Correlation between the genomic o454-nlpD region polymorphisms, virulence gene equipment and phylogenetic group of extraintestinal Escherichia coli (ExPEC) enables pathotyping irrespective of host, disease and source of isolation

Escherichia coli is a normal inhabitant of the gastrointestinal microbiota of mammalians and birds, but at the same time it can cause a variety of diseases relevant for public and animal health such as diarrhoea, bacteraemia, septicaemia, urinary tract infections [1]. From a clinical perspective, E. coli is broadly classified into commensals, intestinal pathogenic E. coli (InPEC) and extraintestinal pathogenic E. coli (ExPEC), the latter group being further divided into uropathogenic E. coli (UPEC), septicaemia-associated E. coli (SEPEC), neonatal meningitis E. coli (NMEC), and avian pathogenic E. coli (APEC). ExPEC strains are normal colonizers of the gut of men and animals, but in contrast to intestinal pathogenic variants, they can cause infections to the urinary tract or the blood stream [2] once they reach the corresponding body site. Although collectively termed ExPEC, simply to reflect their shared ability to express functionally similar virulence factors and to denote considerable overlaps concerning serotypes and phylogenetic background [3],[4], this group of strains exhibits large genome diversity. This has been mainly attributed to the frequent location of virulence associated genes (VAGs) on plasmids, pathogenicity islands, or phages, allowing the VAGs to be highly interchangeable among strains through horizontal gene transfer (HGT) [5],[6]. The population structure of E. coli is characterised by the presence of distinct phylogenetic groups as observed by phylogenetic reconstruction [7],[8] or by the use of specific markers [9]. Based on these approaches, four (A, B1, B2 and D) major phylogroups have been described while according to the method, two minor (E and F) or two hybrid (AxB1 and ABD) phylogroups have been defined in addition, which are not necessarily equivalent [8]-[10]. The distribution (presence/absence) of virulence factors thought to be involved in the ability of a strain to cause diverse diseases also varies among strains of these phylogenetic groups, indicating a role of the genetic background in the expression of virulence [11]-[13].

The high diversity of ExPEC and the difficulty in a clear demarcation of these facultative pathogenic strains from their commensal counterpart poses a huge challenge to infectious medicine in terms of diagnostic and risk assessment. As recently shown, the genetic variability of the mutS-rpoS chromosomal region may serve as indicator and thus as a chromosomal marker for the different virulence potential of E. coli strains [2],[14]-[16]. The crucial genes are mutS, which encodes one of the four proteins required for methyl-directed mismatch repair (MMR) of DNA, and rpoS, which encodes a sigma factor (sigma 38) that regulates many stationary-phase and environmental stress response genes [17]. Although mutS and rpoS are generally conserved in Enterobacteriaceae, the mutS-rpoS intergenic and its adjacent region revealed extensive genetic variability that was subjected to genetic exchange during the evolution of pathogenic lineages. Several studies revealed a pathotype-associated polymorphism in this genetic region [15],[18],[19] suggesting it to be the region owing to HGT and evolutionary processes. In comparison to E. coli K-12, previous studies revealed that enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC) and E. coli group B2 strains harbour specific DNA insertions within the mutS-rpoS intergenic region [15],[16],[19]. An insertion of 2.1 kb, in place of the initially identified 2.9 kb insert at the proximity of E. coli O157:H7 [19] has been found in strains of uropathogenic E. coli[15] and larger intergenic regions exist in strains of EPEC and EHEC [16]. Moreover, phylogenetic analysis of EHEC and EPEC strains, as well as strains of the Ecor collection, revealed that the mutS gene itself may be frequently subject to horizontal transfer and recombination during the evolution of these strains which is consistent with mechanism for stabilizing adaptive changes promoted by mutS mutators with relaxed recombination barriers [14],[20].

In the present study we attempted to assess the importance of the mutS-rpoS intergenic region as a surrogate marker for the rapid identification of highly virulent ExPEC strains and to delineate biological meaningful subgroups among this highly diverse group of strains. In particular, we aimed: (i) to characterize the genetic diversity of the mutS gene and of the o454-nlpD genomic region among 510 E. coli strains obtained from animal and human sources; (ii) to delineate associations between the polymorphism of this region and features such as phylogenetic background of E. coli, bacterial class or pathotype, host species, clinical condition, serogroup and virulence associated genes (VAG)s; and (iii) to identify the most important ExPEC-related VAGs for classification of the o454-nlpD genomic region by using the random forest (RF) algorithm [21]. The results could be a valuable contribution to ongoing analyses on pathoadaptive alterations in ExPEC strains that affect disease severity and may have consequences for diagnostics of E. coli infections (15).

