Molecular Epidemiology of Extraintestinal Pathogenic <i>Escherichia coli</i>

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Molecular Epidemiology of Extraintestinal Pathogenic Escherichia coli

doi: 10.1128/ecosal.8.6.1.4

JAMES R. JOHNSON^1* AND THOMAS A. RUSSO²

[SECTION EDITOR: JAMES KAPER]

Posted November 15, 2004

¹Mucosal and Vaccine Research Center, VA Medical Center, Minneapolis, MN 55417, and Department of Medicine, University of Minnesota, Minneapolis, MN 55455, and ²VA Medical Center, Department of Medicine, Department of Microbiology, and The Witebsky Center for Microbial Pathogenesis and Immunology, University of Buffalo, Buffalo, NY 14214

^*Corresponding author. Phone: (612) 467-4185, Fax: (612) 727-5995, E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

INTRODUCTION

Extraintestinal pathogenic Escherichia coli (ExPEC), the specialized E. coli strains that possess the ability to overcome or subvert host defenses and cause extraintestinal disease, are important pathogens in humans and certain animals. Appreciation of these strains as being distinct from other E. coli (including intestinal pathogenic and commensal variants), and insights into the ecology, evolution, reservoirs, transmission pathways, host-pathogen interactions, and pathogenetic mechanisms of ExPEC, have resulted from molecular epidemiological analysis, which provides an essential complement to experimental assessment of virulence mechanisms. This chapter first reviews the basic conceptual and methodological underpinnings of the molecular epidemiological approach, then summarizes the main aspects of ExPEC that have been investigated using this approach.

METHODOLOGICAL CONSIDERATIONS

Basic Principles of Molecular Epidemiology

Traditional versus Molecular Epidemiology.

Epidemiology is the study of disease as it occurs in populations. In epidemiological studies, structured observations are used to identify host characteristics (that is, risk factors) that predict the occurrence, severity, or clinical manifestations of a particular illness (4, 123, 176, 195, 210, 225). By analogy, molecular epidemiology is the study of an infectious disease in relation to selected genetic characteristics of the causative microorganisms (7, 28, 43, 127, 135, 223). In molecular epidemiological studies, structured observations are used to identify microbial traits (for example, specific genes, phylogenetic background, or clonal identity) that predict the occurrence, severity, or clinical manifestations of a particular infectious disease, or relevant characteristics of the affected hosts, including age, gender, and underlying predisposing conditions. Molecular epidemiological studies seek insights into the molecular basis for the virulence behavior and host predilections of the pathogen and to identify relevant reservoirs and transmission pathways. Such insights can be useful in developing strategies for managing and preventing infections caused by the particular pathogen.

Strengths and Limitations of Molecular Epidemiology.

By virtue of being observational rather than experimental, molecular epidemiological studies exhibit the strengths and limitations inherent to observational studies in general. Their main strength is that they examine "real world" phenomena, that is, wild-type microbes interacting with the natural host in a natural setting, rather than the artificially engineered host-pathogen interactions of experimental studies (63), which can be of uncertain physiological relevance. In addition, molecular epidemiological studies can examine multiple predictor and outcome variables simultaneously, which can be challenging with experimental studies.

The main weakness of molecular epidemiological studies is that they allow the investigator no direct control over the variables analyzed. Consequently, a variable can be isolated only through careful selection of comparison groups so that the groups differ, to the extent possible, only according to the particular variable. Moreover, even with the most carefully selected comparison groups, associations that may emerge still are only that—associations. Although these may reflect causal relationships between the analyzed variables, they also may be due to confounding from other, unmeasured variables (in either the host or the pathogen). Consequently, the better characterized the source subjects and bacteria are, the greater the confidence with which associations between host variables and bacterial traits can be attributed to the particular variables themselves. Additionally, because of the considerable variation within human and bacterial populations, large numbers of subjects are needed per group (relative to the number of replicate determinations needed to address variance in experimental studies), appropriate statistical tests are required to assess the significance of any observed differences between groups, and statistically significant findings require confirmation in different populations.

Finally, molecular epidemiological studies can assess only known microbial characteristics for which appropriate assays are available. That is, they require prior knowledge of the characteristics to be studied. Therefore, in contrast to exploratory methods such as signature-tagged mutagenesis (8) and in vivo expression technology (41), they cannot be used to discover new virulence factors. However, they can provide an important complement to such experimental approaches by assessing the epidemiological (that is, population) relevance of newly identified traits (59, 99, 118, 182). This complementarity between epidemiology and experimentation is implicit in the molecular restatement of Koch's postulates, the first of which is that the trait of interest must be epidemiologically associated with disease (46).

Internal Controls.

Much stronger conclusions can be drawn from observed between-group differences in molecular characteristics if a study includes an internal control group that is tested in parallel with the clinical group of interest, particularly if the controls are temporally, geographically, and demographically matched to the clinical isolates, thereby avoiding some of the problems associated with use of external (that is, historical) control groups (9, 185). Concurrent testing of cases and controls using the same methods and reagents, ideally by an operator who is unaware of sample identity, reduces the likelihood that technical factors or subjective bias could influence the results.

Statistical Considerations.

