The plasmid of Escherichia coli strain S88 (O45:K1:H7) that causes neonatal meningitis is closely related to avian pathogenic E. coli plasmids and is associated with high-level bacteremia in a neonatal rat meningitis model

Abstract

A new Escherichia coli virulent clonal group, O45:K1, belonging to the highly virulent subgroup B2(1) was recently identified in France, where it accounts for one-third of E. coli neonatal meningitis cases. Here we describe the sequence, epidemiology and function of the large plasmid harbored by strain S88, which is representative of the O45:K1 clonal group. Plasmid pS88 is 133,853 bp long and contains 144 protein-coding genes. It harbors three different iron uptake systems (aerobactin, salmochelin, and the sitABCD genes) and other putative virulence genes (iss, etsABC, ompT(P), and hlyF). The pS88 sequence is composed of several gene blocks homologous to avian pathogenic E. coli plasmids pAPEC-O2-ColV and pAPEC-O1-ColBM. PCR amplification of 11 open reading frames scattered throughout the plasmid was used to investigate the distribution of pS88 and showed that a pS88-like plasmid is present in other meningitis clonal groups such as O18:K1, O1:K1, and O83:K1. A pS88-like plasmid was also found in avian pathogenic strains and human urosepsis strains belonging to subgroup B2(1). A variant of S88 cured of its plasmid displayed a marked loss of virulence relative to the wild-type strain in a neonatal rat model, with bacteremia more than 2 log CFU/ml lower. The salmochelin siderophore, a known meningovirulence factor, could not alone explain the plasmid's contribution to virulence, as a salmochelin mutant displayed only a minor fall in bacteremia (0.9 log CFU/ml). Thus, pS88 is a major virulence determinant related to avian pathogenic plasmids that has spread not only through meningitis clonal groups but also human urosepsis and avian pathogenic strains.

FIG. 1.

Circular representation of the Escherichia coli strain S88 plasmid (pS88). Circles display (from the outside) (i) GC percent deviation (GC window − mean GC) in a 1,000-bp window, (ii) predicted ORFs transcribed in the clockwise direction, (iii) predicted ORFs transcribed in the counterclockwise direction, (iv) GC skew (G + C/G − C) in a 1,000-bp window, (v) transposable elements (pink), and (vi) coordinates in kilobase pairs (kbp) from the origin of replication. Genes displayed in circles ii and iii are categorized by color as follows: red, iron uptake systems; orange, other putative virulence factors; yellow, bacteriocin production and immunity; pink, mobile genetic elements; dark blue, plasmid transfer; green, plasmid replication; teal, plasmid maintenance; gray, unknown.

FIG. 2.

Phylogenetic trees generated by the neighbor-joining method for traJ, traM, traS, traT, and traY sequences of several reference E. coli conjugative plasmids. Numbers at the branches are bootstrap proportions (displayed if >75) obtained from 100 replicates. Sequences were extracted from GenBank, except for plasmid pS88: pAPEC-O2-ColV (strain APEC O2; accession number AY545598), pAPEC-O1-ColBM (strain APEC O1; accession number DQ381420), plasmid F (accession number AP001918), p1658-97 (accession number AF550679), p53638 (accession number CP001064), pMAR7 (accession number DQ388534), pRK100 (accession number AY230886), pSMS-3-5 (accession number CP000971), pUTI89 (accession number CP000244), pVM01 (accession number EU330199), and plasmid R1 (accession number X13681) and its derepressed mutant pR1-19 (accession number M19710). Plasmid pS88 and the two APEC plasmids pAPEC-O2-ColV and pAPEC-O1-ColBM are highlighted in bold.

FIG. 3.

Comparison of plasmid pS88 (133,853 bp) with plasmids pAPEC-O1-ColBM (174,241 bp) and pAPEC-O2-ColV (184,501 bp) using the line-plot representation of homologous regions. For convenience, the traM start codon was chosen as the beginning of the three sequences. Strand conservations are indicated in blue and strand inversions in red. Genes displayed are categorized using the color scheme described in Fig. 1.Variable and conserved virulence regions are defined as previously described by Johnson et al. (35).

FIG. 4.

(A) PFGE of undigested DNAs from seven representative strains harboring the 11 ORFs characteristics of pS88. Lane 1, S145; lane 2, Ben1068; lane 3, S4; lane 4, S133; lane 5, S94; lane 6, HN30; lane 7, ECOR62; lane M, molecular size marker (50-kb DNA ladder). (B) Hybridization with iroN-specific probe on a Southern blot.