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Recent application of whole genome sequencing in the investigation of foodborne illness outbreaks has facilitated the identification of Reoccurring, Emerging, or Persistent (REP) bacterial strains that have caused illnesses over extended periods of time. Here, the complete genomes of two Escherichia coli O157:H7 (EcO157) outbreak strains belonging to REPEXH01 and REPEXH02, respectively, were sequenced and annotated. Comparative genomics and phenotypic analyses were carried out to identify REP-associated traits. The REPEXH01 strain PNUSAE013245 belonged to clade 8 and shared >96% CDSs with the 2006 spinach-associated outbreak strain EC4115. A 79-Kb genomic island was identified only in PNUSAE013245, and encoded functions related to heavy metals and antibiotic resistances. The REPEXH02 strain PNUSAE043864 belonged to clade 2, like the 2006 Taco John's restaurants-associated outbreak strain TW14588 and the REPEXH02 strain 2019C-3201. These three strains differed mainly in plasmids and prophages repertoire. Unlike 2019C-3201, PNUSAE043864 carried only the virulence plasmid pO157; unlike TW14588, PNUSAE043864 carried one Stx2a-encoding prophage. Phenotypic assays revealed that all clade 2 strains produced greater amounts of biofilms than the clade 8 strains, although there were no significant differences in adhesion of EcO157 to romaine lettuce. The persistence of EcO157 was further evaluated by quantifying populations of culturable cells, persisters, and Viable But Not Culturable (VBNC) cells for strains inoculated in river water and incubated at 15°C for 14 weeks. The fractions of culturable cells were comparable among the strains tested until 10 weeks post inoculation, when the three clade 2 strains exhibited significantly greater survival than strain PNUSAE013245. The population of persisters for all strains except PNUSAE013245 increased when the incubation time increased and reached up to 24–30% of the total culturable cells. The population of VBNC increased for all strains during the incubation and reached up to 65–80% of the total population. Both persisters and VBNC cells represent the dormancy state of pathogen cells that have increased tolerance to antibiotics and sanitizers. Presence of high portions of persisters and VBNC cells in agricultural environments highlights the need to understand the physiology of bacterial pathogens in leafy greens production environments and the challenges in developing effective control strategies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a major cause of nosocomial infections, particularly in immunocompromised patients or in individuals with cystic fibrosis. The notable ability of P. aeruginosa to inhabit a broad range of environments, including humans, is in part due to its large and diverse genomic repertoire. The genomes of most strains contain a significant number of large and small genomic islands, including those carrying virulence determinants (pathogenicity islands). The pathogenicity island PAPI-1 of strain PAl4 is a cluster of 115 genes, and some have been shown to be responsible for virulence phenotypes in a number of infection models. We have previously demonstrated that PAPI-1 can be transferred to other P. aeruginosa strains following excision from the chromosome of the donor. Here we show that PAPI-1 is transferred into recipient P. aeruginosa by a conjugative mechanism, via a type IV pilus, encoded in PAPI-l by a 10-gene cluster which is closely related to the genes in the enterobacterial plasmid R64. We also demonstrate that the precursor of the major pilus subunit, PilS2, is processed by the chromosomally encoded prepillin peptidase PilD but not its paralog FppA. Our results suggest that the pathogenicity island PAPI-1 may have evolved by acquisition of a conjugation system but that because of its dependence on an essential chromosomal determinant, its transfer is restricted to P. aeruginosa or other species capable of providing a functional prepilin peptidase. doi: 10.1128/JB.00041-10

Recent application of whole genome sequencing in the investigation of foodborne illness outbreaks has facilitated the identification of Reoccurring, Emerging, or Persistent (REP) bacterial strains that have caused illnesses over extended periods of time. Here, the complete genomes of two Escherichia coli O157:H7 (EcO157) outbreak strains belonging to REPEXH01 and REPEXH02, respectively, were sequenced and annotated. Comparative genomics and phenotypic analyses were carried out to identify REP-associated traits. The REPEXH01 strain PNUSAE013245 belonged to clade 8 and shared >96% CDSs with the 2006 spinach-associated outbreak strain EC4115. A 79-Kb genomic island was identified only in PNUSAE013245, and encoded functions related to heavy metals and antibiotic resistances. The REPEXH02 strain PNUSAE043864 belonged to clade 2, like the 2006 Taco John's restaurants-associated outbreak strain TW14588 and the REPEXH02 strain 2019C-3201. These three strains differed mainly in plasmids and prophages repertoire. Unlike 2019C-3201, PNUSAE043864 carried only the virulence plasmid pO157; unlike TW14588, PNUSAE043864 carried one Stx2a-encoding prophage. Phenotypic assays revealed that all clade 2 strains produced greater amounts of biofilms than the clade 8 strains, although there were no significant differences in adhesion of EcO157 to romaine lettuce. The persistence of EcO157 was further evaluated by quantifying populations of culturable cells, persisters, and Viable But Not Culturable (VBNC) cells for strains inoculated in river water and incubated at 15°C for 14 weeks. The fractions of culturable cells were comparable among the strains tested until 10 weeks post inoculation, when the three clade 2 strains exhibited significantly greater survival than strain PNUSAE013245. The population of persisters for all strains except PNUSAE013245 increased when the incubation time increased and reached up to 24-30% of the total culturable cells. The population of VBNC increased for all strains during the incubation and reached up to 65-80% of the total population. Both persisters and VBNC cells represent the dormancy state of pathogen cells that have increased tolerance to antibiotics and sanitizers. Presence of high portions of persisters and VBNC cells in agricultural environments highlights the need to understand the physiology of bacterial pathogens in leafy greens production environments and the challenges in developing effective control strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Carter, Carychao and Bono.)