Abstract
Two novel putative Escherichia coli virulence genes, iha and iroN from E. coli (iroNE. coli), were detected in 55 and 39%, respectively, of 67 E. coli isolates from patients with urosepsis. iha and iroNE. coli exhibited divergent associations with other putative virulence genes, phylogenetic markers, host characteristics, and antimicrobial resistance.
The virulent strains of Escherichia coli that cause urinary tract infections (UTIs) and other extraintestinal infections in humans (i.e., extraintestinal pathogenic E. coli [ExPEC]) owe their pathogenic potential largely to the presence of specialized virulence factors (VFs) which are absent from commensal members of the species and which allow ExPEC strains to colonize host mucosal surfaces, injure and invade host tissues, foil host defense mechanisms, and incite an injurious host inflammatory response (4, 5, 9, 20). Currently recognized putative VFs (PVFs) of ExPEC include adhesins, siderophores, toxins, protectins, and invasins, some of which are encoded on pathogenicity-associated islands (PAIs) (2, 7, 8, 17, 24, 26). PAIs are large blocks of established or suspected virulence genes that are inserted into the E. coli genome (often at tRNA loci) and which may provide a mechanisms for coordinate horizontal transfer of virulence genes between lineages within E. coli and even between species (3, 7, 21, 24, 25).
Two recently described PAI-linked PVF genes of ExPEC, iha and iroN from E. coli (iroNE. coli), are of interest both in their own right and because of their potential utility as markers for their respective PAIs of origin (23, 27). iha, an novel nonhemagglutinating adhesin which in vitro confers HeLa cell adherence capability to (nonadhering) E. coli K-12, was first identified as part of a tellurite resistance-associated PAI (termed TAI, the tellurite resistance-adherence-conferring island) from an E. coli O157:H7 isolate from a patient with hemorrhagic colitis (27). iha exhibits nearly perfect sequence identity with the sequenced portion of an open reading frame (ORF) of unknown function (ORF “R4”) from a PAI in archetypal ExPEC strain CFT073 (8, 17). In addition to the iha homologue and multiple other ORFs of unknown significance, this PAI from strain CFT073 also contains an hly (hemolysin) operon and one of the strain's two pap operons (named for pilus associated with pyelonephritis; P fimbriae), which includes the F7-2 allele of papA (the P fimbrial structural subunit gene) and allele II of papG (the P fimbrial digalactoside-specific adhesin gene) (8, 16, 17). Consistent with the hypothesis that iha is a PVF in ExPEC, probes derived from PAI regions immediately adjacent to iha as it occurs in strain CFT073 hybridized significantly more frequent with UTI or bacteremia isolates of E. coli than with commensal E. coli (8).
iroNE. coli, a novel catechole siderophore receptor which exhibits increased expression in urine, was recently identified in archetypal ExPEC strain CP9 as part of a PAI which also includes one of this strain's two pap operons (i.e., the pap operon containing the F14 papA allele and papG allele III), cnf1 (named for cytotoxic necrotizing factor 1), hly, and foc (F1C fimbriae) (16, 23). iroNE. coli, like iha, was also found by probe hybridization to be more prevalent among E. coli isolates from patients with UTI or bacteremia than among commensal strains, consistent with its being a VF in extraintestinal infections (23).
In the present study, we used PCR- and probe hybridization-based gene detection to determine the prevalence of iha and iroNE. coli within a well-characterized collection of E. coli blood isolates from patients with urosepsis. By comparing newly determined results for iha and iroNE. coli with previously determined extended PVF genotypes (15), O:K:H serotypes (13), carboxylesterase B electrophoretic types (which reflect membership in virulence-associated phylogenetic group B2 versus in other phylogenetic groups) (11), antimicrobial resistance profiles (12), and host compromise data (12), we were able to define and compare the associations of iha and iroNE. coli with these other factors. We present data demonstrating that iha and iroNE. coli exhibit widely divergent associations with all of the above factors and are not consistently linked even with PVFs from their own presumed PAIs of origin.
Strains.
The clinical collection studied comprised 67 well-characterized blood culture isolates of E. coli collected from adults with urosepsis in Seattle, Wash., in the mid-1980s (12). The status of these strains with respect to multiple characteristics has been reported previously (10–13, 15). (Eight strains from the 75-member collection which exhibited amplification fingerprints inconsistent with their putative O:K:H serotype and carboxylesterase B type in experiments not yet published were excluded from the present study, leaving 67 evaluable strains as studied herein.)
Molecular detection of iha and iroNE. coli.
iha and iroNE. coli were each detected in duplicate both by PCR and by dot blot hybridization. Primers for iha, which were based on GenBank accession no. AF126104 (27), were 5′-CTGGCGGAGGCTCTGAGATCA-3′ (IHA-F; forward) and 5′-TCCTTAAGCTCCCGCGGCTGA-3′ (IHA-R; reverse) (827-bp product). Primers for iroNE. coli, which were based on GenBank accession no. AF135597 (23), were 5′-AAGTCAAAGCAGGGGTTGCCCG-3′ (IRONEC-F; forward) and 5′-GACGCCGACATTAAGACGCAG-3′ (IRONEC-R; reverse) (665-bp product). Probes were generated and PCR labeled as previously described (14) using the same primers as for PCR-based gene detection. Amplification and blotting procedures were as previously described (14, 15). Appropriate positive and negative controls were included in each assay. Discrepancies between duplicate blot or PCR determinations were investigated with additional replicates as needed.