Molecular epidemiological studies adhere to the same statistical principles and rely on the same statistical methods as do conventional epidemiological studies (141, 175). Between-group comparisons are tested using standard statistical approaches such as a χ² test, Fisher's exact test, or univariate logistic regression analysis for dichotomous variables, and an unpaired t-test or the Mann-Whitney U test for continuous variables. Multiple independent variables can be assessed simultaneously as predictors of an outcome (-dependent) variable by using appropriate multivariate methods. For comparisons involving multiple testings of an individual (bacterial or human) subject, whether for different traits as assessed at the same time or for a given trait as assessed at different times, appropriate tests for paired comparisons must be used, such as McNemar's test for dichotomous variables and a paired t-test or the Wilcoxon rank-sum test for continuous variables.

Type I errors, which are the false conclusion of a difference when none actually exists, are a hazard of the use of multiple comparisons (since the probability of obtaining a "significant" P value is proportional to the number of comparisons) and of selective testing of associations suggested by post hoc data review (124). However, multiple comparisons are inevitable in molecular epidemiological studies that assess multiple bacterial traits, as increasingly is the practice. Statistical adjustment for multiple comparisons, and/or cautious interpretation of putatively significant associations, can be used to address this problem (188). Likewise, post hoc data review to discover new associations is an important means for generating new hypotheses. Recognition that such hypotheses require independent confirmation provides a helpful safeguard against false conclusions.

Type II errors, which are the false conclusion of absence of a difference when one actually exists, result from insufficient sample size, which limits statistical power for finding differences (50). However, the seemingly obvious remedy of studying large comparison groups may or may not be applicable, depending on the context. This is because, unlike in experimental studies where the number of replicate determinations is largely a matter of investigator choice, in epidemiological studies clinical factors sometimes limit the number of subjects or isolates available for a particular group, thereby imposing insurmountable restrictions on sample size (88). As a consequence, conclusions may need to be tempered to reflect the inherent uncertainty resulting from limited power.

Typing Methods

The various bacterial traits analyzed in molecular epidemiological studies represent a spectrum of levels of organization and complexity, ranging from subgenic DNA sequence (the most basic), through genes, operons, and pathogenicity islands (intermediate), to clones, clonal groups, and phylogenetic groups (the most complex) (24). Each level is important and informative; each requires distinctive typing methods.

Sequence Analysis.

Analysis of sequence diversity within virulence-associated genes or their flanking regions can be done by using restriction fragment length polymorphism (RFLP) analysis or direct DNA sequencing. RFLP analysis can be applied to cloned sequences, total bacterial DNA (with specific detection of target sequences accomplished by Southern hybridization), or gene-specific PCR products (6, 102). In RFLP analysis, sequence variation is identified through its effect on the spacing of recognition sites for restriction endonucleases, as inferred from the size distribution of digestion products (72, 165). Direct sequence analysis is more informative than RFLP analysis for within-gene sequence variation and is increasingly favored as it becomes more readily available (25, 104, 169, 202).

Gene Detection.

Detection of putative virulence markers, perhaps the most familiar application of molecular epidemiology in E. coli, can be done using a variety of methods, with the method selected determining the nature of the results, which in turn shapes the conclusions that can be drawn. Probe hybridization relies on complementarity between the probe, usually several kilobases in length, and the target region (6, 7, 95). Broad genetic regions, such as entire operons, can be screened for readily by using large probes (165). By modifying the degree of stringency, genes with varying degree of homology can be identified. In contrast, PCR detection relies on precise matching between the primers and the target region and, unless special reagents are used, is usually limited to comparatively short targets, typically <2 kb (79). Thus, PCR can differentiate between minor molecular variants of a particular gene and, if multiple primer pairs are used to map an operon, can identify suboperonic deletions (Fig. 1) (104). However, PCR is more vulnerable to false-negative results from sequence polymorphisms or deletions involving the primer binding sites.

Fig. 1PCR analysis of pap operon. Open boxes represent genes within the pap operon (including papA, structural subunit; papC, usher; papEF, minor tip pilins; and papG, adhesin). Forward and reverse primers (right- and left-pointing black triangles, respectively, above and below the pap operon) are used in combinations as shown to yield the indicated PCR products (thin rectangles, below pap operon). Heavily striped rectangles, papA and papG allele PCR products. Solid black rectangles, pap gene PCR products. Finely striped rectangles, long PCR operon fragments (as generated using either flanking or internal allele-specific papG reverse primers, as illustrated for allele I-I').

Source: Reprinted from reference 104, with permission.

Clones, Clonal Groups, and Phylogenetic Groups.

Clones and clonal groups (which are groups of closely related clones) are commonly identified at the molecular level by using typing methods that scan the entire genome. Of the "whole-genome" methods in general use, the most discriminating is pulsed-field gel electrophoresis (PFGE) analysis, which involves electrophoretic separation of total bacterial DNA that has been digested using a restriction enzyme such as XbaI, which in E. coli recognizes a limited number of DNA sites (Fig. 2) (5, 135, 158, 180). Use of a second restriction enzyme can further enhance discrimination (131). Identity of two isolates by PFGE analysis implies that they represent the same strain or clone (215). However, PFGE is so discriminating that beyond a certain limited level of divergence it fails to perceive similarity between isolates.

Fig. 2Pulsed-field gel electrophoresis (PFGE) profiles of E.coli clonal group A (CGA) isolates. Shown are XbaI PFGE profiles for eight CGA pyelonephritis isolates (lanes 1–5 and 8–10; "Py" strain designations, bold) and for two comparison CGA cystitis isolates (lanes 6 and 7: UMN 26, from Minnesota, and UCB 102, from California) (94, 131). Geographical source for the pyelonephritis isolates is indicated above the strain designations.

