand these pathogens on a molecular level as well as those with an applied focus such as disease epidemiology. Cereal rusts can reproduce sexually or asexually, and the emergence of novel lineages has the potential to cause serious epidemics such as the one caused by the 'Warrior' lineage in Europe. In a global context, Pst lineages in Canada were not well-characterized and the origin of foreign incursions was not known. Additionally, while some Pst mating type genes have been identified in published genomes, there has been no rigorous assessment of mating type diversity and distribution across the species. RESULTS: We used a whole-genome/transcriptome sequencing approach for the Canadian Pst population to identify lineages in their global context and evidence tracing foreign incursions. More importantly: for the first time ever, we identified nine alleles of the homeodomain mating type locus in the worldwide Pst population and show that previously identified lineages exhibit a single pair of these alleles. Consistently with the literature, we find only two pheromone receptor mating type alleles. We show that the recent population shift from the 'PstS1' lineage to the 'PstS1-related' lineage is also associated with the introduction of a novel mating type allele (Pst-b3-HD) to the Canadian population. We also show evidence for high levels of mating type diversity in samples associated with the Himalayan center of diversity for Pst, including a single Canadian race previously identified as 'PstPr' (probable recombinant) which we identify as a foreign incursion, most closely related to isolates sampled from China circa 2015. CONCLUSIONS: These data describe a recent shift in the population of Canadian Pst field isolates and characterize homeodomain-locus mating type alleles in the global Pst population which can now be utilized in testing several research questions and hypotheses around sexuality and hybridization in rust fungi.

Robinsoniella peoriensis: an emerging pathogen with few virulence factors.

Robinsoniella peoriensis is a Gram-positive bacterium which is anaerobic, spore-forming, and non-motile. It was initially isolated and characterized from feces and swine manure. Strains of this species have since been identified from different mammalian and non-mammalian gastrointestinal tracts. Strains have also been isolated from a variety of human infections, such as bacteremia, bone infections, and skin structures. R. peoriensis has recently been reported as causative for pyometra, which could result in death in the absence of sufficient antimicrobial treatment. However, to the author's knowledge, there has not been a single virulence factor identified. A major challenge of modern medicine is the failure of conventional procedures to characterize the capability of an emerging pathogen to cause disease. The goal of this study is to initially characterize the pathogenicity of this bacterium using a pathogenomics approach.

Comparative Pathogenomics of Escherichia coli: Polyvalent Vaccine Target Identification through Virulome Analysis.

Comparative genomics of bacterial pathogens has been useful for revealing potential virulence factors. Escherichia coli is a significant cause of human morbidity and mortality worldwide but can also exist as a commensal in the human gastrointestinal tract. With many sequenced genomes, it has served as a model organism for comparative genomic studies to understand the link between genetic content and potential for virulence. To date, however, no comprehensive analysis of its complete "virulome" has been performed for the purpose of identifying universal or pathotype-specific targets for vaccine development. Here, we describe the construction of a pathotype database of 107 well-characterized completely sequenced pathogenic and nonpathogenic E. coli strains, which we annotated for major virulence factors (VFs). The data are cross referenced for patterns against pathotype, phylogroup, and sequence type, and the results were verified against all 1,348 complete E. coli chromosomes in the NCBI RefSeq database. Our results demonstrate that phylogroup drives many of the "pathotype-associated" VFs, and ExPEC-associated VFs are found predominantly within the B2/D/F/G phylogenetic clade, suggesting that these phylogroups are better adapted to infect human hosts. Finally, we used this information to propose polyvalent vaccine targets with specificity toward extraintestinal strains, targeting key invasive strategies, including immune evasion (group 2 capsule), iron acquisition (FyuA, IutA, and Sit), adherence (SinH, Afa, Pap, Sfa, and Iha), and toxins (Usp, Sat, Vat, Cdt, Cnf1, and HlyA). While many of these targets have been proposed before, this work is the first to examine their pathotype and phylogroup distribution and how they may be targeted together to prevent disease.

The Role of Gram-Negative Bacteria in Urinary Tract Infections: Current Concepts and Therapeutic Options.

