Discovery and Phylogenetic Analysis of Novel Members of Class b Enterotoxigenic Escherichia coli Adhesive Fimbriae

Rania A Nada; Hind I Shaheen; Sami B Khalil; Adel Mansour; Nasr El-Sayed; Iman Touni; Matthew Weiner; Adam W Armstrong; John D Klena

doi:10.1128/JCM.02006-10

. 2011 Apr;49(4):1403–1410. doi: 10.1128/JCM.02006-10

Discovery and Phylogenetic Analysis of Novel Members of Class b Enterotoxigenic Escherichia coli Adhesive Fimbriae^{^▿}

Rania A Nada ¹, Hind I Shaheen ¹, Sami B Khalil ¹, Adel Mansour ¹, Nasr El-Sayed ², Iman Touni ¹, Matthew Weiner ^1,^†, Adam W Armstrong ^1,^‡, John D Klena ^1,^*

PMCID: PMC3122862 PMID: 21289147

Abstract

Enterotoxigenic Escherichia coli (ETEC) is recognized to be a common cause of acute watery diarrhea in children from developing countries. Colonization factors (CFAs) have been identified predominantly in ETEC isolates secreting heat-stable enterotoxin (ST) or cosecreting ST with a heat-labile toxin (LT). We hypothesized that LT-only-secreting ETEC produces unique colonization factors not previously described in ST and LTST-secreting ETEC. A set of degenerate primers based on nucleotide sequence similarities between the major structural genes of CS20 (csnA), CS18 (fotA), CS12 (cswA), and porcine antigen 987 (fasA) was developed and used to screen a collection of 266 LT-secreting ETEC isolates in which no known CFA was detected. PCR-amplified products of different molecular masses were obtained from 49 (18.4%) isolates. Nucleotide sequence analysis of the PCR amplicons followed by GenBank nucleotide BLASTn analysis revealed five novel DNA sequences; translated amino acid BLASTx analysis confirmed sequence similarity to class 1b major structural proteins encoded by csnA, fotA, and fasA. Strains expressing the novel CFAs were phylotyped and analyzed using multilocus sequence typing (MLST; Achtman scheme), and the types detected were compared to those of a collection of archived global E. coli strains. In conclusion, application of the degenerate primer sets to ETEC isolates from surveillance studies increased the total number of ETEC isolates with detectable CFAs by almost 20%. Additionally, MLST analysis suggests that for many CFAs, there may be a requirement for certain genetic backgrounds to acquire and maintain plasmids carrying genes encoding CFAs.

INTRODUCTION

Enterotoxigenic Escherichia coli (ETEC) strains are commonly associated with diarrhea in young children in developing countries as well as in travelers to these areas (25, 26, 38). The estimated incidence of ETEC infections in children ranges from 39 to 4,460 per 1,000 persons per year, varying temporally and within different geographies (10). Pathogenesis of ETEC is associated with the expression of two distinct virulence factors. Adhesive fimbriae or colonization factor antigens (CFAs) promote adherence to and colonization of the host small intestine. Watery diarrhea is induced by the production of a heat-stable enterotoxin (ST) and/or a heat-labile enterotoxin (LT) that alters epithelial cell systems (20).

ETEC strains can express 1 or more of at least 22 different CFAs; each fimbria is assembled using multiple copies of a polypeptide known as the major structural fimbrial subunit. In addition, nonstoichiometric amounts of another protein, the minor fimbrial subunit protein, is required. The most common major structural subunit proteins have been isolated from phenotypically characterized reference ETEC strains, and monoclonal antibodies (MAbs) against unique epitopes in these proteins have been produced. Currently, the genetic loci encoding the structural subunit proteins of the most common CFAs have been identified (29).

Genetic differences within the major structural subunits have been detected using DNA hybridization and PCR-based techniques (19, 29, 30). These differences permit CFAs to be grouped into genetically related families on the basis of sequence similarity. The largest CFA genetic family, typified by cfaB (CFA/I), is the class 5 fimbriae. Additional members of this highly related family include csoA (CS1), cotA (CS2), csaB (CS4), csuA (CS14), csbA (CS17), csdA (CS19), and cosA (PCFO71) (1). Separately, type IV pili such as cofA (CS8) and lngA (CS21) are evolutionarily related (9). Other CFAs, such as csfA (CS5) and csvA (CS7), are also genetically related to one another and distantly to cshE (CS13) (8, 36). Three major structural subunits, cswA (CS12), fotA (CS18), and csnA (CS20), share sequence similarity with an ETEC fimbria described from swine, fasA (987P). These fimbriae are referred to as class 1b fimbriae (4, 22, 36). The recently described ETEC fimbria isolated from human children, CS26 (PCFO39), appears also to be related at the amino acid level to the 987P fimbria (14).

