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Medical Service and Geriatric Research, Education, and Clinical Center, VA Medical Center, and Departments of Medicine and Psychiatry, University of Minnesota, Minneapolis
Microbiology Laboratory and Servei de Microbiologia, Instituto Clínico de Infecciones e Inmunología, Hospital Clínic-IDIBAPS, Barcelona, Spain
Molecular analysis of 63 Escherichia coli urine isolates showed that pyelonephritis (n = 23) and prostatitis (n = 17) isolates exhibited more virulence factors (VFs) among the 35 sought than did cystitis isolates (n = 23). Several nontraditional VFsincluding bmaE (M fimbriae), gafD (G fimbriae), fyuA (yersiniabactin receptor), ireA and iroN (novel siderophore receptors), cvaC (colicin V), traT (serum-resistance associated), ibeA (invasion of brain endothelium), ompT (outer membrane protease T), and malX (pathogenicity island marker)either differentiated significantly between syndromes (despite small numbers of isolates and possible multiple-comparison artifacts) or were broadly prevalent. Thus, interventions that target conserved uro-VFs may be possible, despite the likely existence of syndrome-specific pathogenetic mechanisms and/or host defense systems.
Escherichia coli is the most common cause of urinary tract infection (UTI), including acute cystitis, pyelonephritis, and prostatitis, 3 familiar and clinically distinct acute UTI syndromes [13]. The basis for the occurrence of these distinctive clinical syndromes, which presumably arise from localized infection and inflammation within the bladder, kidney, and prostate gland, respectively, is undefined. Conceivably, preventive measures such as vaccines could be devised if the relevant causative factors were known [4, 5]. Possible syndrome-specific bacterial factors among the causative E. coli strains have been sought in a number of studies, with the candidate virulence factors (VFs) including several adhesins, toxins, and/or siderophore systems [610]. The availability of assays for a broader range of VFs than previously assessed in this context [11]and the availability of assays for E. coli phylogenetic group [12]prompted us to revisit the question of syndrome-specific bacterial characteristics among E. coli isolates from patients with cystitis, pyelonephritis, and prostatitis.
PATIENTS, MATERIALS, AND METHODS
Patients and strains.
E. coli isolates from women with acute cystitis (n = 23) were from urine samples submitted to the Clinical Microbiology Laboratory of the Primary Care Center Manso, or the Clinical Microbiology Laboratory of the Hospital Clinic, of Barcelona (Spain). E. coli isolates from women with pyelonephritis (n = 17) and men with prostatitis (n = 23) were from urine samples submitted to the Clinical Microbiology Laboratory of the Hospital Clinic of Barcelona. Only 1 isolate per patient was analyzed. Cystitis was defined by the presence of dysuria and urinary frequency and urgency. Acute pyelonephritis was defined by the presence of fever (>38°C axillary) plus flank pain and/or lumbar tenderness, with or without symptoms of cystitis. Prostatitis was defined by the presence of fever, pyuria, and prostatic tenderness. To avoid possible confounding due to the effects of antimicrobial resistance on the distribution of VFs and/or phylogenetic group [13, 14], the present study was limited to nalidixic acidsusceptible isolates, as determined by E-tests conducted in accordance with the manufacturer's instructions (AB-Biodisk). The study subjects were patients consecutively encountered during the study period (January 2000 through June 2000) who met the inclusion criteria. Group size was determined by the number of available eligible patients. For the pyelonephritis and prostatitis groups, the median ages were 54 years (range, 2485 years) and 55 years (range, 2389 years), respectively, and the prevalence of prior UTI history was 35% and 30%, respectively; such data were unavailable for patients in the cystitis group. The distribution, by syndrome, among the isolates of 7 uro-VFsthat is, papC (P fimbriae assembly), cnf1 (cytotoxic necrotizing factor), iucC (aerobactin synthesis), hly (hemolysin), sat (secreted autotransporter toxin), fimA (type 1 fimbriae structural subunit), and papG (P fimbriae adhesin) allele IIIhas been reported elsewhere [7]. The experimentation guidelines of the authors' institutions were followed in the conduct of clinical research.
Virulence genotypes and phylotyping.
Bacterial DNA was extracted by use of the Wizard Genomic DNA Purification kit (Promega). The presence of 35 VFs was assessed by use of an established multiplex polymerase chain reaction (PCR)based assay [11]. An aggregate VF score was calculated for each isolate as the number of unique VFs detected, with adjustment for multiple detection of the pap (P fimbriae), sfa/foc (S/F1C fimbriae), and kps II (group 2 capsule) operons. The E. coli phylogenetic group of origin (A, B1, B2, or D), as initially defined on the basis of multilocus enzyme electrophoresis [15], was determined by use of a 3-locus PCR-based method [12]. Each isolate was tested in duplicate, with appropriate positive and negative controls.
Statistical analysis.