Urinary tract infections (UTIs) are some of the most common infections in human medicine worldwide, recognized as an important public health concern to healthcare systems around the globe. In addition, urine specimens are one of the most frequently submitted samples for culture to the clinical microbiology laboratory, exceeding the number of most of the other sample types. The epidemiology, species-distribution and susceptibility-patterns of uropathogens vary greatly in a geographical and time-dependent manner and it also strongly correlated with the reported patient population studied. Nevertheless, many studies highlight the fact that the etiological agents in UTIs have changed considerably, both in nosocomial and community settings, with a shift towards "less common" microorganisms having more pronounced roles. There is increasing demand for further research to advance diagnostics and treatment options, and to improve care of the patients. The aim of this review paper was to summarize current developments in the global burden of UTI, the diagnostic aspects of these infectious pathologies, the possible etiological agents and their virulence determinants (with a special focus on the members of the Enterobacterales order), current guidelines and quality indicators in the therapy of UTIs and the emergence of multidrug resistance in urinary pathogens.

Pathogenomics of Emerging Campylobacter Species.

Campylobacter is among the four main causes of gastroenteritis worldwide and has increased in both developed and developing countries over the last 10 years. The vast majority of reported Campylobacter infections are caused by Campylobacter jejuni and, to a lesser extent, C. coli; however, the increasing recognition of other emerging Campylobacter pathogens is urgently demanding a better understanding of how these underestimated species cause disease, transmit, and evolve. In parallel to the enhanced clinical awareness of campylobacteriosis due to improved diagnostic protocols, the application of high-throughput sequencing has increased the number of whole-genome sequences available to dozens of strains of many emerging campylobacters. This has allowed for comprehensive comparative pathogenomic analyses for several species, such as C. fetus and C. concisus These studies have started to reveal the evolutionary forces shaping their genomes and have brought to light many genomic features related to pathogenicity in these neglected species, promoting the development of new tools and approaches relevant for clinical microbiology. Despite the need for additional characterization of genomic diversity in emerging campylobacters, the increasing body of literature describing pathogenomic studies on these species deserves to be discussed from an integrative perspective. This review compiles the current knowledge and highlights future work toward deepening our understanding about genome dynamics and the mechanisms governing the evolution of pathogenicity in emerging Campylobacter species, which is urgently needed to develop strategies to prevent or control the spread of these pathogens.

Community perspectives on the benefits and risks of technologically enhanced communicable disease surveillance systems: a report on four community juries.

BACKGROUND: Outbreaks of infectious disease cause serious and costly health and social problems. Two new technologies - pathogen whole genome sequencing (WGS) and Big Data analytics - promise to improve our capacity to detect and control outbreaks earlier, saving lives and resources. However, routinely using these technologies to capture more detailed and specific personal information could be perceived as intrusive and a threat to privacy. METHOD: Four community juries were convened in two demographically different Sydney municipalities and two regional cities in New South Wales, Australia (western Sydney, Wollongong, Tamworth, eastern Sydney) to elicit the views of well-informed community members on the acceptability and legitimacy of: making pathogen WGS and linked administrative data available for public health researchusing this information in concert with data linkage and machine learning to enhance communicable disease surveillance systems Fifty participants of diverse backgrounds, mixed genders and ages were recruited by random-digit-dialling and topic-blinded social-media advertising. Each jury was presented with balanced factual evidence supporting different expert perspectives on the potential benefits and costs of technologically enhanced public health research and communicable disease surveillance and given the opportunity to question experts. RESULTS: Almost all jurors supported data linkage and WGS on routinely collected patient isolates for the purposes of public health research, provided standard de-identification practices were applied. However, allowing this information to be operationalised as a syndromic surveillance system was highly contentious with three juries voting in favour, and one against by narrow margins. For those in favour, support depended on several conditions related to system oversight and security being met. Those against were concerned about loss of privacy and did not trust Australian governments to run secure and effective systems. CONCLUSIONS: Participants across all four events strongly supported the introduction of data linkage and pathogenomics to public health research under current research governance structures. Combining pathogen WGS with event-based data surveillance systems, however, is likely to be controversial because of a lack of public trust, even when the potential public health benefits are clear. Any suggestion of private sector involvement or commercialisation of WGS or surveillance data was unanimously rejected.

Coagulase-Negative Staphylococci Pathogenomics.