Phylogenetic analysis of PVF distribution.
Strains were sorted according to carboxylesterase B electrophoretic type (i.e., B2 or B1) and then by O:K:H serotype within each carboxylesterase B type. Serotypes with two or more representatives in the study population were considered to exhibit homogeneity with respect to a particular PVF if the PVF was present in or absent from all representatives of the serotype or was present in all representatives but one (if three or more representatives total).
Statistical analyses.
Comparisons of proportions were tested using Fisher's exact test. Comparisons of prevalence within the same population were tested using McNemar's test (6). Comparisons of antibiotic resistance scores and host compromise scores were tested using the Mann-Whitney U test. The criteria for the different categories of statistical significance follow: P > 0.10, indifferent association; P ≤ 0.10, borderline significant trend; P < 0.05, possible statistical significance, and P < 0.01, statistical significance. Associations of iha and iroNE. coli with another PVF were considered to agree when they were in the same direction and exhibited P values of <0.10, to somewhat disagree (regardless of direction) when one exhibited a P value of <0.05 and the other exhibited a P value of >0.10, and to strongly disagree when the associations were in opposite directions and both exhibited P values of <0.10.
Prevalence of iha and iroNE. coli.
iha was detected in 37 (55%) and iroNE. coli in 26 (39%) of the 67 urosepsis isolates (0.05 < P < 0.10, McNemar's test) (Table 1). Probe and PCR yielded identical results for each gene (not shown). Overall, iha and iroNE. coli were indifferently associated with one another (P > 0.10).
TABLE 1.
Prevalence of iha and iroNE. coli and other genes among 67 urosepsis isolatesa
| Strain | CBTb | O:K:H serotype | Adhesin
|
Toxin
|
Siderophore
|
Protectin or invasin
|
Miscellaneous (PAI)
|
|||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| F-type | G allele(s) | sfa/ focDE | sfaS | focG | afa/ draBC | iha | bmaE | gafD | hlyA | cnf1 | cdtB | iroNE. coli | fyuA | iutA | kpsMT II | kpsMT III | K1 | K5 | rfc | cvaC | traT | ibeA | ||||
| H16 | B2 | O1:K1:H7 | F11 | II | − | − | − | − | − | − | − | − | − | − | − | + | − | + | − | + | − | − | − | + | − | + |
| H38 | B2 | O1:K1:H7 | F11 | II | − | − | − | − | − | − | − | − | − | − | − | + | − | + | − | + | − | − | − | + | − | + |
| V15 | B2 | O1:K1:H7 | F11 | II | − | − | − | − | − | − | − | − | − | − | − | + | + | + | − | + | − | − | + | + | − | + |
| V23 | B2 | O1:K1:H7 | F11 | II | − | − | − | − | − | − | − | − | − | − | − | + | − | + | − | + | − | − | − | − | − | + |
| 2H18 | B2 | O1:K1:H7 | F11 | II | − | − | − | − | − | − | − | − | − | − | − | + | − | + | − | + | − | − | − | + | − | + |
| H15 | B2 | O2:K1:H7 | F11 | II | − | − | − | − | − | − | − | − | − | − | + | + | + | + | − | + | − | − | + | + | − | + |
| H25 | B2 | O2:K1:H7 | F11 | II | − | − | − | − | − | − | − | − | − | − | − | + | − | + | − | + | − | − | − | + | − | + |
| PM6 | B2 | O2:K1:H7 | F11 | II | − | − | − | − | − | − | − | − | − | − | + | + | + | + | − | + | − | − | + | + | − | + |
| 2P6 | B2 | O2:K1:H7 | F11 | II | − | − | − | − | − | − | − | − | − | + | + | + | + | + | − | + | − | − | + | + | − | + |
| H35 | B2 | R:K1:H7 | F11 | II | − | − | − | − | − | − | − | − | − | − | − | + | − | + | − | + | − | − | − | + | − | + |
| V27 | B2 | O2:K5:H1 | F10, F14 | II | + | − | + | − | + | − | − | − | − | + | + | + | + | + | − | − | + | − | − | − | − | + |
| H19 | B2 | O2:K7:H− | F10, F14 | II, III | + | − | + | − | + | − | − | + | + | + | + | + | + | + | − | − | + | − | − | − | − | + |
| V6 | B2 | O2:K7:H− | F10, F14 | II | + | − | + | − | + | − | − | + | + | + | + | + | + | + | − | − | + | − | − | − | − | + |
