J Med Microbiol 56 (2007), 1363-1369; DOI: 10.1099/jmm.0.47262-0
© 2007 Society for General Microbiology
ISSN 1473-5644
Genotypic analyses of uropathogenic Escherichia coli based on fimH single nucleotide polymorphisms (SNPs)
Sara Y. Tartof1,
Owen D. Solberg2 and
Lee W. Riley1,3
1 Division of Epidemiology, School of Public Health, University of California, Berkeley, CA, USA
2 Department of Integrative Biology, University of California, Berkeley, CA, USA
3 Division of Infectious Diseases, University of California, Berkeley, CA, USA
Correspondence
Lee W. Riley
lwriley{at}berkeley.edu
Received 28 February 2007
Accepted 25 June 2007
The application of genotyping techniques for subtyping uropathogenic Escherichia coli has contributed to better understanding of the epidemiology of community-acquired urinary tract infection (UTI). However, the current techniques are hampered by limited reproducibility, poor discriminatory power, labour-intensive performance or high cost. A screening test that is sequence-based would provide an inexpensive, reproducible way to subtype E. coli isolates. Such a test, if also discriminatory, would be highly useful for epidemiological studies. The discriminatory ability of 12 putative virulence genes (fimH, fliD, fliM, iha, motA, papA/H, kpsMTII, fepE, fimA, flgA, malG, purD) was evaluated based on single nucleotide polymorphisms (SNPs) in nine uropathogenic E. coli isolates, all previously found to belong to a single multilocus sequence type (MLST) complex (ST69). An additional 25 epidemiologically well-characterized E. coli isolates belonging to 12 distinct MLST clonal complexes were analysed for fimH SNP. None of the 12 genes except fimH were able to further discriminate the nine ST69-complex strains. Isolates belonging to the 12 non-ST69 MLST groups were separated into 10 fimH SNP subgroups. While fimH SNP analysis may not be an appropriate phylogenetic method, it offers discriminatory power similar to that of MLST and could be used as a simple, inexpensive screening test for epidemiological studies of uropathogenic E. coli.
Abbreviations: ERIC-PCR, Enterobacterial Repetitive Intergenic Consensus-PCR; ExPEC, extraintestinal pathogenic Escherichia coli; MLST, multilocus sequence type; SNP, single nucleotide polymorphism; UTI, urinary tract infection.
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INTRODUCTION
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The burden of urinary tract infections (UTIs) in the United States is substantial. From 2001 to 2002, UTIs accounted for 7.88 million ambulatory care visits (Schappert & Burt, 2006). UTI also represents one of the most common hospital-acquired infections. The predominant pathogen in both complicated and uncomplicated UTIs is Escherichia coli (Valiquette, 2001).
Genotyping tests have enhanced our understanding of the epidemiology of UTI caused by uropathogenic E. coli, including helping to characterize modes of transmission, source and risk factors for infection. For example, strain typing techniques have demonstrated that UTIs can occur as community-wide outbreaks, which had not been commonly considered (Phillips et al., 1988). Manges et al. (2001) subtyped uropathogenic E. coli isolates by serotype, Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR), PFGE and virulence factor profile, and found that a single E. coli clonal group (CgA) accounted for nearly half of community-acquired trimethoprim–sufamethoxazole-resistant UTIs in women in one university community (Manges et al., 2001). This and other studies have suggested that a proportion of community-acquired UTIs may be caused by E. coli disseminated from one or more point sources, such as a contaminated food product (Johnson et al., 2002, 2003; Manges et al., 2006).
Both ERIC-PCR and PFGE are based on comparison of gel electrophoresis band patterns, which are often subject to inter-laboratory variability and not amenable to simple standardization. Sequence-based genotyping methods are becoming increasingly used in epidemiological studies of infectious diseases, including UTIs. Multilocus sequence typing (MLST), a sequence-based genotyping technique first introduced by Maiden et al. (1998), provides reproducibility, comparability and transferability between laboratories. MLST is based on sequencing ‘housekeeping’ genes, which are under stabilizing selection. Therefore, this system may not be optimal for distinguishing highly closely related strains. For E. coli, a standardized MLST was shown to be more discriminatory than the ERIC-PCR-based typing method, but less discriminatory than PFGE (Tartof et al., 2005). PFGE is labour-intensive, time-consuming, and suffers drawbacks associated with comparison of band patterns in electrophoresis gels. However, MLST based on multiple gene sequencing would not be amenable for screening a large number of isolates.