The mutS-nlpD region represents a major operational region of genomic evolution in Enterobacteriaceae. Previous studies suggested that the polymorphic nature of this genetic region due to high mutation rate and to loss or acquisition of genes by horizontal gene transfer plays an important role in the constant adaptation of the bacteria to environmental changes and to new ecological niches [2],[14]-[16]. So far, the mosaic structure of the mutS-nlpD region was mainly investigated in intestinal pathogenic strains. By that, RFLP type analysis of the fhlA-nlpD genomic region revealed four different clusters: i.e. K-12 and Ecor group A, EPEC 1 group, EPEC 2 & EHEC 2 groups, and O157:H7 & EHEC 1 group [16]. Soon after, Le Clerc et al. [19] detected a polymorphism at the proximity of the rpoS gene. In particular, a 2.9-kb DNA insertion was identified in E. coli O157:H7 and related enterohaemorrhagic E. coli (EHEC) strains as well as in Shigella dysenteriae[19], while a 2.1-kb DNA insert was observed in E. coli pyelonephritis strain CFT073 [15]. These authors investigated diverse clinical isolates, including 23 from urinary tract infections (UTIs), 26 from infantile diarrhoea and haemorrhagic colitis, and seven from catheter-associated infections. Since the DNA insert previously identified in UPEC strain CFT073 was more common among urinary tract infection isolates (82.6%) than among other clinical E. coli isolates they suggested it to be specifically linked with uropathogens [15]. They also stated, that all B2 strains of the E. coli reference collection harboured this specific DNA insert und concluded that it might also predict the virulence of a strain, as B2 strains are known to be highly virulent in terms of extraintestinal pathogenicity.

From these studies it seemed likely that there might be pathotype- or virulence-associated polymorphisms in the mutS-rpoS region of the E. coli chromosome, although this has not been verified by using a large set of field strains. In the present study, PCR analyses of the o454-nlpD intergenic sequence from 510 predominantly extraintestinal pathogenic and commensal E. coli strains revealed substantial size variation. With only few exceptions, the strains were grouped into four patterns which resembled previously identified groups determined for the O157:H7 & EHEC 1 group (termed as pattern I in our study), K-12 and Ecor A group (pattern II), CFT073 and other uropathogens (pattern III), and the EPEC group (pattern IV) [15]. While patterns I, II, and IV were randomly distributed among all non-B2 group strains, pattern III was exclusively observed in group B2 strains, both of clinical and faecal origin. This is consistent with the genomic variation in selected Ecor strains described by others [15],[19],[23],[24]. With respect to the ongoing evolution and due to observations from previous MLST analyses [8] it is generally recognized that the Ecor collection does not reflect the entire E. coli population. Previous findings about the presence of the rpoS-proximal 2.1-kb insertion in all Ecor group B2 isolates and in a limited number of clinical isolates could thus not be extrapolated to nowadays relevant ExPEC-sequence types, such as ST95, ST127, ST372, and the global emerging multiresistant ST131 clone [25]-[29]. In our strain collection, these sequence types as well as other clinically important B2-STs, including ST73 and ST80 [28],[30] were frequently represented suggesting the existence of a B2-associated mutS-nlpD intergenic region.