| V24 | B2 | O2:K7:H− | F7-1, F10 | II | − | − | − | − | + | − | − | − | − | + | − | + | + | + | − | − | + | − | − | + | − | + |
| H1 | B2 | O2:K7:H1 | F10, F12, F15 | II | − | − | − | − | + | − | − | − | − | + | − | + | + | + | − | − | + | − | − | + | − | + |
| V31 | NAc | O4:K12:H1 | F10, F11 | II | + | − | − | − | − | − | − | + | + | − | + | + | − | + | − | − | + | + | − | + | − | + |
| PM8 | B2 | O4:K12:H− | F10, F11 | II | + | − | − | − | + | − | − | + | − | − | + | + | + | + | − | − | + | + | − | + | − | + |
| V7 | B2 | R:K12:H1 | F10, F11, F16 | II | − | − | − | − | + | − | − | + | + | − | − | + | + | + | − | − | + | + | − | + | − | + |
| H26 | B2 | O6:K2:H1 | F7-2 | II | + | − | + | − | + | − | − | + | − | − | + | + | + | −d | − | − | − | − | − | + | − | + |
| V5 | B2 | O6:K2:H1 | F7-2 | II | − | − | − | − | + | − | − | + | − | − | − | + | + | −d | − | − | − | − | − | + | − | + |
| U3 | B2 | O6:K2:H1 | F7-2 | II | + | − | + | − | + | − | − | + | − | − | + | + | + | −d | − | − | − | − | − | + | − | + |
| PM4 | B2 | O6:K2:H1 | F7-2 | II | + | − | + | − | + | − | − | + | − | − | + | + | + | −d | − | − | − | − | − | − | − | + |
| PM5 | B2 | O6:K2:H1 | F7-2 | II | + | − | + | − | + | − | − | + | − | − | + | + | + | −d | − | − | − | − | − | − | − | + |
| 2H4 | B2 | O6:K2:H1 | F7-2 | II | − | − | − | − | + | − | − | + | − | − | − | + | + | −d | − | − | − | − | − | + | − | + |
| 2H24 | B2 | O6:K2:H1 | F7-2 | II | − | − | − | − | + | − | − | + | − | − | − | + | + | −d | − | − | − | − | − | + | − | + |
| V1 | B2 | O6:H1 | F7-2 | II | + | − | + | − | + | − | − | + | − | − | + | + | + | −d | − | − | − | − | − | − | − | + |
| V3 | B2 | O6:K2:H? | F7-2 | II | − | − | − | − | − | − | − | + | − | − | − | + | + | −d | − | − | − | − | − | − | − | + |
| V16 | B2 | R:K2:H1 | F7-2 | II | + | − | + | − | + | − | − | + | −d | − | + | + | + | −d | − | − | − | − | − | + | − | + |
| V19 | B2 | O6:K5:H− | F10, F13 | II | + | − | + | − | + | − | − | + | + | − | + | + | + | + | − | − | + | − | − | + | − | + |
| V22 | B2 | O6:K5:H− | F10, F13 | II | + | − | + | − | + | − | − | + | + | − | + | + | + | + | − | − | + | − | − | − | − | + |
| H3 | B2 | O6:K53:H7 | F11 | II | − | − | − | − | − | − | − | − | − | − | − | + | − | + | − | + | − | − | − | + | − | + |
| U5 | B2 | O6:K53:H7 | F10, F14 | III | + | − | − | − | − | − | − | + | + | − | + | + | − | + | − | − | + | − | − | − | + | + |
| U7 | B2 | O6:K+?:H− | F48 | III | + | + | − | − | − | − | − | + | + | − | + | + | − | + | − | − | + | − | − | − | − | + |
| H9 | B2 | O12:K1:H6 | F9, F12, F48 | II | − | − | − | − | + | − | − | + | − | − | − | + | + | + | − | + | − | − | − | + | − | + |
| U4 | B2 | O12:K1:H6 | F12, F48 | II | − | − | − | − | + | − | − | + | − | − | − | + | + | + | − | + | − | − | − | + | − | + |
| PM2 | B2 | R:K1:H6 | NEGe | − | + | + | + | − | − | − | − | + | + | − | + | + | − | + | − | + | − | − | − | + | + | + |
| H7 | B2 | O16:K1:H− | F7-1 | II | − | − | − | − | + | − | − | − | − | − | − | + | + | + | − | + | − | − | − | + | + | + |
| 2H25 | B2 | O18ac:K1:H7 | F10 | III | + | + | − | − | − | − | − | + | + | − | + | + | − | + | − | + | − | − | − | + | + | + |
| H5 | B2 | O18ac:K5:H− | F8, F10 | II | + | − | + | − | + | − | − | + | − | − | + | + | + | + | − | − | + | − | − | − | − | + |
| 2H21 | B2 | O21:K100:H5 | F7-2 | II | − | − | − | + | + | − | − | + | − | − | − | + | + | − | − | − | − | − | − | + | − | + |
| V21 | B2 | O75:K5:H5 | F10 | − | − | − | − | − | + | − | − | − | − | − | − | + | + | + | − | − | + | − | − | − | − | + |
| 2H19 | B2 | O75:K5:H5 | F8, F10 | II | − | − | − | − | + | − | − | − | − | − | − | + | + | + | − | − | + | − | − | + | − | + |
| V8 | B2 | O75:K5:H− | F10 | − | − | − | − | − | + | − | − | − | − | − | − | + | + | + | − | − | + | − | − | − | − | + |
| PM3 | B2 | O75:K+:H− | F10 | − | − | − | − | + | + | − | − | − | − | − | − | + | + | + | − | − | + | − | − | + | − | + |