Consequently, other less discriminating whole-genome methods, including ribotyping and PCR-based fingerprinting, are more useful for identifying broader clonal groups. In ribotyping, ribosomal DNA probes are used for Southern hybridization with total DNA that has been restricted with a conventional endonuclease such as HindIII and electrophoretically size-separated (211). PCR-based methods such as random amplified polymorphic DNA (RAPD) analysis, which use random or arbitrary primers (Fig. 3) (12), and repetitive element PCR, which uses primers targeting various known genomic repeat elements (221), generate distinctive banding patterns that reflect the spacing of suitable primer sites in the genome of the particular isolate. For any of these methods, in addition to simple "same-versus-different" comparisons, genomic profiles can be subjected to cluster analysis to define quantitative similarity relationships among isolates. This provides an approximation of broader phylogenetic relationships (Fig. 4) (94, 224). Although such relationships can be more accurately resolved by specialized methods such as multilocus enzyme electrophoresis (MLEE) and multilocus sequence typing (MLST), MLEE and MLST are involved techniques that are not particularly amenable to widespread, large-scale use (44, 67, 129, 193). Isolates nonetheless can be sorted readily into the four major phylogenetic groups of E. coli (A, B1, B2, and D), as originally defined by MLEE (67), by using a simple multiplex PCR-based method (32).

Fig. 3RAPD analysis of E.coli strains 536, NU14, and RS218. RAPD profiles generated by using primer 1247 (12) show E.coli O18:K1:H7 strains NU14 (cystitis: lane 3) and RS218 (neonatal meningitis: lane 4) to be indistinguishable from one another but distinct from strain 536 (O6:K15:H31, pyelonephritis: lane 2). M (lanes 1 and 5), 100-bp marker.

Source: Reprinted from reference 107, with permission.

Fig. 4RAPD-based phylogenetic analysis of E.coli isolates. Genomic profiles (shown in computer reconstruction), as generated for each isolate by using RAPD primers 1247, 1254, 1281, and 1283, were concatenated for cluster analysis. Pyelonephritis isolates ( n = 10; "Py" strain designations) are labeled in bold if from E.coli clonal group A (CGA) ( n = 5) and in lightface italic if non-CGA ( n = 5). CGA isolates (bold) are bracketed and labeled as to syndrome (CY, cystitis; PY, pyelonephritis) and serogroup (O11/O17/O77) (right), with the corresponding cluster shown in bold (left). The two E.coli O15:K52:H1 control strains are bracketed and labeled by serotype. Reference strains from the E.coli Reference (ECOR) collection (bold) are identified as to phylogenetic group (right). The depth of the molecular weight ladder cluster (brackets; MW) reflects the intrinsic variability inherent in gel electrophoresis and image analysis, independent of amplification.

Source: Reprinted from reference 94, with permission.

RESULTS OF MOLECULAR EPIDEMIOLOGICAL STUDIES

VFs: Associations with Clinical Variables and Phylogenetic Background

Comparisons between Clinical Isolates and Controls.

E. coli isolates from the urine, blood, cerebrospinal fluid, etc., of patients with diverse extraintestinal infection syndromes typically exhibit a greater prevalence of specific molecular markers than do fecal isolates from uninfected hosts (7, 16, 78, 134, 136). From an epidemiological perspective such virulence markers can be regarded as "virulence factors" (VFs), although this term must be understood as implying "factors associated with" rather than "contributing to" virulence, since epidemiological associations do not necessarily reflect causality (as discussed above).

These VFs can be grouped by functional category, for example, adhesins (fimbrial and nonfimbrial) (34, 132, 137, 147), siderophore systems (155, 182, 183, 191, 217), toxins (59, 171, 192, 227), surface polysaccharides (29, 181), invasins (69, 70), serum resistance-associated traits (109, 233), and traits of miscellaneous or unknown function (81, 118) (Table 1, Fig. 5). Clinical isolates often contain multiple VFs from a particular functional category (96, 97); this conceivably may allow for redundancy, synergistic interactions among VFs, and/or adaptability to different environmental niches. Conversely, many seemingly virulent strains lack known representatives of one or more of these functional categories (96, 97). Whether such apparent deficits are compensated for by VFs from other functional categories, or these strains actually do contain unrecognized representatives of the "missing" VF categories, is unknown.

Fig. 5Phylogenetic distribution of extraintestinal virulence-associated traits in E.coli. Dendrogram at left depicts phylogenetic relationships for the 72 members of the E.coli Reference (ECOR) collection, as inferred based on multilocus enzyme electrophoresis (67). The four major E.coli phylogenetic groups (A, B1, B2, and D) and the nonaligned strains ("non") are bracketed and labeled. Bullets at right indicate presence of putative virulence genes ( papA, P fimbriae; kpsMT, group II capsule synthesis; sfa/foc, S and F1C fimbriae; iutA, aerobactin system; traT, serum resistance; and fimH, type 1 fimbriae). Horizontal bars at right indicate the 10 ECOR strains isolated from humans with symptomatic UTI. The remaining strains, except for one asymptomatic bacteriuria isolate, are fecal isolates from healthy human or animal hosts. Note the concentration of (chromosomal) virulence genes papA, kpsMT, and sfa/foc within phylogenetic groups B2 and D, but their occasional joint appearance also in distant lineages, consistent with coordinate horizontal transfer. The more scattered phylogenetic distribution of traT is consistent with this gene's typically plasmid location, whereas fimH is nearly universally prevalent, consistent with its presence in other species of Enterobacteriaceae, presumably reflecting an origin in a shared enterobacterial ancestor. Note the concentration of UTI isolates within phylogenetic groups B2 and D and the association of virulence genes with UTI isolates.

