Genome sequencing of strains of the most prevalent clonal group of O1:K1:H7 Escherichia coli that causes neonatal meningitis in France

Escherichia coli is a commensal of the gastrointestinal tract of vertebrates, including humans [1]. It is also involved in intestinal and extraintestinal infections [2]. In particular, E. coli is the most frequent bacteria involved in preterm meningitis and the second most frequent in newborn meningitis, causing a high rate of mortality or sequelae [3, 4].

Sequence types (ST) are grouped into clonal complexes by their similarity to a central allelic profile (genotype). Most E. coli strains responsible for neonatal meningitis belong to Sequence Type complex 95 (STc95) in the Warwick Multi-Locus Sequence Typing (MLST) scheme [5] and are mainly of serotypes O18:K1:H7, O1:K1, O7:K1, O83:K1, and the more recently reported O45_S88:K1:H7 [6–8]. Complete sequencing of strain S88, representative of clone O45_S88:K1:H7, has highlighted the presence of a 134 kb-plasmid, called pS88, encoding three iron capture systems (aerobactin, salmochelin, and SitACBD) and other putative extraintestinal virulence genes. The important role of this plasmid in the experimental pathogenicity of S88 has been demonstrated [8]. Plasmids similar to pS88 (pS88-like plasmid) have also been detected in other clonal groups responsible for meningitis, such as O18:K1, O1:K1 and O83:K1 [8].

Between 2001 and 2013, a French prospective national survey collected data for 325 children hospitalized with E. coli meningitis [5] and 141 E. coli isolates were sent to the national reference center (NRC) and studied. The highly pathogenic phylogenetic subgroup STc95 represented 56% of the 141 strains analyzed and among them, serogroup O1 (27.7%) was the most frequently identified during this period relative to serogroups O18 (19.1%) and O45_S88 (11.3%).

Whole genome sequencing (WGS) of a large collection of strains belonging to STc95 was recently performed [9], but no O1:K1 neonatal meningitis strain belonging to this complex has been comprehensively characterized. We performed WGS of all the 38 O1:K1 STc95 strains isolated from 1998 to 2015 to gain insight into the pathophysiology of neonatal meningitis and their disease potential. Moreover, we analyzed the virulence of representative strains using the Dictyostelium discoideum amoeba model as previously used to evaluate E. coli extraintestinal pathogenic strains [10].

The clonal complex STc95 is one of the major E. coli lineages that causes human extraintestinal infections. The extraintestinal virulence of STc95 E. coli is exemplified by their ability to cause neonatal meningitis. In France, this group represents 56% of the neonatal meningitis strains collected by the national reference center [5]. WGS performed on several hundred STc95 strains from various parts of the world has provided a comprehensive view of the genetic organization of this clonal complex, as well as its geographical distribution and temporal dynamics [9]. However, among the 500 strains investigated in this study, only two were isolated from the CSF of neonates (O18:K1 and O45_S88:K1). During the last 18 years, there has been a change in the relative frequency of major serotypes in STc95 NMEC, with a significant increase of O1:K1:H7 strains that were characterized by WGS.

Among the three major subgroups (A, C, D) that contain O1:H7 strains, described by Gordon et al. [9], strains responsible for meningitis, collected throughout France, belonged exclusively to subgroups A and D. The absence of O1:H7 subgroup C strains in our collection may be related to their lower potential to cause disease relative to other subgroups or their specific geographical distribution, as they were only found in Australia in the world-wide collection [9]. O1:K1:H7 meningitis strains of our study were more frequently found in subgroup D than in subgroup A (n = 34 versus n = 4). These two subgroups were present in all continents studied (USA, Europe, and Australia) with a similar repartition (n = 8 and n = 12 for subgroup A and D, respectively [9]. Subgroup D which predominates among our collection may have a greater potential to cause neonatal meningitis than subgroup A.

Phylogenetic analysis allowed us to distinguish three clusters within subgroup D, called D-1 (n = 28), D-2 (n = 4), and D-3 (n = 2). The closely related clusters D-2 and D-3 are minor relative to cluster D-1 and carry a different ST (ST421) and a different FimH (92) or no FimH gene (D-3). Cluster D-1, which carries FimH30, may represent a group of strains with a high capacity to induce neonatal meningitis. It also carries genetic determinants characteristic of the extraintestinal virulence plasmid pS88, which are absent from clusters D-2 and D-3, and rarely present in subgroup A. This plasmid may be key to the virulence of this group, as shown for the recently described clone O45_S88:K1:H7 [8]. The representative strain of clone O45_S88:K1:H7, S88, which carries the pS88 plasmid, appears to be closely related to D-2 and D-3 strains. Thus, it is possible that clone O45_S88:K1:H7 was derived from D-2/D-3 strains, after switching its O antigen gene cluster and acquiring the pS88 plasmid.