| V14 | B1 | O2:K5:H− | F10, F48 | II | − | − | − | − | + | − | − | + | − | − | − | + | + | + | − | − | + | − | − | + | − | + |
| V20 | B1 | O7:K1:H− | F10 | II | − | − | − | − | + | − | − | − | − | − | − | + | + | + | − | + | − | − | − | − | − | − |
| V29 | B1 | O7:K1:H− | F40 | II | − | − | − | − | + | − | − | − | − | − | − | + | + | + | − | + | − | − | − | − | − | − |
| PM7 | B1 | O7:H− | F10 | II | − | − | − | − | + | − | − | − | − | − | − | + | + | + | − | + | − | − | − | − | − | − |
| H27 | B1 | O8:K27:H− | F11 | − | − | − | − | − | − | + | − | − | − | − | − | + | + | − | − | − | − | − | − | + | − | − |
| U6 | B1 | O8:K27:H− | NEG | II | − | − | − | − | − | + | − | − | − | − | + | + | + | − | − | − | − | − | + | + | − | − |
| U1 | B1 | O8:K44:H9 | NEG | − | − | − | − | − | − | − | − | − | − | − | + | + | + | − | − | − | − | − | + | + | − | − |
| V28 | B1 | O8:K+:H− | NEG | − | − | − | − | − | − | − | − | − | − | − | − | − | + | − | − | − | − | − | − | + | − | − |
| V9 | B1 | O9:K34:H− | NEG | − | − | − | − | − | − | − | − | − | − | − | + | + | + | − | − | − | − | − | + | + | − | − |
| PM9 | B1 | O9:K34:H− | NEG | − | − | − | − | − | − | + | + | − | − | − | + | + | + | − | − | − | − | − | + | + | − | − |
| V26 | B1 | O9:K36:H19 | F11 | II | − | − | − | − | − | − | − | − | − | − | + | + | + | − | − | − | − | − | + | + | − | − |
| V32 | B1 | O15,O40:K+ | F14 | III | − | − | − | − | − | − | − | + | + | − | − | + | + | + | − | − | + | − | − | + | − | − |
| 2H17 | B1 | O15:K52:H1 | F16 | II | − | − | − | − | + | − | − | − | − | − | − | + | + | + | − | − | + | − | − | − | − | − |
| 2P9 | B1 | O15:K52:H1 | F16 | II | − | − | − | − | + | − | − | − | − | − | − | + | + | + | − | − | + | − | − | − | − | − |
| V11 | B1 | O17:K53:H18 | NEG | − | − | − | − | − | − | − | − | − | − | − | − | − | − | + | − | − | + | − | − | − | − | − |
| 2H16 | B1 | O25:K2:H2 | F16 | II | − | − | − | + | + | − | − | + | − | − | − | + | + | −d | − | − | − | − | − | + | − | − |
| H8 | B1 | O25:K2:H2 | F16 | II | − | − | − | − | + | − | − | + | − | − | − | + | + | −d | − | − | − | − | − | − | − | − |
| H17 | B1 | O25:K16:H4 | NEG | − | − | − | − | − | − | − | − | − | − | − | − | + | + | + | − | − | + | − | − | + | − | + |
| PM1 | B1 | O64:H21 | NEG | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − | − |
| V10 | B1 | O77:K54:H18 | NEG | − | − | − | − | − | − | + | − | − | − | − | − | − | + | − | + | − | − | − | − | + | − | − |
| V12 | B1 | O84:H− | NEG | − | − | − | − | − | − | − | − | − | − | − | − | + | − | − | − | − | − | − | − | − | − | − |
| H2 | B1 | O157:H32 | F16 | II | − | − | − | + | + | − | − | − | − | − | − | + | + | + | − | − | + | − | − | + | − | + |
| H18 | B1 | O157:K52:H45 | F16 | II | − | − | − | − | + | − | − | − | − | − | − | + | + | + | − | − | + | − | − | − | − | + |
The presence (+) or absence (−) of the indicated gene in isolates is shown. The iha and iroN data are shown in bold type for emphasis. Data are adapted from reference 15, with permission.
CBT, carboxylesterase B type.
NA, not applicable.
Negative by PCR but positive by probe for kpsMT II.
NEG, no F-type adhesin.
Associations with other PVFs.
iha and iroNE. coli each exhibited positive, negative, and indifferent associations with diverse other PVFs (Table 2). Consistent with the occurrence of iha in archetypal uropathogenic strain CFT073 on a PAI that also contains papG allele II and hly (8, 15), iha was strongly associated with papG allele II (and all other pap elements except papG allele III) and with hlyA (Table 2). Similarly, consistent with the discovery of iroNE. coli in archetypal uropathogenic strain CP9 on a PAI that also contains papG allele III, hly, cnf, and foc, iroNE. coli was positively associated with each of these traits, despite not being significantly associated with other pap elements (Table 2).
TABLE 2.
