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Phylogenetic relationships, virulence factors, alone and in specific combinations, and virulence in a rat meningitis model were examined among 132 isolates of Escherichia coli neonatal meningitis from France and North America. Isolates belonging to phylogenetic groups A (n=11), D (n=20), and B2 (n=99) had similar high prevalence rates of the siderophores aerobactin and yersiniabactin and the K1 capsule (>/=70%) yet induced different level of experimental bacteremia. Ectochromosomal DNA-like domains involved in blood-brain barrier passage (PAI III(536) [sfa/foc and iroN; 34%]; GimA [ibeA and ptnC; 38%]; PAI II(J96) [hly, cnf1, and hra; 10%]) were restricted to B2 isolates. Among group B2 isolates, representatives of the O45:K1 clonal group (n=30), which lacked these domains, were as able as the archetypal O18:K1 strain C5 to cause meningitis. Molecular epidemiology combined with experimental virulence assays demonstrate that known virulence factors are insufficient to fully explain the pathophysiology of ECNM and to allow for rational search for new virulence factors.

Diffusely adhering Escherichia coli strains harboring Afa/Dr adhesins (Afa/Dr DAEC) have been associated with diarrhea and urinary tract infections (UTIs). The present work is the first extensive molecular study of a Afa/Dr DAEC strain using the representational difference analysis technique. We have searched for DNA sequences present in strain C1845, recovered from a diarrheagenic child, but absent from a nonpathogenic K-12 strain. Strain C1845 harbors part of a pathogenicity island (PAI CFT073 ) and several iron transport systems found in other E. coli pathovars. We did not find genes encoding factors known to subvert host cell proteins, such as type III secretion system or effector proteins. Several C1845-specific sequences are homologous to putative virulence genes or show no homology with known sequences, and we have analyzed their distribution among Afa/Dr and non-Afa/Dr clinical isolates and among strains from the E. coli Reference Collection. Three C1845-specific sequences (MO30, S109, and S111) have a high prevalence (77 to 80%) among Afa/Dr strains and a low prevalence (12 to 23%) among non-Afa/Dr strains. In addition, our results indicate that strain IH11128, an Afa/Dr DAEC strain recovered from a patient with a UTI, is genetically closely related to strain C1845.

Specific virulence factors associated with the pathogenesis of Escherichia coli strains causing neonatal meningitis (ECNM), such as the K1 capsular polysaccharide, the S fimbriae, and the Ibe10 protein, have been previously identified. However, some other yet unidentified factors are likely to be involved in the pathogenesis of ECNM. To identify specialized unique DNA regions associated with ECNM virulence, we used the representational difference analysis technique. The genomes of two strains belonging to nonpathogenic phylogenetic group A of the ECOR reference collection were subtracted from E. coli strain C5, isolated from a case of neonatal meningitis. Strain C5 belongs to the phylogenetic group B2 as do the majority of ECNM. We have isolated and mapped 64 DNA fragments which are specific for strain C5 and not found in nonpathogenic strains. Of these clones, 44 were clustered in six distinct regions on the chromosome. The sfa and ibe10 genes were located in regions 2 and 6, respectively. A group of genes ( cnf1 , hra , hly , and prs ) known to be present in a pathogenicity island of the uropathogenic strain E. coli J96 colocalized with region 6. The occurrence of these DNA regions was tested in a set of meningitis-associated strains and in a control group composed of non-meningitis-associated strains belonging to the same B2 group. Regions 1, 3, and 4 were present in 91, 82, and 81%, respectively, of the meningitis strains and in 40, 13, and 47% of the control strains. Together, these data suggest that regions 1, 3, and 4 code for factors associated with the ability of E. coli to invade the meninges of neonates.

International audience; The distribution of the haemoglobin receptor gene (hmbR) was investigated in disease and carried Neisseria meningitidis isolates revealing that the gene occurred at a significantly higher frequency in disease isolates compared to those obtained from carriage. Where hmbR was absent, the locus was occupied by the cassettes exl2 or exl3, or with a "pseudo hmbR" gene designated exl4. The hmbR locus in published N. meningitidis genomes, as well as N. gonorrhoeae and N. lactamica ST-640, exhibited characteristics of a pathogenicity island. These data are consistent with a role for the hmbR gene in meningococcal disease.

Despite being the agent of life-threatening meningitis, Neisseria meningitidis is usually carried asymptomatically in the nasopharynx of humans and only occasionally causes disease. The genetic bases for virulence have not been entirely elucidated and the search for new virulence factors in this species is hampered by the lack of an animal model representative of the human disease. As an alternative strategy we employ a molecular epidemiological approach to establish a statistical association of a candidate virulence gene with disease in the human population. We examine the distribution of a previously-identified genetic element, a temperate bacteriophage, in 1288 meningococci isolated from cases of disease and asymptomatic carriage. The phage was over-represented in disease isolates from young adults indicating that it may contribute to invasive disease in this age group. Further statistical analysis indicated that between 20% and 45% of the pathogenic potential of the five most common disease-causing meningococcal groups was linked to the presence of the phage. In the absence of an animal model of human disease, this molecular epidemiological approach permitted the estimation of the influence of the candidate virulence factor. Such an approach is particularly valuable in the investigation of exclusively human diseases.