Source: Reprinted from reference 81, with permission.

Table 1Virulence-associated traits of ExPEC, by functional category

Associations among VFs.

Certain VFs commonly occur together among clinical isolates in patterns suggesting either coselection or direct genetic linkage (101, 108). Extensive genetic linkage of VFs has been demonstrated within pathogenicity-associated islands (PAIs) and on plasmids (39, 60, 62, 120, 125, 146, 213, 219). Certain VFs typically occur within genomic PAIs (for example, pap, sfa/foc, hly, cnf, and fyuA) (39, 191) and others on plasmids (for example, iss, traT) (20), whereas some occur variably in either context (for example, afa/dra and iuc/iut) (120, 219). ExPEC strains often contain multiple PAIs, each with a distinctive combination of VFs, which sometimes results in a strain having multiple copies of a particular VF, for example, pap (Fig. 6) (23, 39, 226).

Fig. 6Genome map of ExPEC strain 536. The map is based on the chromosome of E.coli MG1655 (K-12). PAIs are indicated according to their chromosomal insertion sites next to tRNA-encoding genes. Contents, by PAI, include: PAI I (α-hemolysin, F17-like fimbriae, CS12-like fimbriae); PAI II (α-hemolysin, P fimbriae with papG III); PAI III (S fimbriae, iro siderophore system, Tsh-like hemoglobin protease); PAI IV (yersiniabactin system). Many additional smaller DNA insertions compared to K-12 are present (not shown).

Source: Reprinted from reference 39, with permission.

This co-occurrence of VFs results in overlapping statistical associations with clinical variables, leading to uncertainty as to which VF is primarily responsible for the association. Multivariate analysis can help in this situation, but is not definitive. Moreover, sequence analysis of PAIs and virulence plasmids (or genomes) that contain known VFs invariably reveals additional genes of unknown function, some of which exhibit homology to known VFs from other species (Fig. 7) (39, 213, 226). This suggests the possibility that the statistical associations of known VFs with virulence may be mediated through various of these as yet uncharacterized VFs, that is, that the known VFs, although useful markers, may not themselves be the actual determinants of virulence.

Fig. 7Maps of PAIs from strain 536. Known or putative open reading frames (ORFs) are grouped according to the following characteristics: blue, functional, known ORFs; green, truncated ORFs with a start codon and a stop codon; gray, as yet unidentified ORFs without homologues on the DNA level. Nonfunctional ORFs (e.g., due to internal stop codons or frameshifts) are indicated by hatched symbols. ORF numbers are indicated below the corresponding ORF symbols. Functional or truncated tRNA-encoding genes are marked in red. Direct repeat (DR) structures flanking PAIs are indicated. Thick black lines below the PAIs represent regions that were detected by PCR. Several PAI-specific PCRs were grouped into PAI regions.

Source: Reprinted from reference 39, with permission.

Phylogenetic Group and Clonal Groups.

According to molecular analyses, certain ExPEC clonal groups, as traditionally identified based on their O:K:H serotypes (for example, O18:K1:H7, O6:K2:H1, and O7:K1:H–) (157), are disproportionately represented among clinical isolates as compared with controls (87, 96, 156). These virulent clonal groups derive primarily from phylogenetic group B2, and to a lesser extent group D, which explains the observed predominance of groups B2 and D among clinical isolates (17, 96, 101, 163). Most of the traditionally recognized extraintestinal virulence markers (for example, pap, sfa/foc, hly, and kps) are typically concentrated within these virulent clonal groups and, hence, within phylogenetic groups B2 and/or D, whereas others (for example, afa/dra, iuc/iut, and traT) are more broadly and/or sporadically distributed across the species (Fig. 5) (24, 86). These divergent patterns of phylogenetic distribution correspond with vertical (within-lineage) versus horizontal (among-lineage) transmission, respectively, and reflect the typically chromosomal versus plasmid location of the respective sequences, as discussed above.

Considerable variation in VF profiles is evident at every level within the phylogenetic tree, including among the major phylogenetic groups, among the various clonal groups within these phylogenetic groups, and even among subclones within individual clonal groups. This is consistent with extensive ongoing remodeling of PAIs and/or virulence plasmids, in addition to acquisition and loss of entire PAIs or plasmids, evolutionary processes that presumably result in the continuous emergence of new pathotypes upon which selective forces can act (18, 96).

Several studies have compared clinical isolates with fecal isolates from the same hosts, as opposed to fecal isolates from a separate control population (90, 166). This strategy ensures a degree of matching for associated host characteristics greater than that provided by a traditional unpaired study design. The results of such studies, like those of most traditional comparison studies, suggest that special pathogenicity (as indicated by presence of multiple VFs) rather than simple prevalence (that is, quantitative predominance in the fecal flora) is necessary for a fecal strain to cause urinary tract infection (UTI).