Distribution of other PVFs according to the presence or absence of iha and iroNE. coli
| Associated PVFa | No. (% of total) | Prevalence (no. [%]) of associated PVF
|
|||||
|---|---|---|---|---|---|---|---|
|
iha
|
iroNE. coli
|
||||||
| Present (n = 37) | Absent (n = 30) | P valueb | Present (n = 26) | Absent (n = 41) | P value | ||
| papAH | 55 (82) | 35 (95) | 20 (67) | 0.004 | 22 (85) | 33 (80) | |
| papG | 53 (79) | 34 (92) | 19 (63) | 0.006 | 22 (85) | 31 (76) | |
| Allele II | 49 (73) | 34 (92) | 15 (50) | <0.001 | 19 (73) | 30 (73) | |
| Allele III | 5 (7) | 1 (5) | 4 (13) | 4 (15) | 1 (2) | 0.07 | |
| sfa/foc | 18 (27) | 13 (35) | 5 (17) | 18 (69) | 0 | <0.001 | |
| sfaS | 3 (4) | 0 | 3 (10) | (0.08) | 3 (12) | 0 | 0.054 |
| focG | 13 (19) | 12 (33) | 1 (3) | 0.004 | 13 (50) | 0 | <0.001 |
| afa/draBC | 4 (6) | 4 (11) | 0 | 0 | 4 (10) | ||
| bmaE | 4 (6) | 0 | 4 (13) | (0.036) | 2 (8) | 2 (5) | |
| gafD | 1 (1) | 0 | 1 (3) | 1 (4) | 0 | ||
| hlyA | 29 (43) | 22 (59) | 7 (23) | 0.006 | 17 (65) | 12 (29) | 0.005 |
| cnf1 | 12 (18) | 6 (16) | 6 (20) | 10 (38) | 2 (5) | <0.001 | |
| cdtB | 6 (9) | 5 (14) | 1 (3) | 4 (15) | 2 (5) | ||
| fyuA | 63 (94) | 37 (100) | 26 (87) | 0.036 | 26 (100) | 37 (90) | |
| iutA | 52 (77) | 37 (100) | 15 (50) | <0.001 | 21 (81) | 31 (76) | |
| iut | |||||||
| Plasmid | 13 (19) | 1 (3) | 12 (40) | (<0.001) | 8 (31) | 5 (12) | |
| Chromosomal | 39 (58) | 36 (97) | 3 (10) | <0.001 | 13 (50) | 26 (63) | |
| kpsMT II | 56 (84) | 36 (97) | 20 (67) | 0.002 | 21 (81) | 35 (85) | |
| K1 | 19 (28) | 6 (16) | 13 (43) | (0.03) | 5 (19) | 14 (34) | |
| “K2” | 12 (18) | 11 (30) | 1 (3) | 0.008 | 6 (23) | 6 (15) | |
| “K5” | 25 (33) | 19 (51) | 6 (20) | 0.01 | 10 (38) | 15 (37) | |
| kpsMT III | 1 (1) | 0 | 1 (3) | 0 | 1 (2) | ||
| rfc | 3 (4) | 2 (5) | 1 (13) | 2 (8) | 1 (2) | ||
| ibeA | 4 (6) | 1 (3) | 3 (10) | 3 (12) | 1 (2) | ||
| cvaC | 9 (13) | 0 | 9 (30) | (<0.001) | 8 (31) | 1 (2) | 0.002 |
| traT | 44 (66) | 21 (57) | 23 (77) | 16 (62) | 28 (68) | ||
| PAI marker | 48 (72) | 30 (81) | 18 (60) | 0.10 | 21 (81) | 27 (66) | |
| iroNE. coli | 29 (39) | 13 (35) | 13 (43) | NAc | NA | ||
As determined by PCR only (K1 and “K5”), probe only (plasmid aerobactin and kpsMT II), or both PCR and probe (all other PVFs). “K2” represents isolates that were blot positive but PCR negative for kpsMT II. “K5” represents isolates with non-K1, non-K2 group II capsules. Plasmid aerobactin status was determined previously (12). Chromosomal aerobactin status was inferred from results for iutA and plasmid aerobactin. No strain had nfaE; all strains had fimH. Results for papC and papEF were similar to those for papAH and papG.
P values (by Fisher's exact test) shown only where <0.10. Parentheses indicate negative association of iha with particular PVF.
NA, not applicable (comparison with self).
However, associations of iha and iroNE. coli with other genes from their respective PAIs in strains CFT073 and CP9 were not categorical. For example, iha exhibited only a marginal statistical association with malX, the PAI marker used in the present study (Table 2), even though in strain CFT073 malX occurs on the same PAI as iha and is separated from iha by <2 kb (8). iha occurred in seven strains that lacked the PAI marker, whereas the PAI marker occurred in 19 strains that lacked iha (Table 1). Similarly, despite its association with papG allele III, iroNE. coli occurred in four pap-negative strains and in 19 strains that contained papG allele II rather than papG allele III, whereas papG allele III occurred in one strain without iroNE. coli (Tables 1 and 2). An analogous discordance was also noted for both iha and iroNE. coli with respect to hly (Tables 1 and 2).
Not only were iha and iroNE. coli inconsistently associated with PVFs from their respective PAIs of origin, but they exhibited divergent associations with other PVFs. iha and iroNE. coli were concordantly positively associated only with focG and hlyA, and exhibited no concordant negative associations (Table 3). In contrast, they exhibited discordant associations with 20 of the remaining PVFs (Table 4). (Of note, whereas iha was strongly negatively associated with plasmid iut [aerobactin] and cvaC [colicin V], iroNE. coli was strongly positively associated with plasmid aerobactin and exhibited a trend toward an association with cvaC [Tables 2 and 3].) Thus, iha and iroNE. coli differed more often than they agreed with respect to associations with other PVF genes (Table 3).