Three types of comparisons were made, including comparisons between each clinical group (or phylogenetic group) and the rest of the population, comparisons across the 3 clinical groups (or 4 phylogenetic groups), and, if a statistically significant among-group difference was detected for a particular variable, pairwise comparisons between individual clinical groups (or phylogenetic groups). Comparisons of proportions were tested by Fisher's exact test (2-tailed). The distribution of VF scores was examined in each clinical and phylogenetic group and was found to be nonnormally distributed (P < .05, by Lilliefor's test). Therefore, the Mann-Whitney U test was used to compare each clinical or phylogenetic group with the rest of the population, and nonparametric analysis of variance (Kruskal-Wallis) followed by Mann-Whitney post-hoc pairwise 2-group comparisons were used to assess between-group differences in VF scores. The threshold for statistical significance was considered to be P < .05.
RESULTS
Overall prevalence of VFs.
Among the 63 E. coli urine isolates from patients with cystitis (n = 23), pyelonephritis (n = 17), and prostatitis (n = 23), the various VFs ranged in prevalence from 3% (kpsMT III [group 3 capsule]) to 95% (fimH [type 1 fimbriae]), with all except papG allele I being detected in 1 isolate each (table 1). Overall, the 8 most prevalent VFs, in order of descending frequency, were fimH (95%), fyuA (yersiniabactin receptor; 94%), ompT (outer membrane protease T; 83%), iroN (novel siderophore receptor; 78%), malX (pathogenicity island marker; 76%), kpsM II (group 2 capsule; 75%), sfa/foc (67%), and traT (serum-resistance associated; 65%). Also occurring in >50% of isolates were hlyD (hemolysin; 57%), cnf1 (54%), and iutA (aerobactin receptor; 54%).
Syndrome-specific distribution of VFs.
Of the 31 markers detected (with papA, C, EF, and G [i.e., P fimbriae subunits and assembly] counted as a single marker and the papG alleles [i.e., adhesin variants] counted separately), 11 (35%) were significantly distributed according to clinical syndrome (table 1). Each syndrome differed significantly from the others when analyzed either individually or collectively, according to the prevalence of 57 markers. For example, cystitis isolates differed significantly from all other isolates by their lower prevalence of papA/C/EF/G, papG allele II, and the papG allele II + III combination. Additionally, cystitis isolates differed specifically from pyelonephritis isolates by their lower prevalence of ireA (novel siderophore receptor) and cvaC (colicin V) and from prostatitis isolates by their lower prevalence of hlyD and cnf1. Pyelonephritis isolates differed significantly from all other isolates by their higher prevalence of papG allele II, bmaE (M fimbriae), gafD (G fimbriae), ireA, the K2 kpsM variant, cvaC, and ibeA (invasion of brain endothelium). Additionally, pyelonephritis isolates differed from prostatitis isolates (with borderline statistical significance) by their lower prevalence of hlyD and cnf1 and higher prevalence of iss (increased serum survival). Finally, prostatitis isolates differed significantly from all other isolates by their higher prevalence of papA/C/EF/G, hlyD, and cnf1 and lower prevalence of ibeA (table 1).
The differences between syndromes according to individual VFs were reflected in aggregate VF scores, which, on average, were lowest among cystitis isolates, intermediate among prostatitis isolates, and highest among pyelonephritis isolates (table 2). The pyelonephritis and prostatitis isolates alike had significantly higher aggregate VF scores than did the cystitis isolates, whereas these 2 groups did not differ significantly by VF score from one another (table 2).
Phylogenetic groups.
The 4 major E. coli phylogenetic groups differed greatly in overall prevalence, with group B2 accounting for 71% of the population versus 13% for group A, 10% for group D, and 6% for group B1 (for group B2 vs. others, P < .001). Aggregate VF scores varied significantly by phylogenetic group, with groups B1 and B2 exhibiting the highest scores and groups A and D exhibiting the lowest (table 2). Despite this, the 3 UTI syndromes did not differ significantly by distribution of phylogenetic groups (data not shown).
DISCUSSION
In the present genetic analysis of E. coli urine isolates from adult patients with cystitis, pyelonephritis, and prostatitis, we have documented syndrome-specific differences in distribution for certain VFs but conservation across syndromes for others, including some of the most prevalent VFs. This finding suggests that, among these syndromes, although there may be syndrome-specific VFs (which implies possible syndrome- or gender-specific differences in pathogenesis and/or host defense systems), there also may be conserved VFs, including certain newly discovered traits. Conceivably, these could be exploited as targets for broadly active preventive measures, such as vaccines.