Phylogenetic Background versus VFs.

The overlapping associations of VFs and phylogenetic background with clinical virulence call into question which of these bacterial characteristics, VFs or phylogenetic background, more directly determines virulence. Several studies in which both phylogenetic group and VF profiles were analyzed have shown that VFs are statistically more closely associated with clinical virulence (90, 91, 164). However, phylogenetic group exhibits a residual association with virulence even after known VFs are accounted for (91). This is consistent with the existence of as-yet-undefined VFs that are both phylogenetically distributed and incompletely linked with known VFs.

Comparisons among Syndromes and Host Groups.

Molecular epidemiological comparisons are not limited to E. coli populations from infected versus uninfected hosts. Comparisons also can be made between isolates (i) from patients with different clinical syndromes (to identify syndrome-specific, versus conserved, VFs or clonal groups) (22, 108, 177), (ii) from infected hosts who possess or lack particular predisposing conditions (to identify bacterial traits that may interact with specific host defense mechanisms or host receptors) (92, 134), and (iii) from different host species (to identify species-specific, versus broad-host-range, VFs or pathogens) (103, 228).

The results of such studies support certain general conclusions. First, invasive clinical syndromes such as pyelonephritis, bacteremia, prostatitis, and meningitis, as compared with less invasive syndromes such as cystitis and asymptomatic bacteriuria, on average usually involve strains with greater molecular virulence, as reflected in the number of VFs and a group B2 background. Second, various forms of host compromise significantly decrease the requirement for bacterial virulence within a defined clinical syndrome. This is exemplified by the reduced prevalence of pap among pyelonephritis isolates from patients with, versus those without, vesicoureteral reflux, that is, spontaneous retrograde flow of urine from the bladder back up to the kidneys (130), and the reduced prevalence of pap and chromosomal aerobactin determinants among blood isolates from patients with urosepsis who have, versus those who lack, underlying anatomical or medical conditions predisposing to UTI (95). Third, although there is some syndrome and host specificity of VFs and clonal groups, there also is considerable commonality among syndromes and host groups, whereas tremendous diversity is apparent within each syndrome and host group. Examples of relative syndrome and host specificity that have been identified include the statistical association of sfaS (S fimbriae) with neonatal meningitis (16, 97); of pap with pyelonephritis (71); of papG allele III, hly, and cnf with canine UTI (103); and of the F11 variant of papA (P fimbriae structural subunit) with avian septicemic E. coli (42). However, each of these associations is incomplete, since the same VFs or clonal groups occur to various degrees also in other syndromes and host groups, as exemplified by the prominence of the O18:K1:H7 clonal group in both neonatal meningitis (as traditionally recognized) and uncomplicated cystitis in women (as recently appreciated) (Fig. 3) (1, 87, 117).

Regulation of Expression.

In addition to the simple presence or absence of a particular gene, molecular epidemiological studies may also need to consider gene expression, since expression obviously is required if the genotype is to influence the virulence phenotype. Expression can be assessed through a variety of phenotypic tests, which moves beyond the realm of strict molecular epidemiology. However, in the instance of the fim operon, expression is regulated by an invertible switch element in the promoter region, the position of which ("on" versus "off") can be defined for a bacterial population via a simple PCR assay (56, 122). Differences between UTI versus control isolates, and between cystitis versus pyelonephritis isolates, with respect to their fim switching bias can be demonstrated, supporting the concept that regulation of fim expression may influence not only overall pathogenicity but also anatomical site tropism (56, 122). Such a molecular epidemiological approach is particularly relevant for fim since, although expression of type 1 fimbriae appears from experimental studies to be quite important for UTI pathogenesis, the nearly uniform presence of fim in E. coli (and other enterobacteria) all but precludes demonstration of a virulence association for fim through conventional presence-absence comparisons between clinical isolates and controls (86).

Molecular Variants.

Another potentially important consideration is the particular variant of a virulence gene present in an isolate. Molecular variation within a gene may produce pathogenetically important phenotypic alterations in the encoded peptide, such as the shifts in preferred receptor sugars or glycolipids that are associated with polymorphisms in fimH (type 1 fimbrial adhesin) and papG, respectively. Diverse single-nucleotide polymorphisms (SNPs) in fimH, which can be detected by sequence analysis or with SNP-specific PCR primers, cause single-amino-acid changes in the FimH peptide that produce a shift from a (commensal-associated) trimannose binding phenotype to a (UTI-associated) monomannose binding phenotype (199). Interestingly, the monomannose binding variants, although at an advantage within the pathogenic niche (for example, because of their enhanced binding to bladder epithelium), also are more susceptible to inhibition by monomannose residues, such as are found in salivary glycoproteins; this presumably makes them less effective as gut colonization factors (199). An additional point mutation in a monomannose binding fimH variant, resulting in a single FimH amino acid substitution (Ser-62-Ala), can further strengthen monomannose binding and also confer type IV collagen binding (Fig. 8), which may be important in the pathogenesis of neonatal meningitis (107, 167). Such mutations have been termed pathoadaptive, since they represent minor modifications of genes already present in nonpathogenic members of the species, with the mutations conferring enhanced fitness in the pathogenic niche (200).