TABLE 3.
Summary of agreement and disagreement between iha and iroNE. coli with respect to associations with other PVFs:a
| Agree (positive vs positiveb) | Somewhat disagree
|
Strongly disagree (Negative vs positivef) | ||
|---|---|---|---|---|
| Positive vs indifferentc | Indifferent vs positived | Negative vs indifferente | ||
| focG, hlyA | papAH, papG allele II, fyuA, iutA, kpsMT II, “K2,” “K5,” PAI | papG allele IIIg, sfa/foc, cnf1 | bmaE, K1, traTh, plasmid iut | sfaSg,h, cvaC |
Of the PVFs present in two or more strains each, iha and iroNE. coli were both indifferently associated with afaBC, cdtB, gafD, rfc, and ibeA but exhibited opposed associative trends with all but rfc. PVFs which were present in 0, 1, or all 75 of the urosepsis isolates, including gafD, nfaE, fimH, and kpsMT III, were excluded from the present analysis.
Positive associations of both iha and iroNE. coli with other PVFs.
Association with other PVFs: positive (iha), indifferent (iroNE. coli) (this also applies to papC papEF, and papG [not shown]).
Association with other PVFs: indifferent (iha), positive (iroNE. coli).
Association with other PVFs: negative (iha), indifferent (iroNE. coli).
Association with other PVFs: negative (iha), positive (iroNE. coli).
For iroNE. coli, 0.05 < P < 0.10.
For iha, 0.05 < P < 0.10.
TABLE 4.
Distribution of papA alleles according to the presence or absence of iha and iroNE. coli
| Associated papA allelea | No. (% of total) | Prevalence (no. [%]) of associated papA allele
|
|||||
|---|---|---|---|---|---|---|---|
|
iha
|
P valueb |
iroNE. coli
|
P value | ||||
| Present (n = 37) | Absent (n = 30) | Present (n = 26) | Absent (n = 41) | ||||
| F7-1 | 2 (3) | 2 (5) | 0 | 0 | 2 (5) | ||
| F7-2 | 11 (16) | 10 (27) | 1 (3) | 0.017 | 6 (23) | 5 (12) | |
| F8 | 2 (3) | 2 (5) | 0 | 1 (4) | 1 (2) | ||
| F9 | 1 (1) | 1 (3) | 0 | 0 | 1 (2) | ||
| F10 | 18 (27) | 15 (41) | 3 (10) | 0.006 | 9 (35) | 9 (22) | |
| F11 | 15 (22) | 1 (3) | 14 (47) | (<0.001)c | 5 (19) | 10 (24) | |
| F12 | 3 (4) | 3 (8) | 0 | 0 | 3 (7) | ||
| F13 | 2 (3) | 2 (5) | 0 | 2 (8) | 0 | ||
| F14 | 6 (9) | 4 (11) | 2 (7) | 5 (19) | 1 (2) | 0.029 | |
| F15 | 1 (1) | 1 (3) | 0 | 0 | 1 (2) | ||
| F16 | 8 (12) | 8 (22) | 0 | 0.007 | 1 (4) | 7 (17) | |
| F48 | 4 (6) | 3 (8) | 1 (3) | 1 (4) | 3 (7) | ||
As determined by PCR (16). papA alleles F7-1 through F16 correspond with the traditionally recognized P fimbrial F serotypes. F48 is a recently described novel papA allele.
P values (by Fisher's exact test) shown only where <0.10.
Parentheses indicate a negative association of iha with F11.
Associations with papA alleles.
iha and iroNE. coli differed substantially with respect to their associations with individual papA alleles (Table 4). iha was significantly associated with the F7-2 allele, which is present in the PAI of strain CFT073 that contains iha, and with the F10 and F16 alleles, whereas it was significantly negatively associated with the F11 allele (Table 4). In contrast, iroNE. coli was positively associated with the F14 allele (P = 0.029), which is present in the iroNE. coli-containing PAI from strain CP9. iha and iroNE. coli exhibited (nonsignificant) opposing associative trends with all the remaining papA alleles except F13 (Table 4).
Phylogenetic distribution of iha and iroNE. coli.
Although iha and iroNE. coli were both somewhat more prevalent among carboxylesterase B2 strains, they exhibited divergent patterns of distribution with respect to individual O:K:H serotypes (Table 1). In contrast to the more homogeneously distributed iha, iroNE. coli was sporadically distributed, occurring in many different serotypes but rarely in all members of any one serotype (Table 1). Although both PVFs occurred in a similar proportion of the 14 most prevalent O:K:H serotypes (i.e., 10 of 14 for iha versus 9 of 14 for iroNE. coli), iha satisfied criteria for homogeneous distribution (as defined above [phylogenetic analysis]) in all 14 serotypes, as compared with only seven for iroNE. coli (P < 0.02, McNemar's test) (Table 1).