It has been suggested that the E. coli strains that cause most UTIs constitute genetically distinct groups of E. coli, characterized by the presence of multiple virulence factors (reviewed by Johnson, 1991; Mobley et al., 1994). These virulence factors include adhesins, siderophores, toxins, polysaccharides and other products (Eden et al., 1976; Guyer et al., 2002; Johnson & Kuskowski, 2000; Johnson & Stell, 2000; Johnson, 2003; Russo & Johnson, 2000; Russo et al., 2001; Timmis et al., 1985). These properties help the bacteria circumvent host defences to allow entry into a normally sterile site, replicate, and stimulate an inflammatory response. Virulence factors are frequently under selective pressure driven by host innate and acquired immunity. This may contribute to antigenic diversification of proteins directly exposed to host defence systems. Therefore, genes encoding such factors are more likely to undergo mutations over a short period of time compared with genes necessary for basic metabolic processes, such as the ‘housekeeping’ genes used in MLST. We reasoned that such genes may be more useful for studying epidemiological events that occur over a short time period or geographical distance.
Other investigators have applied this idea to distinguish between so-called extraintestinal pathogenic E. coli (ExPEC) and non-ExPEC strains. These applications have generally made dichotomous determinations (presence or absence) of these targets by DNA hybridization probes or by PCR (Johnson et al., 2001, 2005b). In this study, we sought to determine whether a sequence-based comparison of these so-called virulence factor genes could further discriminate uropathogenic and other ExPEC organisms, which may then be applied to better elucidate modes of transmission of community-acquired E. coli infections.
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METHODS
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Selection of genes.
Twelve genes (fimH, fliD, fliM, iha, motA, papA/H, kpsMTII, fepE, fimA, flgA, malG, purD) were selected as targets for sequence analysis. Four were previously identified as putative virulence genes of uropathogenic E. coli (fimH, iha, papA/H, kpsMTII) (Johnson et al., 2000; Johnson & Stell, 2000; Sokurenko et al., 2004), five genes were associated with flagella or type 1 fimbriae shown to contribute to the virulence of uropathogenic E. coli (fliD, fliM, motA, fimA, flgA) (Connell et al., 1996; Francis et al., 1994; Iino et al., 1988; Ikeda et al., 1985; Lane et al., 2005; Macnab, 1987) and three additional genes were included based on their relatively low level of nucleotide sequence conservation observed in alignments of these genes in strains CFT073 and F11 (fepE, malG, purD). Genes were also selected based on the likelihood of their being present as a single copy in uropathogenic strains of E. coli. Eight new primer pairs were designed for this study (fepE, fimA, flgA, fliD, fliM, malG, motA, purD). Primers were designed to amplify a product of approximately 650–1100 bp, and to include the highest number of observed single nucleotide polymorphisms (SNPs) in a sequence alignment of K12, CFT073 and O157 : H7 E. coli reference strains. Four additional primer pairs were previously reported (Johnson et al., 2000; Johnson & Stell, 2000). Nucleotide sequence and citation information for the primers used to amplify the selected gene segments and the expected PCR product lengths are shown in Table 1
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Bacterial isolates and analysis.
Six human uropathogenic isolates (102, 220, PY3, A925, A1707, G), two human blood isolates (W55291, X19714) and one animal E. coli isolate (An559), all previously shown to belong to clonal group CgA by ERIC-PCR and as ST69 complex by MLST, comprised a core group; they were sequenced for each of the 12 selected genes (Table 2). The CgA ERIC-PCR electrophoretic pattern has been described previously as four predominant bands of approximately 1145, 1029, 908 and 720 bp (Manges et al., 2001). These isolates were selected from varying geographical and host sources to maximize the potential to detect differences. Six of these isolates (102, W55291, 220, PY3, X19714, A925) were identical by MLST (designated ST69) (Tartof et al., 2005). Three isolates (A1707, An559, G) differed by one nucleotide in one of the seven MLST genes. All nine isolates grouped together in an SNP dendrogram based on concatenated sequences of the MLST alleles (Tartof et al., 2005). All isolates had been previously typed by PFGE, and all were distinguishable by this method (Manges et al., 2001; Ramchandani et al., 2005). Sequence information for CFT073 and K12 was obtained from GenBank.
fimH.