Fig. 8Receptor binding specificity of type 1 fimbrial adhesin (FimH) variants. In vitro binding of isogenic recombinant strains expressing the Ala-62 or Ser-62 FimH variants (from strains NU14 and 536, respectively) to (A) a trimannose substrate (bovine RNAse B), (B) human collagen type IV, and (C) a monomannose substrate (yeast mannan). Both variants bind equally well to trimannose, but the Ala-62 variant exhibits stronger type IV collagen and monomannose binding than does the Ser-62 variant. (Commensal-associated FimH variants exhibit equally strong trimannose binding but minimal binding to type IV collagen or monomannose [not shown].) Open columns, bacteria incubated without α-methyl mannoside (αmM); solid columns, bacteria incubated with 50 mM αmM. Data are mean + SEM ( n = 4) of number of bacteria bound per well.

Source: Reprinted from reference 107, with permission.

More extensive alterations within papG, which can be detected by using specific primers or probes, are evident among the several known allelic variants of papG (78, 84, 104, 110, 132). The respective peptide products of the papG alleles bind preferentially to various forms of Gal(α1-4)Gal-containing glycolipids (196, 206, 208, 209). Because of the varied distribution of these particular glycolipids by anatomical site and host species, the distinctive binding preference of the PapG variants may underlie their somewhat divergent associations with clinical syndromes and host groups, such as the associations of PapG II with pyelonephritis and of PapG III with cystitis, with dogs and cats, and with compromised hosts with urosepsis (77, 78, 82, 98, 103, 160, 197, 208). Other examples of epidemiologically significant molecular variation within a particular VF, or VF family, include the sfa/foc (S and F1C fimbriae) family (159), the afa/dra (afimbrial and Dr-binding adhesins) family (121, 147, 237), the group 2 and group 3 kps (capsule) families (172, 184), and cnf1 and cnf2 (cytotoxic necrotizing factor I and II; associated with extraintestinal and intestinal virulence, respectively) (21). Likewise, papA (P fimbriae structural subunit) exhibits 13 known alleles (100, 105). These tend to be clonally distributed, such that they can be used, in conjunction with other VFs or seroantigens, as markers for particular clones (96, 105). They also exhibit somewhat distinctive associations with specific host groups or syndromes, for example, F10, F12, and F48 with canine UTI (51, 90, 103).

VFs as Predictors of Clinical Outcomes.

Apart from their role in pathogenesis, VFs also have been studied as potential clinical predictors that could be used to guide patient management. For example, P fimbriae (or pap) testing has been proposed as a way to identify boys at risk for renal scarring (37), adults with pyelonephritis or urosepsis who have unrecognized predisposing conditions (40), and patients whose household members should be screened for colonization with the patient's UTI strain (173). Other proposed clinical applications of such testing include determining length of therapy for children with UTI (214) and identifying pregnant women at high risk for developing pyelonephritis (207). However, the true clinical utility and cost-effectiveness of such clinical applications of VF testing are unconfirmed (80), such that at present they cannot be recommended outside of a research setting.

Antimicrobial Resistance and Virulence.

Acquired resistance to therapeutically important antimicrobial agents is increasingly prevalent among clinical E. coli isolates, making management of E. coli infections more difficult and costly (57, 232). The relationship between resistance and virulence or phylogenetic background has been explored in several molecular epidemiological studies. Older data suggest that among E. coli isolates from patients with urosepsis, resistance to historical antimicrobial agents such as ampicillin, sulfonamides, tetracycline, and streptomycin is negatively associated with virulence and a group B2 phylogenetic background but is positively associated with host compromise (89). This is consistent with a scenario wherein resistance provides a greater fitness advantage than do traditional VFs or a group B2 background for infections in compromised hosts, who have weakened defenses but are frequently exposed to antimicrobial agents.

More recent data regarding fluoroquinolone agents demonstrate similar negative associations between resistance and VFs or a B2 phylogenetic background (93, 106, 220, 222). This has been interpreted as suggesting that VFs may be lost concomitant with mutation to fluoroquinolone resistance (222). However, this hypothesis does not account for the observed phylogenetic shifts (away from group B2), which suggest instead that resistant isolates derive predominantly from distinct, less virulent bacterial populations (93, 106). Therefore, selection for antimicrobial resistance within different populations rather than loss of VFs in exchange for resistance may account for the observed virulence differences between susceptibility groups. Indeed, fluoroquinolone resistance has been shown to be associated with host compromise. Thus, among clinical isolates the same selection factors that produce statistical associations of low virulence with resistance to older antimicrobial agents may be operative also with fluoroquinolones.

However, antimicrobial resistance clearly does not equate with reduced virulence in all circumstances. For example, among fecal E. coli from diseased cattle and swine, a setting in which most of the organisms probably are not pathogens, resistance to extended-spectrum cephalosporins or fluoroquinolones is associated with minimal shifts in VF profile (93). Likewise, the recently described E. coli "clonal group A," although exhibiting multidrug resistance and accounting for 33 to 50% of recent trimethoprim-sulfamethoxazole-resistant E. coli isolates among women with acute uncomplicated cystitis or pyelonephritis in the United States, is replete with VFs, which presumably contribute to this clonal group's success as a pathogen among otherwise healthy hosts (94, 131). Likewise, certain recent human clinical isolates from the O15:K52:H1 clonal group, representatives of which caused a large community-wide outbreak of drug-resistant UTI and septicemia in London in 1986 and 1987, have acquired fluoroquinolone resistance yet exhibit the clonal group's full characteristic VF profile (93, 168). Resistance and virulence presumably are uncoupled in the first instance (for the animal commensal isolates) by the absence of selection for virulence, and in the later instances (for human clinical isolates from clonal group A and the O15:K52:H1 clonal group) by the uniform requirement for virulence, irrespective of resistance. Further studies clearly are needed to clarify the relationship between virulence and resistance, taking into account ecological source and relevant selection factors.