Among the B2 strains, iha was almost universally prevalent except among O1:K1:H7 and O2:K1:H7 strains, from which it was notably absent (P < 0.001 versus other B2 strains). In contrast, although iroNE. coli was also absent from the five O1:K1:H7 strains, it occurred in three of the five (iha-negative) O2:K1:H7 strains and was absent from all four (iha-positive) O75 strains (Table 1). Likewise, among the B1 strains, whereas iha was largely confined to serotypes O7:K1, O15:K52, O25:K2, and serogroup O157 (P < 0.001 versus other B1 strains), iroNE. coli was confined to strains of serogroups O8 and O9 (P < 0.001 versus other B1 strains), which resulted in a significant negative association of iha with iroNE. coli among B1 strains (P = 0.004) (Table 1).
Host compromise and antimicrobial resistance.
iha and iroNE. coli differed with respect to their associations (or lack thereof) with host compromise and antimicrobial resistance. iha was consistently more prevalent among strains from noncompromised hosts than those from compromised hosts, with the strength of the association depending on the type of host compromise (Table 5). In contrast, iroNE. coli was more prevalent among strains from compromised hosts (Table 5). Host compromise scores were significantly lower among iha-positive strains (mean, 0.8 [range, 0–2]) than among iha-negative strains (mean, 1.4 [range, 0–3]; P = 0.002) but did not differ between iroNE. coli-positive and -negative strains (means of 1.0 and 1.1, respectively [range for both groups, 0–3]; P > 0.10). Among strains from noncompromised hosts, iha was always somewhat or significantly more prevalent than was iroNE. coli, whereas among strains from compromised hosts, iha and iroNE. coli were similarly prevalent (Table 5).
TABLE 5.
Distribution of iha and iroNE. coli according to the presence or absence of host compromise
| Host compromise condition | Prevalence (%) of ihaa
|
Prevalence (%) of iroNE. coli
|
||||
|---|---|---|---|---|---|---|
| Host condition
|
P valueb | Host condition
|
P value | |||
| Present | Absent | Present | Absent | |||
| Medical illnessc | 40 | 64f | (0.076) | 48 | 33f | |
| Diabetes mellitus | 50 | 56g | 50 | 37g | ||
| Cancer | 31 | 61f | (0.065) | 54 | 35f | |
| Immunosuppression | 46 | 57f | 69 | 31f | 0.02 | |
| Uremia | 25 | 59g | 38 | 39g | ||
| Urinary tract abnormalityd | 47 | 62 | 37 | 41 | ||
| Upper urinary tract | 13 | 56h | (0.018) | 38 | 39h | |
| Urinary tract instrumentation | 25 | 65h | (0.009) | 38 | 39h | |
| Any of abovee | 44 | 77f | (0.018) | 42 | 32f | 0.03 |
The denominator for prevalence values was the number of patients with or without the indicated compromising condition.
For comparisons between patients with versus patients without the indicated type of host compromise, P values (by Fisher's exact test) shown only where <0.10. (Parentheses indicate negative associations of iha with host compromise.)
Including diabetes mellitus, cancer, immunosuppression, and uremia.
Preexisting functional or anatomic abnormality of the urinary tract.
Any of the above conditions, i.e., medical illness or urinary tract abnormality or instrumentation.
Among patients without the indicated host compromise condition, for prevalence of iha versus iroNE. coli, P < 0.02 (McNemar's test). Among patients with the indicated host compromise condition, P > 0.10 for all comparisons of iha versus iroNE. coli.
Among patients without the indicated host compromise condition, for prevalence of iha versus iroNE. coli, 0.05 < P < 0.10 (McNemar's test). Among patients with the indicated host compromise condition, P > 0.10 for all comparisons of iha versus iroNE. coli.
Among patients without the indicated host compromise condition, for prevalence of iha versus iroNE. coli, and P < 0.05 (McNemar's test). Among patients with the indicated host compromise condition, P > 0.10 for all comparisons of iha versus iroNE. coli.
Resistance to one or more antimicrobial agent was less common among iha-positive strains than among iha-negative strains (38 versus 67%; P = 0.03), whereas it was comparably prevalent among iroNE. coli-positive and -negative strains (46 versus 54%; P > 0.10). Similarly, antimicrobial resistance scores were lower among iha-positive strains than among iha-negative strains (mean of 0.8 [range, 0–7] versus 3.0 [range, 0–10]; P = 0.002) but were similar among iroNE. coli-positive and -negative strains (mean of 2.0 [range, 0–10] versus 1.7 [range, 0–9]; P > 0.10).
Implications of findings.
That iha and iroNE. coli were both significantly associated with other PVF genes from their respective PAIs in archetypal ExPEC strains CFT073 (iha) and CP9 (iroNE. coli) suggests, as a first approximation, that iha and iroNE. coli occur consistently on the same (or similar) PAIs throughout the ExPEC population. Consequently, the striking differences documented in the present study between iha and iroNE. coli with respect to their individual associations with other bacterial and host characteristics demonstrate that such associations are likely to be specific to individual PAIs and/or PVF genes rather than being common to all PAIs or to PAI-linked PVF genes in general. This implies that discovery that a particular PVF of ExPEC is linked to a PAI is insufficient to predict its associations with other bacterial and host characteristics, which instead can be discovered only through specific study of the particular PVF itself or the individual PAI.