Since pattern III was found to be highly associated to the strain category ExPEC and with a higher number of ExPEC-related VAGs we could speculate a potential role of these genes with the ability of the strain to invade extraintestinal tissues. However, replacement of slyA with other genes such as o347 and o183 in pattern III and their roles in pathogenesis remain to be investigated. SlyA, found exclusively in the EHEC- and EPEC-related patterns I and IV plays an important role in the invasion and survival of Salmonella in macrophage cells [31]. The relevance of this transcriptional regulator for the pathogenesis of non-B2 ExPEC strains has not been explored yet. Similarly, the putative role of the o347-encoded factor, showing a limited level of similarity to enzymes implicated in antibiotic hydrolysis, remains to be determined [15],[32]. Girardeau et al. [33] investigated common traits between animal and human ExPEC isolates positive for afimbrial adhesin gene afa-8. Interestingly, they found a significant proportion of human pyelonephritis-associated isolates showing the mutS-rpoS intergenic region found in EPEC and EHEC isolates. Furthermore, a putative function of SlyA in the pathogenesis of certain extraintestinal E. coli isolates which largely lack ExPEC-associated traits, such as S-fimbrial gene sfa, alpha-hemolysin gene hly, cytotoxic necrotizing factor gene cnf, was suggested. In accordance with our data, Girardeau et al. [33] and others observed mutS-rpoS intergenic patterns among ExPEC strains, which were previously associated with intestinal pathotypes [15],[33]. Thus, with respect to the group of ExPEC and its various pathotypes this genetic region may not be regarded as pathotype-associated but more likely as a marker to reflect their diversity and probably to predict highly virulent members of this group, as different patterns are obviously linked with single ExPEC-related virulence genes.

Advance statistics like Random Forest (RF) algorithm was used to resolve difficult tasks such as the identification of the most important VAGs for the prediction of the o454-nlpD patterns. The RF classification of the o454-nlpD patterns estimated an OOB (out-of-bag) error rate of 18.4% when using 46 virulence-associated genes (VAGs). This relatively high error rate clearly indicated that not all VAGs used were strongly associated with a specific o454-nlpD pattern as shown in Table 3 where only pattern III is associated to specific genes. Other genes where associated to at least two different patterns. However, since the error rate in predicting the pattern III was zero (Table 5 and Additional file 4) we can conclude that RF performed very well for pattern III prediction. Furthermore, RF allowed us to identify for the first time the top-ranked VAGs for pattern III prediction. The most predictive indicators, the genes csgA, malX, chuA, vat and sitD_chromosomal were also statistically significant predictors, and thus worthy of further investigation. Since pattern III was nearly exclusively linked with group B2 strains in our study, these genes may also be regarded as predictive for highly virulent members of the ExPEC group and of commensal E. coli harbouring virulence potential, respectively. Indeed, the heme binding protein encoding gene chuA is one of the genetic regions targeted in the PCR-based approach for rapid phylogenetic typing of E. coli strains and is said to occur regularly in group B2 and D strains [9],[34]. ChuA, together with vat, which encodes an autotransporter serine protease toxin, fyuA, which encodes the yersiniabactin receptor and finally a gene (yfcV), which encodes the major subunit of a putative chaperone-usher fimbria, have previously been included in a diagnostic multiplex PCR to identify strains of the UPEC pathotype [35]. In their study, Spurbeck et al. [35] suggested that E. coli isolates that encode these four genes are correlated with high numbers of other VAGs, are able to colonize the bladder in higher numbers than strains lacking these genes, and are nearly 10 times more likely to represent UPEC or NMEC strains than faecal commensal strains [35]. Likewise positively linked with pattern III strains is malX, which codes for a phosphotransferase system enzyme II that recognizes maltose and glucose [36] and is frequently present in ExPEC strains [37]-[39]. Östblom et al. [40] could demonstrate, that malX was among those genes that were associated with fitness of E. coli in the infant bowel microbiota. Here, carriage of various pathogenicity island markers and particularly malX correlated positively with the time of persistence of individual strains in the colon, supporting their role to increase the fitness of E. coli in its natural niche, the colon [38].

