Escherichia albertii is an emerging foodborne pathogen. We previously reported that some avian Shiga toxin-producing E. albertii strains exhibited higher or comparable cytotoxicity in Vero-d2EGFP cells with several enterohemorrhagic E. coli (EHEC) outbreak strains. To better understand the environmental persistence of this pathogen, comparative genomics and phenotypic assays were applied to assess adhesion capability, motility, and biofilm formation in E. albertii. Among the 108 adherence-related genes, those involved in biogenesis of curli fimbriae, hemorrhagic E. coli pilus, type 1 fimbriae, and Sfm fimbriae were conserved in E. albertii. All 20 E. albertii strains carried a complete set of primary flagellar genes that were organized into four gene clusters, while five strains possessed genes related to the secondary flagella, also known as lateral flagella. Compared to EHEC strain EDL933, the eight chemotaxis genes located within the primary flagellar gene clusters were deleted in E. albertii. Additional deletion of motility genes flhABCD and motBC was identified in several E. albertii strains. Swimming motility was detected in three strains when grown in LB medium, however, when grown in 5% TSB or in the pond water-supplemented with 10% pigeon droppings, an additional four strains became motile. Although all E. albertii strains carried curli genes, curli fimbriae were detected only in four, eight, and nine strains following 24, 48, and 120 h incubation, respectively. Type 1 fimbriae were undetectable in any of the strains grown at 37°C or 28°C. Strong biofilms were detected in strains that produced curli fimbriae and in a chicken isolate that was curli fimbriae negative but carried genes encoding adhesive fimbriae K88, a signature of enterotoxigenic E. coli strains causing neonatal diarrhea in piglets. In all phenotypic traits examined, no correlation was revealed between the strains isolated from different sources, or between the strains with and without Shiga toxin genes. The phenotypic variations could not be explained solely by the genetic diversity or the difference in adherence genes repertoire, implying complex regulation in expression of various adhesins. Strains that exhibited a high level of cytotoxicity and were also proficient in biofilm production, may have potential to emerge into high-risk pathogens.

Shiga toxin-producing Escherichia coli (STEC) O145 is a major serotype associated with severe human disease. Production of Shiga toxins (Stxs), especially Stx2a, is thought to be correlated with STEC virulence. Since stx genes are located in prophages genomes, induction of prophages is required for effective Stxs production. Here, we investigated the production of Stxs in 12 environmental STEC O145:H28 strains under stresses STEC encounter in natural habitats and performed comparative analysis with two O145:H28 clinical strains, one linked to a 2010 U.S. lettuce-associated outbreak (RM13514) and the other linked to a 2007 Belgium ice cream-associated outbreak (RM13516). Similar to the outbreak strains, all environmental strains belong to Sequence Type (ST)-78 using the EcMLST typing scheme. Although all Stx1a-prophages were grouped together, variations in Stx1a production were observed prior to or following the inductions. Among all stx(2a) positive environmental strains, only the Stx2a-prophage in cattle isolate RM9154-C1 was clustered with the Stx2a-prophages in RM13514, the Stx2a-phage induced from a STEC O104:H4 strain linked to the 2011 outbreak of enterohemorrhagic infection in Germany, and the Stx2a-prophage in STEC O157:H7 strain EDL933, a prototype of enterohemorrhagic E. coli. Furthermore, the Stx2a-prophage in RM9154-C1 shared the same chromosomal insertion site and carried the same antiterminator Q gene and the late promoter P-R' as the Stx2a-prophage in RM13514. Following mitomycin C or enrofloxacin treatment, the production of Stx2a in RM9154-C1 was the highest among all environmental strains tested. In contrast, following acid challenge and recovery, the production of Stx2a in RM9154-C1 was the lowest among all the environmental strains tested, at a level comparable to the clinical strains. A significant increase in Stx2a production was detected in all strains when exposed to H2O2, although the induction fold was much lower than those by other inducers. This low-efficiency induction of Stx-prophages by H2O2, a natural inducer of Stx-prophages, supports the hypothesis of bacterial altruism in controlling Stxs production, a strategy that assures the survival of the STEC population as a whole by sacrificing a small fraction of cells for Stxs production and release. Differential induction of Stxs among strains carrying nearly identical Stx-prophages suggests a role of host bacteria in regulating Stxs production. Our study revealed diverse Stx-prophages in STEC O145:H28 strains that were genotypically indistinguishable. Identification of a cattle isolate harboring a Stx2a-prophage associated with high virulence supports the premise that cattle, a natural reservoir of STEC, serve as a source of hypervirulent STEC strains.

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.

We previously reported a distinct methylome between the two Shiga toxin-producing Escherichia coli (STEC) O145:H28 strains linked to the 2010 U.S. lettuce-associated outbreak (RM13514) and the 2007 Belgium ice cream-associated outbreak (RM13516), respectively. This difference was thought to be attributed to a prophage encoded type II restriction-modification system (PstI R-M) in RM13514. Here, we characterized this PstI R-M system in comparison to DNA adenine methylase (Dam), a highly conserved enzyme in γ proteobacteria, by functional genomics. Deficiency in Dam led to a differential expression of over 1000 genes in RM13514, whereas deficiency in PstI R-M only impacted a few genes transcriptionally. Dam regulated genes involved in diverse functions, whereas PstI R-M regulated genes mostly encoding transporters and adhesins. Dam regulated a large number of genes located on prophages, pathogenicity islands, and plasmids, including Shiga toxin genes, type III secretion system (TTSS) genes, and enterohemolysin genes. Production of Stx2 in dam mutant was significantly higher than in RM13514, supporting a role of Dam in maintaining lysogeny of Stx2-prophage. However, following mitomycin C treatment, Stx2 in RM13514 was significantly higher than that of dam or PstI R-M deletion mutant, implying that both Dam and PstI R-M contributed to maximum Stx2 production.

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 (stx_1a), RM10410 (stx_1a + stx_2d), and RM14516 (stx_2a). 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.