VFs versus Colonization Factors.

Interestingly, certain molecular epidemiological data suggest that at least some of what traditionally have been regarded as VFs in ExPEC may also promote intestinal colonization (149, 150, 229). Some experimental support also exists for this hypothesis (2, 65, 66, 128, 230). This provides a more plausible mechanism for the evolution of these traits than does the enhanced pathogenicity the traits confer, since the ability to persist and flourish within the host intestinal tract represents a more obvious survival advantage than does the ability to cause sporadic and usually self-limited or even fatal disease. Moreover, although many of the traditionally recognized extraintestinal VFs are encoded on what have been designated PAIs, which implies that pathogenicity is their raison d'être, this terminology is undergoing revision. The newer, more inclusive designation "fitness island" reflects the recognition that similar accretions of genes encoding related functions, with associated insertion sequences and other mobility-promoting elements, occur in nonpathogens, including even environmental (non-host-associated) organisms (61). However, the hypothesis that VFs have evolved primarily as colonization factors does not explain why ExPEC are not the dominant clone(s) within the intestinal tract in most human hosts, as would be expected if they truly have a fitness advantage in this niche (108, 198). Additional epidemiological and experimental studies are needed to clarify the relationship between specific bacterial traits, including recently discovered putative VFs, and intestinal colonizing ability.

Molecular Fingerprinting

In most of the studies discussed above, E. coli isolates were analyzed in the aggregate, with conclusions being based on comparisons between groups of isolates, without particular regard to the constituent clones. In contrast, molecular fingerprinting allows individual clones or strains to be resolved and analyzed. This approach underlies a distinct branch of molecular epidemiology, one that focuses on the individual clone rather than on the group. Topics that have been addressed using this approach include the reservoirs, colonization patterns, and transmission pathways of ExPEC.

Fecal-Vaginal-Urethral Hypothesis.

According to the fecal-vaginal-urethral hypothesis, E. coli strains causing UTI usually derive immediately from the host's own fecal and perineal flora. This model, which was first suggested by O serotype data (55), is now supported also by molecular data showing that in most episodes of acute cystitis or pyelonephritis in women, prostatitis in men, or UTI in dogs, the urine organism is also the host's predominant fecal strain (90, 180, 216, 235). This is relevant to prevention efforts, since it suggests that fecal (and vaginal) colonization with a urovirulent organism is a potentially modifiable risk factor for subsequent UTI. This provides a rationale for studying the determinants of intestinal (and vaginal) colonization with particular E. coli strains and for searching for external reservoirs of virulent E. coli that might be acquired by the host as intestinal (and vaginal) colonizers.

Longitudinal studies have shown that the fecal and vaginal E. coli populations are highly dynamic and that the correlation between antecedent (as opposed to concurrent) fecal and/or vaginal colonization with a uropathogenic strain (as defined, for example, by the presence of pap) and subsequent UTI or asymptomatic bacteriuria due to that strain is limited (187, 204). This suggests that either strain detection methods are insensitive, leading to a false conclusion of absence of a particular strain prior to a UTI episode when it actually is present, or the interval between strain acquisition and UTI is often shorter than the sampling interval used in these studies, such that the strain appears to occur in the urine simultaneous with its first detection in stool and vagina.

Same- versus Different-Strain Recurrent UTI.

Molecular fingerprinting also has been used to assess the same- versus different-strain nature of recurrent UTI isolates, in comparison with index isolates. The findings have been quite variable, with same-strain episodes accounting for from 25 to 100% of recurrences in different studies (73, 76, 111, 180, 236), with differences in selection criteria and patient populations contributing to the variability. In one study, 30/44 (68%) of recurrent UTIs were caused by a strain previously identified in that person (180). This percentage was 55% (6/11) among patients with two recurrences per person-year, and 72% (17/24) and 78% (7/9), respectively, among those with four or five recurrences and six or more recurrences. Analysis of a subset of subjects established that the majority of recurrent UTIs were due to reinfection, not overt persistence within the urinary tract, and suggested that the colonic flora was the reservoir for these reinfecting strains (although intracellular persistence within the urinary tract, as discussed below, could not be ruled out) (180). As observed in this study and others, some patients experience multiple same-strain recurrences, some of which can occur months or years after the initial episode, occasionally with intervening UTI episodes due to unrelated strains (26, 73, 74, 76, 180).

The biological relevance of this sort of analysis is that different-strain recurrence implies an independent infection episode whereas same-strain recurrence implies either relapse from a persisting endogenous focus or reintroduction of the strain from a persisting external reservoir in the host or the environment. This distinction may have clinical relevance for prevention and treatment efforts, since the occurrence of multiple independent infection episodes suggests an underlying host predisposition to infection (which may be amenable to intervention, for example, through a change in contraceptive method) (205), whereas the presence of a persisting reservoir suggests a need to identify and eradicate the reservoir.