fimH was analysed for an additional 25 previously well-characterized E. coli isolates. This target was specifically selected for analyses of all the study isolates for the following reasons: (a) it is present in both commensal and pathogenic variants of E. coli, thus satisfying the typeability criterion for its use for genotyping; (b) sequences of its alleles are well-represented in GenBank; and (c) it is known to be under strong selective pressure and thus likely to show a high degree of sequence heterogeneity. The 25 isolates (including K12 and CFT073) included strains which had been previously typed by ERIC-PCR and MLST (Table 2
). The isolates were from the following sources: (a) eight isolates (six CgA, two non-CgA) from women from California, USA, with symptoms of UTI, who were seen at a university health service (consecutively enrolled into a study between 11 October 1999 and 31 January 2000), and five trimethoprim–sulfamethoxazole-resistant isolates (two CgA, three non-CgA) from Minnesota, USA (obtained from students who were seen at university health services with uncomplicated cystitis and were enrolled in a study between June 1998 and August 1999); (b) five animal isolates belonging to CgA by ERIC2 PCR provided by the Gastroenteric Disease Center at Pennsylvania State University (University Park, PA, USA); (c) nine human bacteraemia isolates (six CgA, three non-CgA) from San Francisco General Hospital provided by Dr Francoise Perdreau-Remington; and (d) five other isolates recovered from humans (three CgA, two non-CgA) for geographical comparison and provided by James R. Johnson, MD of Minneapolis VA Medical Center, University of Minnesota, USA. Sequence information for CFT073 and K12 was obtained from GenBank.
DNA isolation and PCR.
E. coli isolates were cultured and DNA was extracted as previously described (Tartof et al., 2005). Amplifications were carried out in a total volume of 25 µl with 1 µl template DNA, 2 µl of each 10 µM primer (Table 1), 2.5 µl 10x buffer, 15 mM MgCl2, 2.5 µl 2 mM dNTP mix, and 0.1 µl AmpliTaq Gold (Applied Biosystems). Reaction conditions used were 2 min denaturation at 95 °C, 33 cycles of 30 s denaturation at 94 °C, 30 s annealing at 58 °C, 1 min extension at 72 °C, and a final additional 7 min at 72 °C in an Applied Biosystems GeneAmp PCR System 2400 thermocycler.
Sequencing and sequence analysis.
All PCR products were purified for sequencing with the Qiagen QIAquick PCR Purification kit. The forward and reverse PCR primers were used for sequencing of the fimH sequences. Sequencing was performed at the University of California at Berkeley Sequencing Facility. The facility runs a 25 cycle sequencing reaction with the following program: 96 °C for 10 s, 50 °C for 5 s, 60 °C for 4 min.
Forward and reverse strand DNA sequence traces were visually inspected and edited with Chromas and BioEdit version 7.0.1. Finished sequences were aligned with CLUSTAL W. Nucleotide sequences from two complete E. coli genomes, K12 (NC_000913) and CFT073 (NC_004431), were trimmed and included in the alignment.
Dendrogram construction.
The 422 bp alignment of a portion of the fimH gene for 32 pathogenic E. coli strains and of the two reference sequences was imported into MEGA version 3.1 (Kumar et al., 2004) for phylogenetic analysis. The fimH sequence used to create the dendrogram is located at position 4 547 233–4 547 654 on the K12 genome. For the same strains, an MLST alignment was made from the 3423 bp concatenation of sequences of the seven genes used in standardized E. coli MLST (adk, fumC, icd, purA, gyrB, recA, mdh) (Tartof et al., 2005). For both alignments, neighbour-joining trees were produced by the Kimura two-parameter model of nucleotide substitution with uniform rates. Bootstrap values were computed by MEGA with 500 replications.
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RESULTS AND DISCUSSION
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The first part of the study was to find sequence variability in a selected set of genes for a panel of closely related clinical isolates from various sources. Nine E. coli isolates (102, W55291, 220, PY3, X19714, A925, A1707, G, An559), identified as CgA by ERIC-PCR and as ST69-complex members by MLST but as distinguishable by PFGE, were analysed for sequence variation in the 12 candidate genes (fimH, fliD, fliM, iha, motA, papA/H, kpsMTII, fepE, fimA, flgA, malG, purD) (Table 1). We found that the selected gene segments, except fimH, had identical sequences among the nine strains, indicating that these gene targets did not contain any SNPs that could discriminate these strains beyond those discriminated by the standardized MLST procedure.
At the fimH locus, we compared nucleotide sequence variation in fimH in 34 (32 clinical and 2 reference strains) epidemiologically well-characterized isolates. These strains consisted of 16 ST69-complex and 18 non-ST69-complex members from various sources. This set of 34 strains was previously typed by the standardized seven-gene MLST procedure and reported by Tartof et al. (2005). We found that the SNP analysis of fimH could separate the 34 isolates into subgroups similar to those separated by the seven-gene-based MLST procedure.
In the fimH-sequence-based dendrogram, 10 distinct terminal groups representing 10 unique sequences were found. By comparison, the dendrogram constructed from seven concatenated MLST genes distinguished 16 distinct terminal groups. However, three of those sequences differed by only one SNP from the ST69 sequence type and are included in the same MLST complex. The 16 isolates identified as ST69 complex by MLST were mostly retained as a single clade group in the fimH dendrogram (Fig. 1). The differences included a cow isolate (An559) which left the group, and a UTI isolate (no. 111) and the K12 strain which joined the group. In both dendrograms, three animal CgA isolates constituted a distinct branch (An300, An298, An45), Minnesota UTI CgA E. coli isolates (J and K) remained clustered, and two San Francisco human bacteraemia CgA isolates (H42937 and M32569) formed another cluster. With the exception of the animal isolate (An559), all CgA isolates which grouped with ST69 by MLST constituted a clade by the fimH SNP analysis.