Analysis of the clinical features of infected neonates did not provide evidence that cluster D-1 is more virulent than D-2/D-3, despite the large number of strains of cluster D1 and the presence of plasmid pS88. However, this cluster appeared to elicit a larger inflammatory response.

Several factors known to be involved in the pathophysiology of neonatal meningitis were completely absent from our collection, i.e. ibeA, cnf1, and sfa. This highlights the variation in the virulence factor repertoire that leads to acute bacteremia and crossing of the blood brain barrier, the two major steps of this infection. Several studies have attempted to define a potential NMEC pathotype [12, 13]. For example, Wijetunge et al. compared 26 genes encoding virulence factors between 53 NMEC strains and 48 fecal strains of healthy individuals and found that the combination of K1 capsule, aerobactin siderophore, vacuolating cytotoxin (Vat), and the iron-binding protein (Sit) are typical traits of NMEC [13]. Among toxins, only vacuolating cytotoxin was present in almost all O1:K1 NMEC strains, irrespective of genetic subgroup, reinforcing the potential role of this toxin in the physiopathology of neonatal meningitis.

We assessed the experimental virulence of O1:K1:H7 and representative and control strains in the amoeba D. discoideum model, previously used to assess E. coli resistance to phagocytosis [10]. Our aim was to analyze possible fine differences between meningitis-causing clones and not to simulate the global pathophysiology of meningitis. This model avoids the use of animals and is of interest because it is performed at a low temperature (22 °C). At this temperature, the K1 capsule, the major virulence factor of NMEC, which may mask other bacterial traits involved in virulence, is inactivated [14]. Its inactivation may facilitate analysis of the potential role of other factors. Indeed, we assessed the production of the capsule of our O1:K1 strains by the agglutination test after culture at 22 °C and 35 °C and found that the K1 capsule was undetectable at 22 °C, but present at 35 °C (data not shown). Generally, the O1:K1 strains appeared to be less virulent than representative strains of O45_S88:H7 and O18:H7 serotypes and the control virulent strains. However, we found that they were not equally virulent upon analysis of each subclone. Among the D-1 strains, closely related subclones, with an identical repertoire of virulence genes behaved differently in the D. discoideum model. This highlights the complexity of the regulation of virulence and the involvement of various factors that are currently not known. Moreover, since most virulence factors implicated in human pathogeny are more efficient at 37 °C, it is also likely that the amoeba model underestimates their role. Nevertheless, we were able to show that the pS88-like plasmid plays a role in the resistance against phagocytosis using isogenic strains, thus complementing its previously described role in survival to bactericidal activity of serum [8].

Another notable difference between the O1:K1 clusters is the adhesin FimH. It binds specifically to D-mannose residues attached to the surface of glycoproteins that line vaginal, perineal, and bladder cells, as well as enterocytes [15]. FimH is expressed by more than 95% of E. coli and genetic variation can change its tropism [16]. It also plays an important role in the adhesion and invasion of endothelial cells of brain capillaries by NMEC in humans [17]. However it may be dispensable, since we found two meningitis-causing strains, S229 and S245, with no fimH gene (this was confirmed by fimH-specific PCR, data not shown).

The strains of NMEC O1:K1:H7 described here, carry mostly the fimH30 allele, whereas the other main serotypes responsible for meningitis carry the FimH54 (O45:K1:H7) and FimH18 (O18:K1:H7) alleles. Of note, most strains of the multi-resistant epidemic clonal group of E. coli ST131 O25b:H4, found throughout the world, carry FimH30 [18]. A recent study has shown that this clone display a greater adherence to CaCo2 enterocytes compared to other ESBL-producing E. coli isolates, although the specific role of FimH30 was not assessed [19]. It is possible that FimH30 allele confer an advantage to strains of the D-1 cluster for gut colonization, thus aiding their expansion.

Antimicrobial resistance was limited to the production of penicillinase (encoded by blaTEM-1 except for one strain), resistance to streptomycin, tetracyclin and sulphonamid and about one third of strains were devoid of any acquired resistance mechanism. We found no association between resistance and a particular WGS-subgroup or with the presence of pS88 virulence factors, suggesting that virulence and resistance genes are harbored by different plasmids or genomic islands such as Integrative and Conjugative Elements (ICE).

This work describes the recent dominance of the meningitis-causing E. coli O1:K1:H7, belonging to the highly virulent STc95, in France. We used WGS to describe its genetic diversity and found the predominance of a major cluster (D-1), characterized by the presence of the pS88-like plasmid. Clinical data and an ex vivo model failed to show higher pathogenicity of strains belonging to this major cluster D-1. Whether its high prevalence results from a high capacity to colonize the gut, due to adhesion through the FimH30 allele and/or the presence of the pS88-like plasmid, are yet to be determined.