Genomic and Phenotypic Characterization of Shiga Toxin-Producing Escherichia albertii Strains Isolated from Wild Birds in a Major Agricultural Region in California

Abstract

Escherichia albertii is an emerging foodborne pathogen. To better understand the pathogenesis and health risk of this pathogen, comparative genomics and phenotypic characterization were applied to assess the pathogenicity potential of E. albertii strains isolated from wild birds in a major agricultural region in California. Shiga toxin genes stx_2f were present in all avian strains. Pangenome analyses of 20 complete genomes revealed a total of 11,249 genes, of which nearly 80% were accessory genes. Both core gene-based phylogenetic and accessory gene-based relatedness analyses consistently grouped the three stx_2f-positive clinical strains with the five avian strains carrying ST7971. Among the three Stx2f-converting prophage integration sites identified, ssrA was the most common one. Besides the locus of enterocyte effacement and type three secretion system, the high pathogenicity island, OI-122, and type six secretion systems were identified. Substantial strain variation in virulence gene repertoire, Shiga toxin production, and cytotoxicity were revealed. Six avian strains exhibited significantly higher cytotoxicity than that of stx_2f-positive E. coli, and three of them exhibited a comparable level of cytotoxicity with that of enterohemorrhagic E. coli outbreak strains, suggesting that wild birds could serve as a reservoir of E. albertii strains with great potential to cause severe diseases in humans.

Keywords: Escherichia albertii; Shiga toxin; cytolethal distending toxin; cytotoxicity; foodborne pathogen; intimin; pangenome; pathogenicity; phage integration; stx2f.

Figure 1

Pangenome of E. albertii strains. (A) Core and accessory genes of E. albertii. The numbers of genes in the core and accessory genomes were calculated using Roary (V3.13.0), as detailed in the Materials and Methods section. The core genes refer to all genes shared by all input genomes (n = 20); the soft-core genes refer to all genes present in all 19 input genomes; the shell genes refer to all genes present in at least 3 but less than 19 input genomes; the cloud genes refer to all genes present in less than 3 input genomes. (B) Change in the total number of new genes and unique genes as genomes are added in random orders. As the number of genomes used for the pangenome analyses increased, the total number of new genes (genes not found in the previously compared genomes) decreased, while the total number of unique genes (genes unique to individual strains) increased. (C) Core genome-based phylogeny of the E. albertii strains. The Roary matrix contained a total of 11,249 gene clusters. Strain names in red are clinical strains, and strain names in red bold are stx_2f-positive clinical isolates. (D) The accessory genome-based strain relatedness. The number in the parentheses represents the number of strain-specific genes. Strain names in red are clinical strains, and strain names in red bold are stx_2f-positive clinical strains.

Figure 2

Sequence analyses of LEE in E. albertii. (A) Phylogeny of E. albertii LEEs. The LEEs in the E. albertii strains were identified by BLASTn search of a database containing all genomes examined in this study using DNA sequence of EDL933 LEE as a query in Geneious Prime^®. The sequences of the LEEs were extracted from corresponding genomes and aligned using Clustal Omega alignment in Geneious Prime^®. A neighbor-joining consensus tree was constructed using the LEE in strain EDL933 as an outgroup with the following parameters: Genetic Distance Model, Jukes-Cantor; Resampling tree method: Bootstrap; Number of Replicates: 10,000; Support Threshold: 50%. The length of each LEE is indicated in parentheses. Subtypes of intimin gene eae were identified by BLASTn search of a database with 19 subtypes of eae. (B) Alignment of representative E. albertii LEEs. Representative E. albertii LEEs were aligned with the EDL933 LEE using Clustal Omega method. Green triangles represent the annotated CDSs, and red blocks represent LEE polycistronic operons. The identity of the consensus sequence is color-coded (green: 100% identity; brown: at least 30% and under 100% identity; and red: below 30% identity).

Figure 3

Sequence analyses of ETT2 in E. albertii. (A) Phylogeny of E. albertii ETT2s. The ETT2s in E. albertii strains were identified by BLASTn search of a database containing all genomes examined in this study using DNA sequence of EDL933 ETT2 as a query in Geneious Prime^®. The sequences of the ETT2s were extracted from corresponding genomes and aligned using Clustal Omega alignment, and a neighbor-joining consensus tree was constructed using EDL933 ETT2 as an outgroup with the following parameters: Genetic Distance Model, Jukes-Cantor; Resampling tree method: Bootstrap; Number of Replicates: 10,000; Support Threshold: 50%. (B) Alignment of representative E. albertii ETT2s. Six representative E. albertii ETT2s were aligned using Clustal Omega method. Green triangles represent the annotated CDSs, and red blocks represent genes with annotated functions. Grey triangles represent hypothetical genes. The triangles with an overlapping smaller triangle represent genes carrying mutations that result in a premature stop codon. The identity of the consensus sequence is color-coded (green: 100% identity; brown: at least 30% and under 100% identity; and red: below 30% identity).

Figure 4

Sequence analyses of OI-122 and HPI in E. albertii. (A) Pairwise comparison of OI-122 in strains EDL933 and 2010C-3449. The sequences were aligned using Clustal Omega in Geneious Prime^®. Green arrows represent genes, while red arrows represent virulence genes located on three modules (purple blocks). (B) Pairwise comparison of HPI in strains EDL933 and 2010C-3449. The sequences were aligned using Clustal Omega in Geneious Prime^®. The identity of the consensus sequence is color-coded (green: 100% identity; brown: at least 30% and under 100% identity; and red: below 30% identity).

Figure 5

T6SSs in E. albertii strains. Phylogenetic analyses of T6SS-2. Sequences of E. albertii T6SS gene clusters and the T6SS gene cluster from EHEC strain EDL933 were aligned using Clustal Omega in Geneious Prime^® software Version 2023.2.1. A consensus tree was constructed using Geneious Tree Builder with the following settings: Genetic Distance Model: Jukes-Cantor; Tree Build Method: Neighbor-Joining; Outgroup: EDL933 T6SS; Resampling Method: Bootstrap; Random Seed: 326,544; Number of Replicates: 10,000. The length in parentheses represents the length of DNA fragment carrying T6SS genes. Strain RM9974 was not included in the analyses due to absence of T6SS gene cluster.

Figure 6

Sequence analyses of Stx2f-converting prophages in E. albertii. (A) Relatedness and chromosomal locations of Stx2f prophages. The genome size of each prophage is indicated in parentheses. The chromosomal insertion sites of Stx2f prophage are indicated with the corresponding positions in the clinical strain 05-3106. The putative integration sites are indicated by the red arrows. Yellow blocks refer to tmRNA and tRNA genes. Blue blocks refer to annotated genes. (B) Sequence analyses of antitermination protein Q genes located upstream of stx_2f coding sequences. The coding sequences of antitermination protein Q were aligned using Clustal Omega. A consensus tree was generated using neighbor-joining method with Jukes-Cantor for genetic distance model and rooted with the Q protein encoded by the Stx2a-prophage in EHEC strain EDL933. The Q protein size is indicated in parentheses. An asterisk indicates a truncated Q protein.