ShiF acts as an auxiliary factor of aerobactin secretion in meningitis Escherichia coli strain S88.

BACKGROUND: The neonatal meningitis E. coli (NMEC) strain S88 carries a ColV plasmid named pS88 which is involved in meningeal virulence. Transcriptional analysis of pS88 in human serum revealed a strong upregulation of an ORF of unknown function: shiF, which is adjacent to the operon encoding the siderophore aerobactin. The aim of this work is to investigate the role of shiF in aerobactin production in strain S88. RESULTS: Study of the prevalence of shiF and aerobactin operon in a collection of 100 extra-intestinal pathogenic E. coli strains (ExPEC) and 50 whole genome-sequenced E. coli strains revealed the colocalization of these two genes for 98% of the aerobactin positive strains. We used Datsenko and Wanner's method to delete shiF in two S88 mutants. A cross-feeding assay showed that these mutants were able to excrete aerobactin meaning that shiF is dispensable for aerobactin excretion. Our growth assays revealed that the shiF-deleted mutants grew significantly slower than the wild-type strain S88 in iron-depleted medium with a decrease of maximum growth rates of 23 and 28% (p < 0.05). Using Liquid Chromatography-Mass Spectrometry, we identified and quantified siderophores in the supernatants of S88 and its shiF deleted mutants after growth in iron-depleted medium and found that these mutants secreted significantly less aerobactin than S88 (- 52% and - 49%, p < 0.001). CONCLUSIONS: ShiF is physically and functionally linked to aerobactin. It provides an advantage to E. coli S88 under iron-limiting conditions by increasing aerobactin secretion and may thus act as an auxiliary virulence factor.

Enteric fever among children: 50 cases in a French tertiary care centre.

Background: Enteric fever in France is primarily travel-associated. Characteristics of paediatric cases are scarce and information from field studies in endemic countries might not be generalizable to non-endemic countries. Methods: In this retrospective study, we reviewed all cases of typhoid and paratyphoid fever treated in a French paediatric tertiary care centre from 1993 to 2015. Results: Fifty cases of enteric fever due to Salmonella enterica serovar Typhi (n = 44) and Paratyphi (n = 6) were identified. Sixty-one percent of the children had travelled to Africa and 34% to the Indian subcontinent. Among travel-associated cases, 85% were visiting friends and relatives (VFR). Ninety-six percent had high fever associated with gastrointestinal symptoms. Anaemia (66%), elevated C-reactive protein (80%), transaminitis (87%) and mild hyponatremia (50%) were the main biological findings. Blood cultures were positive in 90% of cases. Twelve strains (24%) were resistant at least to one antibiotic, and all of them had been isolated since 2003, increasing the resistance rate during this last period to 43% (12/28). Ceftriaxone was administered to 71 patients for a median duration of 6 days (interquartile range (IQR): 4-8). The median time to apyrexia after the onset of treatment was 4 days (IQR: 2-5 days). Complications occurred in nine children with five (10%) presenting neurologic disorders. All 50 patients recovered. Conclusion: In France, paediatric enteric fever is mainly a travel-associated disease and occurs in patients returning from a prolonged stay in an endemic area. Children VFR are at high risk and should be a priority target group for pre-travel preventive measures. The increase in antibiotic resistance reflects the situation in endemic countries and is a major concern.

The ssbL gene harbored by the ColV plasmid of an Escherichia coli neonatal meningitis strain is an auxiliary virulence factor boosting the production of siderophores through the shikimate pathway.

The ability to capture iron is a challenge for most bacteria. The neonatal meningitis Escherichia coli strain S88 possesses several iron uptake systems, notably including siderophores. Transcriptional analysis of the ColV plasmid pS88 has shown strong induction of a previously undescribed gene with low identity to three E. coli chromosomal genes encoding phospho-2-dehydro-3-deoxyheptonate aldolases involved in aromatic amino acid and catecholate/phenolate siderophore biosynthesis through the shikimate pathway. Here, we investigated the role of this gene, ssbLp (ssbL carried on the plasmid), in siderophore biosynthesis and, consequently, in S88 virulence. We constructed an S88 mutant designated S88 ΔssbLp, which exhibited reduced growth under low-iron conditions compared to the wild-type strain. Liquid chromatography-mass spectroscopy analysis of culture supernatants showed that the mutant secreted significantly smaller amounts of enterobactin, salmochelin SX, and yersiniabactin than the wild-type strain. The mutant was also less virulent in a neonatal rat sepsis model, with significantly lower bacteremia and mortality. Supplementation with chorismate, the final product of the shikimate pathway, restored the wild-type phenotype in vitro. In a collection of human extraintestinal E. coli isolates, we found that ssbL was present only in strains harboring the iro locus, encoding salmochelins, and was located either on the chromosome or on plasmids. Acquisition of the iro locus has been accompanied by acquisition of the auxiliary gene ssbL, which boosts the metabolic pathway essential for catecholate/phenolate siderophore biosynthesis and could represent potential therapeutic targets.