The mutS chromosomal region has long been identified as the location for the insertion of blocks of VAGs e.g. in the case of the two widely diverged pathogens, Salmonella Typhimurium and Haemophilus influenzae. In S. Typhimurium, a 40 kb pathogenicity island (SPI-1) is inserted 5? to the mutS gene [40] and in H. influenzae, a 3.1 kb tryptophanase gene cluster (tna) is inserted on the 3? side of the mutS gene in strains that cause spinal meningitis in infants [41]. This insertion allows the utilization of tryptophan and, thus, provides a growth advantage for the pathogen, particularly in the tryptophan-rich environment of cerebrospinal fluid. In our study, there was no indication for an insertion of PAI-like structures or of larger blocks of VAGs in the intergenic mutS-rpoS region in any of the 510 E. coli strains under investigation. Other studies raised the hypothesis that a certain o454-nlpD pattern, reflecting distinct evolutionary E. coli lineages, might be linked with the acquisition of VAGs at chromosomal sites outside this genetic region by HGT [2],[15],[42]. Culham and Wood [15] suggested that the 2.1-kb insertion upstream of rpoS arrived earlier than certain virulence determinants linked with urinary tract infections, such as genes for P-fimbriae (pap), S-fimbriae (sfa), a polyketide synthetase (pks), and ?-hemolysin (hly) during the evolution of group B2. Basically, the polymorphism in this genetic region is considered to result from the close linkage of mutS and rpoS genes which are frequently mutated in E. coli evolution due to ecological specialization upon repeated shuttles between different environments [2]. Here, their inactivation as well as the re-acquisition of functional alleles might have been of selective advantage, e.g. in terms of stress resistance, higher mutation rates, genome plasticity, and stabilization of beneficial adaptive mutations [5],[43].

Indeed, in addition to the findings of genetic variability, phylogenetic analysis of EHEC and EPEC pathogens, as well as strains of the Ecor collection, revealed that an unexpected level of recombination between mutS genes has occurred during the evolution of these strains [14],[20]. In a comparison of mutS phylogeny against predicted E. coli ‘whole-chromosome’ phylogenies, derived from multilocus enzyme electrophoresis (MLEE) and mdh sequences, Brown et al. [14] observed striking levels of phylogenetic discordance among mutS alleles and their host strains, which basically represented the Ecor collection. To investigate whether this is also true for a greater collection of strains and using concatenated MLST gene sequences instead of single genes or MLEE as comparison, we extended this approach on 177 ExPEC and commensal strains. Here, many of the mutS alleles clustered according to the population structure given from the housekeeping genes phylogeny, indicating a low frequency of recombination events across phylogenetic groups. However, for a number of strains we also found incongruence between these two phylogenies, which is likely due to recombination of mutS between different phylogenetic groups. By investigating the molecular phylogeny of MMR (methyl-directed mismatch repair) genes from natural E. coli isolates Denamur et al. [20] could show that, compared to two housekeeping genes, individual functional MMR genes exhibit high sequence mosaicism derived from diverse phylogenetic lineages. They suggested that the MMR functions have frequently been lost and reacquired in the evolution of E. coli. To which extent mutS and other genes of the MMR system as well as the mutS-nlpD intergenic region represent a hallmark of a mechanism of adaptive evolution in ExPEC and in other E. coli pathotypes has been scarcely investigated. Certainly, mutS has a unique role in the formation of mutators with relaxed recombination barriers, and bacteria with a defect in their MMR system, e.g. by a temporary loss of mutS, are more prone to genetic variations and HGT and, consequently, have an increased capacity to adapt to the host environment or acquire new VAGs, respectively [2],[44]. This phenomenon was recently described for UPEC strains, where mutS and other genes of the MMR system were found to be involved in the reciprocal control of motility and adherence of UPEC due to an increased expression of flagellin [44]. The authors discussed a possible relationship between MutS and UPEC pathogenesis in general as urinary tract isolates exhibit a higher occurrence of mutator strains than commensal E. coli or any other E. coli pathotype [42].

The mutS-rpoS intergenic region of ExPEC and commensal E. coli strains resembles a great phylogenetic diversity of these strains as exemplified by the presence of four different patterns. The grouping based on o454-nlpD amplicons and RFLP patterns of the entire mutS-rpoS region was in accordance with other genotype-based methods, like MLST and Ecor typing. Significant associations were determined between pattern III and phylogenetic group B2 strains, representing the most virulent members of the ExPEC group. MutS alleles revealed an evolution parallel to their phylogenetic background with few exceptions. The random forest algorithm allowed for the first time the identification of the most important virulence genes for prediction of the most virulent ExPEC strains.

Our results suggest the mutS-rpoS region to be of great value in identifying highly extraintestinal virulent strains among the mixed population of E. coli promising to be the basis of a typing tool for ExPEC and their reservoir.

CE designed the experiments. CE, ID, HD, AF, and NN performed the experiments and contributed to acquisition and interpretation of data. CE and FD structured and prepared the manuscript. FD, TS and CE performed biostatistics. LHW and TS revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.