However, our findings also suggest that the concept of an individual PAI as a discrete entity which can be tracked through the ExPEC population may itself be illusory (1, 2, 7, 8, 23, 26). The associations observed in the present study of iha and iroNE. coli with other PVFs from their respective PAIs in source strains CFT073 and CP9, although statistically significant, were not absolute, evidence which suggests that the corresponding PAIs may have undergone considerable recombination over time, with acquisition or deletion of various PVF genes, or that each PVF has independently entered multiple different PAIs. Specific evidence of the fluidity of PAIs was found in the discordant occurrence of iha and the PAI marker malX (Tables 1 and 2), genes which on their shared PAI in strain CFT073 are located within 2 kb of one another (8). Thus, it is likely that at the same time that PAIs are generating diversity within the species by introducing new genetic material into disparate lineages via horizontal transfer (7), PAIs themselves are undergoing diversification through deletion and insertion of PVF genes, some of which may be more mobile (or labile) than are the PAIs themselves. PAIs thus may exist as continuously evolving mosaic constructs, a phenomenon which would be predicted to confound efforts to define a reliable marker for the PAIs of ExPEC in general or for any particular PAI. An example of this phenomenon may be the apparent insertion of the group III capsule genes into the group II capsule locus in strain CP9 (24). Similarly, TAI, the PAI on which iha was first identified, was conserved in structure in some, but not all, diarrheagenic E. coli tested (27).
The respective patterns of phylogenetic distribution of iha and iroNE. coli suggest divergent evolutionary histories and mechanisms of horizontal mobility, iha was present in almost all strains of carboxylesterase B type B2 except for those of serotypes O1:K1:H7 and O2:K1:H7 and was also highly prevalent in selected lineages among the non-B2 strains. This is consistent with early acquisition of iha by the B2 phylogenetic group soon after the differentiation of B2 strains from other E. coli (18) but after the O1:K1:H7 and O2:K1:H7 clonal group (which constitutes a discrete evolutionary cluster within the B2 group [19]) had split off from a common B2 ancestor. In this scenario, the appearance of iha among non-B2 strains would require the independent acquisition of iha by non-B2 lineages through horizontal transfer either from a B2 source or from an unknown external source. As an alternative scenario, iha could have entered E. coli prior to the differentiation of the B2 phylogenetic group and subsequently have been deleted from the ancestor of the O1:K1:H7 and O2:K1:H7 branch of the B2 group after the differentiation of this branch from other B2 strains. In this scenario, iha could have been inherited vertically from a common ancestor by both B2 and certain non-B2 strains, for example, those of closely related phylogenetic group D (18).
In contrast to iha, the sporadic appearance of iroNE. coli in many different serotypes strongly suggests either multiple horizontal acquisition events or multiple scattered deletions of iroNE. coli within otherwise iroNE. coli-positive lineages. Although in strain CP9 iroNE. coli occurs on a genomic PAI (23), the possibility of plasmid-associated transfer of iroNE. coli in some strains was suggested in the present study by the striking statistical association of iroNE. coli with cvaC (colicin V), which occurs on large conjugative plasmids (28–30). Precedent in ExPEC for a siderophore system which occurs variously on colicin V plasmids and on the genome is provided by the aerobactin system, which has been postulated to represent an extinct transposon (22, 28). The finding that in strain CP9 iroNE. coli is flanked by IS1230 sequences (23) suggests the possibility of independent mobility of iroNE. coli, which would be consistent with exchange of iroNE. coli between genome and plasmids.
Although direct genetic linkage is a plausible explanation for the observed statistical associations between different putative virulence genes, other explanations should be considered. Alternative mechanisms include coselection for the associated traits (independent of these traits' genetic relationship to one another), and coselection for unmeasured traits that are genetically linked to the statistically associated putative virulence genes. Confirmation of actual genetic linkage between putative virulence genes would require direct genetic analyses. The results of the present study can assist the design of such analyses by suggesting which linkages should be sought experimentally.
The significantly higher prevalence of iha found in the present study among E. coli bacteremia isolates from noncompromised hosts than in those from compromised hosts suggests that iha (or VFs linked to iha) may assist in overcoming host defenses that are breached in patients with the types of compromise studied (12). In contrast, our observation that the prevalence of iroNE. coli increased in the presence of host compromise suggests that unlike iha, iroNE. coli should be as good a target for an anti-VF intervention among compromised hosts as among noncompromised hosts. The absence of an association of iroNE. coli with antimicrobial resistance suggests that iroNE. coli should also remain as effective a target for an anti-VF intervention among multidrug-resistant E. coli as among susceptible strains, whereas iha, which was negatively associated with antimicrobial resistance, would be most useful as a target among antimicrobial-susceptible strains.
It should be noted that the present study population represented a single geographical locale, a single clinical syndrome, and a time period of several years in the mid-1980s. Analysis of strains from other locales, clinical syndromes, and time periods for iha and iroNE. coli would provide a valuable complement to the present study.
Acknowledgments
This work was supported in part by VA Merit Review (J.R.J. and T.A.R.); National Institutes of Health grants DK-47504 (J.R.J.), AI-42059 (T.A.R.), and AI-38419 (P.I.T.); United States Department of Agriculture grant 94-03953 (P.I.T.); and a National Cattlemen's Beef Association grant (P.I.T.).
Diana Owensby and Ann Emery helped prepare the manuscript.
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