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Fig. 1. Neighbour-joining trees constructed from the sequences of the fimH gene subsegment (a) and concatenated sequences of the seven standard MLST genes (b) described in the text. The 22 isolates previously identified as CgA are shown with shaded backgrounds. Bootstrap values over 50 % are indicated on the corresponding nodes. *An559 strain fell out of the ST69 complex when analysed for fimH SNP.
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FimH is a mannose-binding subunit protein located at the tip of type 1 fimbriae (Abraham et al., 1988; Klemm & Christiansen, 1987; Krogfelt et al., 1990). Phenotypic variants of FimH are predominantly the product of SNPs in fimH (Hommais et al., 2003; Schembri et al., 2000; Sokurenko et al., 1997, 1998). We selected fimH for our initial analysis for several reasons. (a) FimH is a critical determinant of tropism for the urinary tract and vaginal epithelium for extraintestinal E. coli (Connell et al., 1996; Hung et al., 2002; Klumpp et al., 2002). A urovirulent phenotype is associated with genetic variants of this protein and hence is potentially clinically relevant (Hommais et al., 2003). (b) The vast majority of both intestinal and extraintestinal E. coli express type 1 fimbriae. In one study, among 31 putative virulence factor genes examined in a collection of 63 E. coli urine isolates from patients with cystitis, pyelonephritis and prostatitis, fimH was the most prevalent (Johnson et al., 2005a). Thus fimH satisfies the typeability criteria of a genotyping test. (c) It has been reported that non-synonymous mutations accumulate at the fimH locus at a high rate (Sokurenko et al., 2004). Therefore, the level of discrimination at this locus is likely to be sufficiently high enough for studying uropathogenic E. coli UTI epidemiological events that occur over short time periods or in restricted geographical settings.
Of course, the other 11 genes examined in this study may offer a similar level of discrimination as did fimH for the same 34 isolates. However, not all of the other 11 genes are necessarily present in both commensal and ExPEC isolates. Unlike fimH, some of the other genes are not restricted to the members of the Enterobacteriaceae (e.g. fliD, fliM, motA, flgA, malG, purD). Finally, within a clonal complex, fimH acquires point mutations more frequently than fimA or any of the genes used in MLST (Weissman et al., 2006), and many fimH sequences from a variety of E. coli isolates are deposited in the nucleic acid sequence database for comparison. Since none of the 11 genes studied were able to further discriminate the ST69-complex strains, there is no added advantage of using the other genes.
Interestingly, by fimH SNP analysis, strain An559 isolated from a cow in 1988 in the USA fell into a distinct cluster group outside of the ST69 complex, whereas by MLST it is considered a member of the ST69 complex (ST408) (Fig. 1b). This strain was also previously shown to be 94 % similar by PFGE to one of the human UTI CgA isolates (Ramchandani et al., 2005). Thus it appears that in some of the strains, fimH SNP analysis provides more information than MLST.
One limitation of this study is that the fimH allele is prone to horizontal transfer, as evidenced by the comparison of the two dendrograms (Fig. 1). Thus fimH SNP-based groupings would not be considered phylogenetically meaningful taxa as are the groupings based on MLST. However, this fimH SNP analysis is not meant to provide yet another phylogenetic tool. Rather, the main goal of this study was to develop a simple, sequence-based screening test that could be applied to a large number of UTI E. coli isolates to characterize recent epidemiological events. Plasmids are also subject to frequent horizontal transfers, and yet plasmid profile analyses have been shown to be highly useful for epidemiological studies of enteric pathogens (Holmberg et al., 1984).
The results from this study suggest that this fimH SNP-based genotyping method could serve as a relatively inexpensive, highly reproducible screening test for epidemiological studies of uropathogenic E. coli. A large collection of E. coli isolates could initially be screened for fimH sequence. Those that are indistinguishable can then be tested further by MLST or PFGE. This test could replace ERIC-PCR for screening E. coli isolates, especially in laboratories with access to a sequencing facility.
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ACKNOWLEDGEMENTS
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We thank the staff of Tang Health Center, James R. Johnson, Francoise Perdreau-Remington, Amee Manges and Chitrita DebRoy for providing isolates. We thank Remi Ajiboye for technical support. This work was supported by the NIH grant number RO1 AI059523.
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INTRODUCTION
METHODS