A conserved virulence plasmidic region contributes to the virulence of the multiresistant Escherichia coli meningitis strain S286 belonging to phylogenetic group C.

Recent isolation of the non-K1 Escherichia coli neonatal meningitis strain S286, belonging to phylogroup C, which is closely related to major group B1, and producing an extended-spectrum beta-lactamase, encouraged us to seek the genetic determinants responsible for its virulence. We show that S286 belongs to the sequence O type ST23O78 and harbors 4 large plasmids. The largest one, pS286colV (~120 kb), not related to resistance, contains genes characteristic of a Conserved Virulence Plasmidic (CVP) region initially identified in B2 extra-intestinal avian pathogenic E. coli (APEC) strains and in the B2 neonatal meningitis E. coli strain S88. The sequence of this CVP region has a strong homology (98%) with that of the recently sequenced plasmid pChi7122-1 of the O78 APEC strain Chi7122. A CVP plasmid-cured variant of S286 was less virulent than the wild type strain in a neonatal rat sepsis model with a significant lower level of bacteremia at 24 h (4.1 ± 1.41 versus 2.60 ± 0.16 log CFU/ml, p = 0.001) and mortality. However, the mortality in the model of adult mice was comparable between wild type and variant indicating that pS286colV is not sufficient by itself to fully explain the virulence of S286. Gene expression analysis of pS286colV in iron depleted environment was very close to that of pS88, suggesting that genes of CVP region may be expressed similarly in two very different genetic backgrounds (group C versus group B2). Screening a collection of 178 human A/B1 extraintestinal pathogenic E. coli (ExPEC) strains revealed that the CVP region is highly prevalent (23%) and MLST analysis indicated that these CVP positive strains belong to several clusters and mostly to phylogroup C. The virulence of S286 is explained in part by the presence of CVP region and this region has spread in different clusters of human A/B1 ExPEC, especially in group C.

Necrotizing pneumonia in children: report of 41 cases between 2006 and 2011 in a French tertiary care center.

Forty-one children hospitalized for necrotizing pneumonia were retrospectively analyzed. Necrotizing pneumonia represented 0.8% of community-acquired pneumonia and 6% of hospitalized community-acquired pneumonia. The chest radiograph revealed necrosis on admission in onethird of cases. Twenty-one cases (51%) were documented, including 13 Staphylococcus aureus, all Panton-Valentine leukocidin positive, 7 Streptococcus pneumoniae and 1 Fusobacterium nucleatum.

Transcriptional analysis of the Escherichia coli ColV-Ia plasmid pS88 during growth in human serum and urine.

BACKGROUND: The sequenced O45:K1:H7 Escherichia coli meningitis strain S88 harbors a large virulence plasmid. To identify possible genetic determinants of pS88 virulence, we examined the transcriptomes of 88 plasmidic ORFs corresponding to known and putative virulence genes, and 35 ORFs of unknown function. RESULTS: Quantification of plasmidic transcripts was obtained by quantitative real-time reverse transcription of extracted RNA, normalized on three housekeeping genes. The transcriptome of E. coli strain S88 grown in human serum and urine ex vivo were compared to that obtained during growth in Luria Bertani broth, with and without iron depletion. We also analyzed the transcriptome of a pS88-like plasmid recovered from a neonate with urinary tract infection. The transcriptome obtained after ex vivo growth in serum and urine was very similar to those obtained in iron-depleted LB broth. Genes encoding iron acquisition systems were strongly upregulated. ShiF and ORF 123, two ORFs encoding protein with hypothetical function and physically linked to aerobactin and salmochelin loci, respectively, were also highly expressed in iron-depleted conditions and may correspond to ancillary iron acquisition genes. Four ORFs were induced ex vivo, independently of the iron concentration. Other putative virulence genes such as iss, etsC, ompTp and hlyF were not upregulated in any of the conditions studied. Transcriptome analysis of the pS88-like plasmid recovered in vivo showed a similar pattern of induction but at much higher levels. CONCLUSION: We identify new pS88 genes potentially involved in the growth of E. coli meningitis strain S88 in human serum and urine.