Coagulase-negative Staphylococci (CoNS) are skin commensal bacteria. Besides their role in maintaining homeostasis, CoNS have emerged as major pathogens in nosocomial settings. Several studies have investigated the molecular basis for this emergence and identified multiple putative virulence factors with regards to Staphylococcus aureus pathogenicity. In the last decade, numerous CoNS whole-genome sequences have been released, leading to the identification of numerous putative virulence factors. Koch's postulates and the molecular rendition of these postulates, established by Stanley Falkow in 1988, do not explain the microbial pathogenicity of CoNS. However, whole-genome sequence data has shed new light on CoNS pathogenicity. In this review, we analyzed the contribution of genomics in defining CoNS virulence, focusing on the most frequent and pathogenic CoNS species: S. epidermidis, S. haemolyticus, S. saprophyticus, S. capitis, and S. lugdunensis.

Pathogenomics of Uterine Fibroids Development.

We review recent studies dealing with the molecular genetics and basic results of omics analysis of uterine leiomyoma (LM)-a common benign muscle tumor of the uterus. Whole genome studies of LM resulted in the discovery of many new gene nets and biological pathways, including its origin, transcriptomic, and epigenetic profiles, as well as the impact of the inter-cell matrix in LM growth and involvement of microRNA in its regulation. New data on somatic cell mutations ultimately involved in the origin, distribution and growth of LM are reviewed. Putative identification of LM progenitor SC (stem cells) giving rise to maternal fibroid nodes and junctional zones provide a new clue for hypotheses on the pathogenomics of LM. The reviewed data are consistent with at least two different but probably intimately interacted molecular mechanisms of LM. One of them (the genetic hypothesis) is focused primarily on the MED12 gene mutations and suggests its onset in the side population of embryonic myoblasts of the female reproductive system, which later gave rise to multiple small and medium fibroids. The single and usually large-size fibroids are induced by predominantly epigenetic disorders in LM SC, provoked by enhanced expression of the HMGA2 gene caused by its hypomethylation and epigenetic deregulation enhanced by hypoxia, muscle tension, or chromosome instability/aberrations. The pathogenomics of both genetic and epigenetic programs of LM with many peculiarities at the beginning later became rather similar and partly overlapped due to the proximity of their gene nets and epigenetic landscape. Pathogenomic studies of LM open ways for elaboration of novel strategies of prevention and treatment of this common disease.

Pathogenomics of Endometriosis Development.

For over 100 years, endometriosis, as a chronic, estrogen-dependent, inflammatory, heritable disease affecting approximately 5⁻10% of women in reproductive age has been the focus of clinicians and scientists. In spite of numerous environmental, genetic, epigenetic, endocrine, and immunological studies, our knowledge of endometriosis is still fragmentary, and its precise pathophysiology and pathogenomics remain a mystery. The implementation of new technologies has provided tremendous progress in understanding the many intrinsic molecular mechanisms in the development of endometriosis, with progenitor and stem cells (SCs) of the eutopic endometrium as the starting players and endometriotic lesions as the final pathomorphological trait. Novel data on the molecular, genetic, and epigenetic mechanisms of the disease are briefly outlined. We hypothesize the existence of an endometriosis development genetic program (EMDP) that governs the origin of endometrium stem cells programmed for endometriosis (1), their transition (metaplasia) into mesenchymal SCs (2), and their invasion of the peritoneum and progression to endometriotic lesions (3). The pros and cons of the recent unifying theory of endometriosis are also discussed. Complex genomic and epigenetic interactions at different stages of the endometriosis process result in different forms of the disease, with specific features and clinical manifestations. The significance of the EMDP in elaborating a new strategy for endometriosis prediction, prevention, and treatment is discussed.

Emergence of wheat blast in Bangladesh was caused by a South American lineage of Magnaporthe oryzae.

BACKGROUND: In February 2016, a new fungal disease was spotted in wheat fields across eight districts in Bangladesh. The epidemic spread to an estimated 15,000 hectares, about 16 % of the cultivated wheat area in Bangladesh, with yield losses reaching up to 100 %. Within weeks of the onset of the epidemic, we performed transcriptome sequencing of symptomatic leaf samples collected directly from Bangladeshi fields. RESULTS: Reinoculation of seedlings with strains isolated from infected wheat grains showed wheat blast symptoms on leaves of wheat but not rice. Our phylogenomic and population genomic analyses revealed that the wheat blast outbreak in Bangladesh was most likely caused by a wheat-infecting South American lineage of the blast fungus Magnaporthe oryzae. CONCLUSION: Our findings suggest that genomic surveillance can be rapidly applied to monitor plant disease outbreaks and provide valuable information regarding the identity and origin of the infectious agent.