International audience; Cerebrospinal meningitis is a feared disease that can cause the death of a previously healthy individual within hours. Paradoxically, the causative agent, Neisseria meningitidis, is a common inhabitant of the human nasopharynx, and as such, may be considered a normal, commensal organism. Only in a small proportion of colonized people do the bacteria invade the bloodstream, from where they can cross the blood–brain barrier to cause meningitis. Furthermore, most meningococcal disease is caused by bacteria belonging to only a few of the phylogenetic groups among the large number that constitute the population structure of this genetically variable organism. However, the genetic basis for the differences in pathogenic potential remains elusive. By performing whole genome comparisons of a large collection of meningococcal isolates of defined pathogenic potential we brought to light a meningococcal prophage present in disease-causing bacteria. The phage, of the filamentous family, excises from the chromosome and is secreted from the bacteria via the type IV pilin secretin. Therefore, this element, by spreading among the population, may promote the development of new epidemic clones of N. meningitidis that are capable of breaking the normal commensal relationship with humans and causing invasive disease.

Neisseria meningitidis is a Gram-negative bacterium which is an important causative agent of septicaemia and meningitis. LuxS has been shown to be involved in the biosynthesis of a quorum sensing molecule, autoinducer-2 (AI-2), known to play a role in virulence in Escherichia coli, as well as other bacteria. Evidence that serogroup B of N. meningitidis produces AI-2, along with the observation that a luxS mutant of this strain had attenuated virulence in an infant rat model of bacteraemia, led to further investigation of the role of this quorum sensing molecule in N. meningitidis. In this study, it is demonstrated that AI-2 is not involved in regulating growth of meningococci, either in culture or in contact with epithelial cells. Furthermore, transcriptional profiling using DNA microarrays shows an absence of the concerted regulation seen in other bacteria. Taken together, these data suggest that in N. meningitidis, AI-2 may be a metabolic by-product and not a cell-to-cell signalling molecule.

Neisseria meningitidis colonizes the nasopharynx and, unlike commensal Neisseria species, is capable of entering the bloodstream, crossing the blood-brain barrier, and invading the meninges. The other pathogenic Neisseria species, Neisseria gonorrhoeae , generally causes an infection which is localized to the genitourinary tract. In order to investigate the genetic basis of this difference in disease profiles, we used a strategy of genomic comparison. We used DNA arrays to compare the genome of N. meningitidis with those of N. gonorrhoeae and Neisseria lactamica , a commensal of the nasopharynx. We thus identified sequences conserved among a representative set of virulent strains which are either specific to N. meningitidis or shared with N. gonorrhoeae but absent from N. lactamica . Though these bacteria express dramatically different pathogenicities, these meningococcal sequences were limited and, in contrast to what has been found in other pathogenic bacterial species, they are not organized in large chromosomal islands.

The pathogenic species Neisseria meningitidis and Neisseria gonorrhoeae cause dramatically different diseases despite strong relatedness at the genetic and biochemical levels. N. meningitidis can cross the blood-brain barrier to cause meningitis and has a propensity for toxic septicemia unlike N. gonorrhoeae . We previously used subtractive hybridization to identify DNA sequences which might encode functions specific to bacteremia and invasion of the meninges because they are specific to N. meningitidis and absent from N. gonorrhoeae . In this report we show that these sequences mark eight genetic islands that range in size from 1.8 to 40 kb and whose chromosomal location is constant. Five of these genetic islands were conserved within a representative set of strains and/or carried genes with homologies to known virulence factors in other species. These were deleted, and the mutants were tested for correlates of virulence in vitro and in vivo. This strategy identified one island, region 8, which is needed to induce bacteremia in an infant rat model of meningococcal infection. Region 8 encodes a putative siderophore receptor and a disulfide oxidoreductase. None of the deleted mutants was modified in its resistance to the bactericidal effect of serum. Neither were the mutant strains altered in their ability to interact with endothelial cells, suggesting that such interactions are not encoded by large genetic islands in N. meningitidis .

Neisseria meningitidis and Neisseria gonorrhoeae give rise to dramatically different diseases. Their interactions with the host, however, do share common characteristics: they are both human pathogens which do not survive in the environment and which colonize and invade mucosa at their port of entry. It is therefore likely that they have common properties that might not be found in nonpathogenic bacteria belonging to the same genetically related group, such as Neisseria lactamica . Their common properties may be determined by chromosomal regions found only in the pathogenic Neisseria species. To address this issue, we used a previously described technique (C. R. Tinsley and X. Nassif, Proc. Natl. Acad. Sci. USA 93:11109–11114, 1996) to identify sequences of DNA specific for pathogenic neisseriae and not found in N. lactamica . Sequences present in N. lactamica were physically subtracted from the N. meningitidis Z2491 sequence and also from the N. gonorrhoeae FA1090 sequence. The clones obtained from each subtraction were tested by Southern blotting for their reactivity with the three species, and only those which reacted with both N. meningitidis and N. gonorrhoeae (i.e., not specific to either one of the pathogens) were further investigated. In a first step, these clones were mapped onto the chromosomes of both N. meningitidis and N. gonorrhoeae . The majority of the clones were arranged in clusters extending up to 10 kb, suggesting the presence of chromosomal regions common to N. meningitidis and N. gonorrhoeae which distinguish these pathogens from the commensal N. lactamica . The sequences surrounding these clones were determined from the N. meningitidis genome-sequencing project. Several clones corresponded to previously described factors required for colonization and survival at the port of entry, such as immunoglobulin A protease and PilC. Others were homologous to virulence-associated proteins in other bacteria, demonstrating that the subtractive clones are capable of pinpointing chromosomal regions shared by N. meningitidis and N. gonorrhoeae which are involved in common aspects of the host interaction of both pathogens.