Potential endogenous reservoirs include the long-term intracellular persistence of a strain within the bladder epithelium that seems to underlie the intermittent episodes of recurrent bacteriuria that occur in experimentally infected mice following apparent resolution of the initial infection (142). Clinical studies are under way to assess for this phenomenon in humans (W. E. Stamm, personal communication). For external reservoirs, the host may be persistently colonized with a strain in the intestine and/or vagina or may repeatedly reacquire it from the (animate or inanimate) external environment (73). In either situation, efforts to identify and eliminate the external reservoir conceivably could be protective.

Strain Sharing among Associated Hosts.

Environmental sources of uropathogenic or antimicrobial-resistant E. coli have been investigated by using molecular fingerprinting, with or without VF detection and phylogenetic analysis. Within-household strain sharing has been demonstrated between adult sex partners (in some instances accompanied by symptomatic UTI in one or both individuals) and between parents and children, siblings, and even humans and pets (Fig. 9) (10, 31, 49, 83, 85, 231). Likewise, several hospital-based pyelonephritis outbreaks have been documented in which a virulent strain seemingly was transmitted to patients by health care workers (112, 212, 218). Interestingly, some evidence suggests that what classically have been regarded as VFs also predict cocolonization of epidemiologically associated hosts, implying that these traits may promote person-to-person transmission as well as infection (48).

Fig. 9PFGE profiles and colonization patterns of E.coli isolates from three household members (man, woman, and pet cat). (A) PFGE profiles. Lane numbers are shown below gel images. Lanes 1 through 10, profiles of nine of the unique strains, with strain designations shown above gel lanes, plus subtype 1″ (lane 9). Lanes 11 through 16, profiles of independent isolates of strain 1, as recovered from various anatomical sites from the woman (lanes 11–13), man (lanes 14 and 15), and cat (lane 16). (B) Distribution of 14 unique E.coli strains over time (week of sampling shown below grid), as recovered from various anatomical sites from the three household members. Female symbol, woman; male symbol, man; NG, no growth; •, no sample. Strains isolated more than once appear in colored boxes, with a unique color for each strain. Strains isolated only once appear in colorless boxes. Week 12, which coincided with symptoms of acute UTI in the woman, yielded strain 1 from the woman's urine specimen (boldface box). There is no strain 7.

Source: Reprinted from reference 144, with permission.

However, that person-to-person transmission actually occurs outside of the hospital setting is inferential: the underlying mechanism for the observed within-household strain sharing remains to be defined. Sexual transmission, a favored hypothesis, is supported by epidemiological evidence associating certain sexual practices with cocolonization of adult sex partners and by anatomical coincidences such as colonization of the male partner's urethra or glans penis with a strain also found in the female partner's vagina and urine (48, 49, 203). However, sexual transmission is unlikely to explain cocolonization involving children and pets (31, 83, 144); other modes of host-to-host transmission (whether direct or indirect) must be considered, as must possible coordinate acquisition from a common external source, such as the food supply.

Community-Wide Dissemination of ExPEC.

Transmission pathways of ExPEC within the larger community are relevant to the dissemination of virulent clonal groups within the human population. Recent examples of this phenomenon include the O15:K52:H1 clonal group, which (as mentioned above) first came to attention when it caused the mysterious South London outbreak of 1986 and 1987 (45, 153, 162), and E. coli clonal group A, which (as also mentioned above) has emerged as a prominent cause of drug-resistant UTI across the United States (28, 94, 131) and appears to have undergone point-source spread within at least one community (131). The latter observation prompted the suggestion that, by analogy to E. coli O157:H7, Campylobacter, and Salmonella, contaminated products could serve as a transmission vehicle also for ExPEC (131). Indeed, retail foods are commonly contaminated with antibiotic-resistant E. coli and ExPEC, along a descending prevalence gradient from poultry (highest) through meats (beef and pork) to produce and miscellaneous foods (lowest) (J. R. Johnson, submitted for publication). The human health implications of such contamination remain to be defined. Insights into this question conceivably could derive from molecular epidemiological comparisons of ExPEC isolates from retail foods versus colonized and infected humans, to ascertain the extent of commonality between these populations, and by longitudinal molecular epidemiological surveillance of individuals and the foods they consume, to identify temporal patterns suggesting food-borne transmission.

SUMMARY AND CONCLUSIONS

Molecular epidemiological analyses of ExPEC, which are based on structured observations of E. coli as they occur in the wild, provide an important complement to experimental assessment. Fundamental to the success of molecular epidemiological studies are the careful selection of subjects and the use of appropriate methods for genotyping and statistical analysis. To date, molecular epidemiological studies have yielded numerous important insights into host-pathogen relationships, phylogenetic background, reservoirs, and transmission pathways of ExPEC and have delineated areas in which further study is needed. The rapid pace of discovery of new putative VFs and the increasing awareness of the importance of VF expression and molecular variants of VFs provide abundant stimulus for future molecular epidemiological investigation, while the continued evolution of molecular typing methodologies provides improved tools for such studies and allows entirely new questions to be addressed.

Acknowledgments

This material is based upon work supported by Office of Research and Development, Medical Research Service, Department of Veterans Affairs (J.R.J., T.A.R.). Ann Emery and Dave Prentiss (both at the Minneapolis VA Medical Center) helped prepare the manuscript and figures, respectively.

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