Host specialization of the blast fungus Magnaporthe oryzae is associated with dynamic gain and loss of genes linked to transposable elements.

BACKGROUND: Magnaporthe oryzae (anamorph Pyricularia oryzae) is the causal agent of blast disease of Poaceae crops and their wild relatives. To understand the genetic mechanisms that drive host specialization of M. oryzae, we carried out whole genome resequencing of four M. oryzae isolates from rice (Oryza sativa), one from foxtail millet (Setaria italica), three from wild foxtail millet S. viridis, and one isolate each from finger millet (Eleusine coracana), wheat (Triticum aestivum) and oat (Avena sativa), in addition to an isolate of a sister species M. grisea, that infects the wild grass Digitaria sanguinalis. RESULTS: Whole genome sequence comparison confirmed that M. oryzae Oryza and Setaria isolates form a monophyletic and close to another monophyletic group consisting of isolates from Triticum and Avena. This supports previous phylogenetic analysis based on a small number of genes and molecular markers. When comparing the host specific subgroups, 1.2-3.5 % of genes showed presence/absence polymorphisms and 0-6.5 % showed an excess of non-synonymous substitutions. Most of these genes encoded proteins whose functional domains are present in multiple copies in each genome. Therefore, the deleterious effects of these mutations could potentially be compensated by functional redundancy. Unlike the accumulation of nonsynonymous nucleotide substitutions, gene loss appeared to be independent of divergence time. Interestingly, the loss and gain of genes in pathogens from the Oryza and Setaria infecting lineages occurred more frequently when compared to those infecting Triticum and Avena even though the genetic distance between Oryza and Setaria lineages was smaller than that between Triticum and Avena lineages. In addition, genes showing gain/loss and nucleotide polymorphisms are linked to transposable elements highlighting the relationship between genome position and gene evolution in this pathogen species. CONCLUSION: Our comparative genomics analyses of host-specific M. oryzae isolates revealed gain and loss of genes as a major evolutionary mechanism driving specialization to Oryza and Setaria. Transposable elements appear to facilitate gene evolution possibly by enhancing chromosomal rearrangements and other forms of genetic variation.

Overview of genomic and bioinformatic resources for Zymoseptoria tritici.

Zymoseptoria tritici (syn. Mycosphaerella graminicola, Septoria tritici) is a haploid fungus belonging to the class Dothideomycetes. It is the causal agent of septoria leaf blotch - one of the world's most significant diseases of wheat. Here we review the genomic and bioinformatic resources that have been generated for Z. tritici. These include the whole-genome reference assembly for isolate IPO323, genome resequencing of alternate isolates, mitochondrial genome sequences, transcriptome sequences and expression data, and annotations of gene structure and function. We also highlight important advances in our fundamental knowledge of genome evolution and its effects on adaptation and pathogenicity in Z. tritici that have been facilitated by these resources.

COMPARATIVE WHOLE METAGENOME ANALYSIS IN LESIONAL AND NON-LESIONAL SCALP AREAS OF PSORIASIS CAPITIS PATIENTS AND HEALTHY INDIVIDUALS.

Psoriasis is an immune-mediated inflammatory disorder, where the majority of the patients suffer from psoriasis capitis or scalp psoriasis. Current therapeutics remain ineffective to treat scalp lesions. Here, we present a whole-metagenome characterisation of the scalp microbiome in psoriasis capitis. We investigated how changes in the homeostatic cutaneous microbiome correlate with the condition and identified metagenomic biomarkers (taxonomic, functional, virulence factors, antimicrobial resistance genes) that could partly explain its emergence. Within this study, 83 top and back scalp samples from healthy individuals and 64 lesional and non-lesional scalp samples from untreated psoriasis capitis subjects were analysed. Using qPCR targeting the 16S and 18S rRNA genes, we found a significant decrease in microbial load within scalp regions affected by psoriasis compared to their non-lesional counterparts. Metagenomic analysis revealed that psoriatic lesions displayed significant lower Cutibacterium species (incl. C. modestum, C. namnetense, C. granulosum, C. porci), along with an elevation in Staphylococcus aureus. A heightened relative presence of efflux pump protein-encoding genes was detected, suggesting potential antimicrobial resistance mechanisms. These mechanisms are known to specifically target human antimicrobial peptides (incl. cathelicidin LL-37) which are frequently encountered within psoriasis lesions. These shifts in microbial community dynamics may contribute to psoriasis disease pathogenesis.