The cystitis isolates exhibited lower VF scores and similar or lower prevalences for the individual VFs, compared with the pyelonephritis and prostatitis isolates. This finding suggests that (1) cystitis can be caused by strains that are less virulent than those that typically cause the more-invasive syndromes of pyelonephritis and prostatitis and (2) none of the VFs studied here are specifically associated with cystitis. (Of note, differences in fimH sequence [16] or in fim operon regulation [17], which may function as cystitis-specific factors, were not evaluated here.) In contrast, UTI-associated E. coli strains appear to require special features to cause pyelonephritis or prostatitis, with somewhat different VFs being associated with either syndrome. This conclusion is largely consistent with prevailing concepts, according to which a gradient of virulence exists from fecal strains, to those causing cystitis, and to those causing more-invasive UTI syndromes, such as pyelonephritis and prostatitis [7, 9, 10, 18, 19]. The present study extends this paradigm by identifying bmaE, gafD, the K2 kpsM variant, ireA, cvaC, and ibeA as novel pyelonephritis-associated VFs (together with the previously recognized pap and papG allele II) and by confirming pap, hlyD, and cnf1 as prostatitis-associated VFs [9].
Of the 11 VFs that occurred in >50% of isolates, 5 (fyuA, ompT, iroN, malX, and traT) are nontraditionalthat is, they have been recognized only recently and/or have received comparatively little attention to date as possible uro-VFs [11, 2022]. Their broad prevalence, like that of certain of the more-traditional VFs (e.g., fim, kpsM II, sfa/foc, hly, cnf1, and iutA), suggests that they might also make useful targets for protective interventions. However, it should be noted that, for most of the markers studied here, it remains to be determined experimentally whether the traits themselves or others associated with them are actually responsible for virulence.
Interestingly, despite the quite strong associations found between VFs and both syndrome and phylogenetic group, the distribution of phylogenetic groups did not vary significantly by syndrome. This finding suggests that there may be different virulence strata within the phylogenetic groups, with more-virulent strains being selectively concentrated in pyelonephritis and prostatitis and less-virulent strains being selectively concentrated in cystitis, which would be analogous to similar findings among E. coli bacteremia and fecal isolates [23]. This observation also provides further evidence of the supremacy of VF repertoire over phylogenetic background in predicting pathogenic behavior [24]. Also of note, phylogenetic group B1, which within the E. coli reference collection is associated with the absence of most extraintestinal VFs [25], here exhibited the second highest overall virulence level (as reflected in VF scores), nearly equaling that of group B2. This finding is consistent with strong selection factors (i.e., the requirement that isolates have been able to cause acute UTI in an intact host), effectively excluding from the study group B1 strains other than those uncommon variants that have acquired sufficient extraintestinal VFs via horizontal transfer [26].
It is possible that sex differences contributed to the observed among-syndrome differences, since all of the cystitis and pyelonephritis isolates were from women, whereas the prostatitis isolates were from men. Future comparisons of cystitis or pyelonephritis isolates from women versus men could help clarify this issue.
Limitations of the present study include its small numbers of isolates, which limited the power to detect differences. For example, on the basis of the observed prevalence values, the power for the detection of a statistically significant difference in prevalence between cystitis isolates and other isolates was only 63% for iutA (30% observed difference), 52% for ompT (20% observed difference), and 57% for malX (24% observed difference). Future similar studies involving larger populations would be desirable. Another limitation is that the use of multiple comparisons increased the chance of type I errors (i.e., the spurious classification of chance differences as real). We guarded against this by testing pairwise comparisons between individual clinical or phylogenetic groups only for those variables that exhibited a nonrandom distribution according to an initial 3-group (for syndrome) or 4-group (for phylogenetic group) screen. Moreover, in comparisons of individual clinical groups versus all other isolates combined, 11 of the 13 significant differences for individual VFs and both significant differences for VF score yielded P .017, which is the Bonferroni 3-comparisonadjusted significance threshold. Likewise, in comparisons of VF scores involving individual phylogenetic groups versus all other isolates combined, 2 of the 3 significant comparisons yielded P .0125, which is the Bonferroni 4-comparisonadjusted significance threshold. The absence of a fecal control group left undetermined which of the high-prevalence markers were actually more prevalent among the tested isolates than among commensal E. coli and, conversely, whether some of the low-prevalence markers nonetheless might have been more prevalent among the tested isolates than among such controls. Finally, the isolates were from a single locale, from adults who met specific inclusion criteria, and from a limited time interval, possibly limiting generalizability.
In summary, we found in the present study that E. coli isolates from adults with cystitis, pyelonephritis, and prostatitis differed significantly by syndrome in their extended VF profiles, with pyelonephritis and prostatitis isolates exhibiting more VFs overall, plus a higher prevalence of certain specific VFs, than cystitis isolates. Still, some VFs were highly prevalent irrespective of syndrome, and several nontraditional VFs were among those that either differentiated among syndromes or were most broadly prevalent. Notwithstanding the limitations imposed by sample size and multiple comparisons, these findings suggest that, despite the probable existence of syndrome-specific pathogenetic mechanisms and/or host defense systems, conserved VFs may make it possible to develop broadly active preventive measures against UTI.
Acknowledgment
Ann Emery helped to prepare the manuscript.
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