A Single Nucleotide Polymorphism in lptG Increases Tolerance to Bile Salts, Acid, and Staining of Calcofluor-Binding Polysaccharides in Salmonella enterica Serovar Typhimurium E40.

The outer membrane of Salmonella enterica plays an important role in combating stress encountered in the environment and hosts. The transport and insertion of lipopolysaccharides (LPS) into the outer membrane involves lipopolysaccharide transport proteins (LptA-F) and mutations in the genes encoding for these proteins are often lethal or result in the transport of atypical LPS that can alter stress tolerance in bacteria. During studies of heterogeneity in bile salts tolerance, S. enterica serovar Typhimurium E40 was segregated into bile salts tolerant and sensitive cells by screening for growth in TSB with 10% bile salts. An isolate (E40V) with a bile salts MIC >20% was selected for further characterization. Whole-genome sequencing of E40 and E40V using Illumina and PacBio SMRT technologies revealed a non-synonymous single nucleotide polymorphism (SNP) in lptG. Leucine at residue 26 in E40 was substituted with proline in E40V. In addition to growth in the presence of 10% bile salts, E40V was susceptible to novobiocin while E40 was not. Transcriptional analysis of E40 and E40V, in the absence of bile salts, revealed significantly greater (p < 0.05) levels of transcript in three genes in E40V; yjbE (encoding for an extracellular polymeric substance production protein), yciE (encoding for a putative stress response protein), and an uncharacterized gene annotated as an acid shock protein precursor (ASPP). No transcripts of genes were present at a greater level in E40 compared to E40V. Corresponding with the greater level of these transcripts, E40V had greater survival at pH 3.35 and staining of Calcofluor-binding polysaccharide (CBPS). To confirm the SNP in lptG was associated with these phenotypes, strain E40E was engineered from E40 to encode for the variant form of LptG (L26P). E40E exhibited the same differences in gene transcripts and phenotypes as E40V, including susceptibility to novobiocin, confirming the SNP was responsible for these differences.

Chronological set of E. coli O157:H7 bovine strains establishes a role for repeat sequences and mobile genetic elements in genome diversification.

BACKGROUND: Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a significant foodborne pathogen that resides asymptomatically within cattle and other ruminants. The EHEC genome harbors an extensive collection of mobile genetic elements (MGE), including multiple prophage, prophage-like elements, plasmids, and insertion sequence (IS) elements. RESULTS: A chronological collection of EHEC strains (FRIK804, FRIK1275, and FRIK1625) isolated from a Wisconsin dairy farm (farm X) comprised a closely related clade genetically differentiated by structural alterations to the chromosome. Comparison of the FRIK804 genome with a reference EHEC strain Sakai found a unique prophage like element (PLE, indel 1) and an inversion (1.15 Mb) situated symmetrically with respect to the terminus region. Detailed analysis determined the inversion was due to homologous recombination between repeat sequences in prophage. The three farm X strains were distinguished by the presence or absence of indel 3 (61 kbp) and indel 4 (48 kbp); FRIK804 contained both of these regions, FRIK1275 lacked indel 4, and indels 3 and 4 were both absent in FRIK1625. Indel 3 was the stx2 prophage and indel 4 involved a deletion between two adjacent prophage with shared repeat sequences. Both FRIK804 and FRIK1275 produced functional phage while FRIK1625 did not, which is consistent with indel 3. Due to their involvement in recombination events, direct and inverted repeat sequences were identified, and their locations mapped to the chromosome. FRIK804 had a greater number and overall length of repeat sequences than E. coli K12 strain MG1655. Repeat sequences were most commonly associated with MGE. CONCLUSIONS: This research demonstrated that three EHEC strains from a Wisconsin dairy farm were closely related and distinguished by variability within prophage regions and other MGE. Chromosome alterations were associated with recombination events between repeat sequences. An inventory of direct and inverted repeat sequences found a greater abundance and total length of repeat sequences in the EHEC strains compared to E. coli strain MG1655. The locations of the repeat sequences were biased towards MGE. The findings from this study expand our understanding of the precise molecular events and elements that contributed to genetic diversification of wild-type EHEC in the bovine and farm environments.

Genome Sequences of 14 Escherichia coli O157:H7 Strains Isolated before and during the Time Frame of the 2018 Multistate Outbreak Associated with Romaine Lettuce.

Several outbreaks of Escherichia coli O157:H7 associated with contaminated leafy green vegetables have been documented. Here, we report the draft genome sequences of 14 strains isolated from human patients in the state of Wisconsin during a multistate outbreak in early 2018 that was linked to consumption of romaine lettuce.

Phylogenetic characterization of Escherichia coli O157 : H7 based on IS629 distribution and Shiga toxin genotype.

Shiga toxin (stx)-producing Escherichia coli O157 : H7 is a prominent food-borne pathogen. Symptoms in human infections range from asymptomatic to haemorrhagic colitis and haemolytic uraemic syndrome, and there is a need for methods that yield information that can be used to better predict clinical and epidemiological outcomes. IS629 is an insertion sequence notable for its prevalence and variable distribution in the chromosome of E. coli O157 : H7, which has been exploited for subtyping and strain characterization. In particular, IS629 distribution is closely aligned with the major phylogenetic lineages that are known to be distinctive in their genome structure and virulence potential. In the present study, a comprehensive subtyping method in which IS629-typing was combined with stx genotyping was developed using a conventional PCR approach. This method consisted of a set of 32 markers based on the unique distribution of IS629 in the three major phylogenetic lineages of E. coli O157 : H7 and six additional markers to determine the stx genotype, a key virulence signature associated with each lineage. The analysis of IS629 loci variation with the 32 markers allowed us to determine the IS629 distribution profile (IDP), phylogenetic lineage and genetic relatedness of the 31 E. coli O157 : H7 strains examined. An association between IDP typing and stx genotype was observed. The use of both IDP and the stx genotype for strain characterization provided confirmative and complementary data in support of lineage placement of closely related strains. In addition, IS629/stx profiles were in agreement with strain segregation based on LSPA-6 (lineage-specific polymorphism assay) and PFGE subtyping, demonstrating its potential as a subtyping and strain tracking method.

Evolution of the Stx2-encoding prophage in persistent bovine Escherichia coli O157:H7 strains.

Escherichia coli O157:H7 is a human pathogen that resides asymptomatically in its bovine host. The level of Shiga toxin (Stx) produced is variable in bovine-derived strains in contrast to human isolates that mostly produce high levels of Stx. To understand the genetic basis for varied Stx production, chronological collections of bovine isolates from Wisconsin dairy farms, R and X, were analyzed for multilocus prophage polymorphisms, stx(2) subtypes, and the levels of stx(2) transcript and toxin. The E. coli O157:H7 that persisted on both farms were phylogenetically distinct and yet produced little to no Stx2 due to gene deletions in Stx2c-encoding prophage (farm R) or insertional inactivation of stx(2a) by IS1203v (farm X). Loss of key regulatory and lysis genes in Stx2c-encoding prophage abolished stx(2c) transcription and induction of the prophage and stx(2a)::IS1203v in Stx2a-encoding prophage generated a truncated stx(2a) mRNA without affecting phage production. Stx2-producing strains were transiently present (farm R) and became Stx2 negative on farm X (i.e., stx(2a)::IS1203v). To our knowledge, this is the first study that details the evolution of E. coli O157:H7 and its Stx2-encoding prophage in a chronological collection of natural isolates. The data suggest the bovine and farm environments can be niches where Stx2-negative E. coli O157:H7 emerge and persist, which explains the Stx variability in bovine isolates and may be part of an evolutionary step toward becoming bovine specialists.