Multimodal artificial intelligence-based pathogenomics improves survival prediction in oral squamous cell carcinoma.

In this study, we aimed to develop a novel prognostic algorithm for oral squamous cell carcinoma (OSCC) using a combination of pathogenomics and AI-based techniques. We collected comprehensive clinical, genomic, and pathology data from a cohort of OSCC patients in the TCGA dataset and used machine learning and deep learning algorithms to identify relevant features that are predictive of survival outcomes. Our analyses included 406 OSCC patients. Initial analyses involved gene expression analyses, principal component analyses, gene enrichment analyses, and feature importance analyses. These insights were foundational for subsequent model development. Furthermore, we applied five machine learning/deep learning algorithms (Random Survival Forest, Gradient Boosting Survival Analysis, Cox PH, Fast Survival SVM, and DeepSurv) for survival prediction. Our initial analyses revealed relevant gene expression variations and biological pathways, laying the groundwork for robust feature selection in model building. The results showed that the multimodal model outperformed the unimodal models across all methods, with c-index values of 0.722 for RSF, 0.633 for GBSA, 0.625 for FastSVM, 0.633 for CoxPH, and 0.515 for DeepSurv. When considering only important features, the multimodal model continued to outperform the unimodal models, with c-index values of 0.834 for RSF, 0.747 for GBSA, 0.718 for FastSVM, 0.742 for CoxPH, and 0.635 for DeepSurv. Our results demonstrate the potential of pathogenomics and AI-based techniques in improving the accuracy of prognostic prediction in OSCC, which may ultimately aid in the development of personalized treatment strategies for patients with this devastating disease.

Comparative genomics, pangenomics, and phenomic studies of Pectobacterium betavasculorum strains isolated from sugar beet, potato, sunflower, and artichoke: insights into pathogenicity, virulence determinants, and adaptation to the host plant.

Introduction: Bacteria of genus Pectobacterium, encompassing economically significant pathogens affecting various plants, includes the species P. betavasculorum, initially associated with beetroot infection. However, its host range is much broader. It causes diseases of sunflower, potato, tomato, carrots, sweet potato, radish, squash, cucumber, and chrysanthemum. To explain this phenomenon, a comprehensive pathogenomic and phenomic characterisation of P. betavasculorum species was performed. Methods: Genomes of P. betavasculorum strains isolated from potato, sunflower, and artichoke were sequenced and compared with those from sugar beet isolates. Metabolic profiling and pathogenomic analyses were conducted to assess virulence determinants and adaptation potential. Pathogenicity assays were performed on potato tubers and chicory leaves to confirm in silico predictions of disease symptoms. Phenotypic assays were also conducted to assess the strains ability to synthesise homoserine lactones and siderophores. Results: The genome size ranged from 4.675 to 4.931 kbp, and GC % was between 51.0% and 51.2%. The pangenome of P. betavasculorum is open and comprises, on average, 4,220 gene families. Of these, 83% of genes are the core genome, and 2% of the entire pangenome are unique genes. Strains isolated from sugar beet have a smaller pangenome size and a higher number of unique genes than those from other plants. Interestingly, genomes of strains from artichoke and sunflower share 391 common CDS that are not present in the genomes of other strains from sugar beet or potato. Those strains have only one unique gene. All strains could use numerous sugars as building materials and energy sources and possessed a high repertoire of virulence determinants in the genomes. P. betavasculorum strains were able to cause disease symptoms on potato tubers and chicory leaves. They were also able to synthesise homoserine lactones and siderophores. Discussion: The findings underscore the adaptability of P. betavasculorum to diverse hosts and environments. Strains adapted to plants with high sugar content in tissues have a different composition of fatty acids in membranes and a different mechanism of replenishing nitrogen in case of deficiency of this compound than strains derived from other plant species. Extensive phenomics and genomic analyses performed in this study have shown that P. betavasculorum species is an agronomically relevant pathogen.

Clonal reproduction of Moniliophthora roreri and the emergence of unique lineages with distinct genomes during range expansion.

The basidiomycete Moniliophthora roreri causes frosty pod rot of cacao (Theobroma cacao) in the western hemisphere. Moniliophthora roreri is considered asexual and haploid throughout its hemibiotrophic life cycle. To understand the processes driving genome modification, using long-read sequencing technology, we sequenced and assembled 5 high-quality M. roreri genomes out of a collection of 99 isolates collected throughout the pathogen's range. We obtained chromosome-scale assemblies composed of 11 scaffolds. We used short-read technology to sequence the genomes of 22 similarly chosen isolates. Alignments among the 5 reference assemblies revealed inversions, translocations, and duplications between and within scaffolds. Isolates at the front of the pathogens' expanding range tend to share lineage-specific structural variants, as confirmed by short-read sequencing. We identified, for the first time, 3 new mating type A locus alleles (5 in total) and 1 new potential mating type B locus allele (3 in total). Currently, only 2 mating type combinations, A1B1 and A2B2, are known to exist outside of Colombia. A systematic survey of the M. roreri transcriptome across 2 isolates identified an expanded candidate effector pool and provided evidence that effector candidate genes unique to the Moniliophthoras are preferentially expressed during the biotrophic phase of disease. Notably, M. roreri isolates in Costa Rica carry a chromosome segment duplication that has doubled the associated gene complement and includes secreted proteins and candidate effectors. Clonal reproduction of the haploid M. roreri genome has allowed lineages with unique genome structures and compositions to dominate as it expands its range, displaying a significant founder effect.

Pathogenicity assessment of Shiga toxin-producing Escherichia coli strains isolated from wild birds in a major agricultural region in California.

Shiga toxin-producing Escherichia coli (STEC) consists of diverse strains differing in genetic make-up and virulence potential. To better understand the pathogenicity potential of STEC carried by the wildlife, three STEC and one E. coli strains isolated from wild birds near a major agricultural region in California were selected for comparative pathogenomic analyses. Three American crow (Corvus brachyrhynchos) strains, RM9088, RM9513, and RM10410, belonging to phylogroup A with serotypes O109:H48, O9:H30, and O113:H4, respectively, and a red-winged blackbird (Agelaius phoeniceus) strain RM14516 in phylogroup D with serotype O17:H18, were examined. Shiga toxin genes were identified in RM9088 (stx1a), RM10410 (stx1a + stx2d), and RM14516 (stx2a). Unlike STEC O157:H7 strain EDL933, none of the avian STEC strains harbored the pathogenicity islands OI-122, OI-57, and the locus of enterocyte effacement, therefore the type III secretion system biogenesis genes and related effector genes were absent in the three avian STEC genomes. Interestingly, all avian STEC strains exhibited greater (RM9088 and RM14516) or comparable (RM10410) cytotoxicity levels compared with EDL933. Comparative pathogenomic analyses revealed that RM9088 harbored numerous genes encoding toxins, toxins delivery systems, and adherence factors, including heat-labile enterotoxin, serine protease autotransporter toxin Pic, type VI secretion systems, protein adhesin Paa, fimbrial adhesin K88, and colonization factor antigen I. RM9088 also harbored a 36-Kb high pathogenicity island, which is related to iron acquisition and pathogenicity in Yersinia spp. Strain RM14516 carried an acid fitness island like the one in EDL933, containing a nine gene cluster involved in iron acquisition. Genes encoding extracellular serine protease EspP, subtilase cytotoxin, F1C fimbriae, and inverse autotransporter adhesin IatC were only detected in RM14516, and genes encoding serine protease autotransporter EspI and P fimbriae were only identified in RM10410. Although all curli genes were present in avian STEC strains, production of curli fimbriae was only detected for RM9088 and RM14516. Consistently, strong, moderate, and little biofilms were observed for RM9088, RM14516, and RM10410, respectively. Our study revealed novel combinations of virulence factors in two avian strains, which exhibited high level of cytotoxicity and strong biofilm formation. Comparative pathogenomics is powerful in assessing pathogenicity and health risk of STEC strains.