Although detection of enterotoxins is sufficient to verify the presence of ETEC in clinical samples, identification of CFAs is important for epidemiological studies and the development of CFA-based vaccines against ETEC infection (31). However, nearly 50% of clinically derived ETEC isolates lack an identifiable CFA, as determined using a panel of 12 MAbs raised against CFA/1, CS1 to CS8, CS12, CS14, and CS17 (11, 23, 28). In particular, LT- and, to a lesser extent, LTST-secreting ETEC isolates more often lack a phenotypically detectable colonization factor than ST-secreting isolates, determined using available immunological reagents (19). Since class 1b fimbriae are primarily associated with LT-secreting ETEC, we designed a set of degenerate primers that targeted relatively conserved regions within the major structural subunit of CS12, CS18, CS20, and 987P to detect and investigate the occurrence of uncharacterized but genetically related CFAs.

MATERIALS AND METHODS

ETEC reference strains.

Twenty-one ETEC reference strains expressing various ETEC CFAs were used in this study. Strains expressing the following CFAs were kindly provided by A. M. Svennerholm (Göteborg University Vaccine Research Institute, Göteborg, Sweden): CFA/I (strain 258909-3), CS1 and CS3 (strain E-1392), CS2 and CS3 (strain 278485-2), CS3 (strain VM73494), CS4 and CS6 (strain E11881-9), CS5 and CS6 (strain E17018A), CS7 (strain E29101A), CS6 and CS8 (strain E34420A), CS12 (strain 350C1A), CS14 (strain E7476A), CS17 (strain E20738A), and 987P. Strains expressing CS10 (strain 2230), CS11 (strain 148B7A), CS13 (strain PE360), CS15 (strain 8786), CS18 (strain ARG-2), CS20 (strain H721AB), CS21 (strain E9034), and CS22 (strain ARG-3) were kindly provided by S. J. Savarino (Enteric Diseases Department, Naval Medical Research Center, Silver Spring, MD). Strains expressing CS19 (strain WS-1513B), CS26 (WS5874A), and PCFO71 (strain WS-2173A) were from the Naval Medical Research Unit No. 3 (NAMRU3) collection of ETEC isolates.

Bacterial isolates.

E. coli isolates used in this study were originally obtained from pediatric cases with diarrhea that were enrolled in a hospital-based study conducted for the surveillance of severe diarrheal diseases in children seeking medical care in Abu-Homos, Egypt, a rural district located in the Nile Delta, and Manshayet Nasser, an urban slum in Cairo, Egypt (39). No CFAs were detected after this collection of ETEC (266 clinical LT-associated isolates) was screened using an established panel of 12 monoclonal antibodies (2, 17); 259 secreted LT only and 7 secreted LTST, determined using a ganglioside M1 enzyme-linked immunosorbent assay for ETEC enterotoxins (27, 32).

mPCR for detection of ETEC enterotoxins.

In addition to phenotypic identification of ETEC isolates, we also used a recently developed multiplex PCR (mPCR) to confirm the presence or absence of genes encoding previously described CFAs and the LT, STa, or STp types.

Degenerate primer design and PCR conditions.

A pair of degenerate oligonucleotide primers (dCS20F and dCS20R) was developed on the basis of the similarities of published sequence data between the fimbrial major structural genes of CS20 (csnA; GenBank accession number AF438155), CS18 (fotA; GenBank accession number U31413), and the porcine antigen 987 (fasA; GenBank accession number U50547). DNA sequences were aligned using the Clustal X application (34) within the BioEdit software package, version 7.0.1 (13), and primers were designed manually. In order to identify additional putative major structural subunit genes related to class b fimbriae, a second PCR was performed using primer CS12R, which recognizes the major fimbrial structural gene of CS12 (cswA; GenBank accession number AY009096), with primer dCS20F. Both simplex PCRs were performed in a 50-μl reaction mixture containing 1.2 μM dCS20F (5′-KCC AGY CTW TGC CAR GT-3′)/dCS20R (5′-YAC AGT ACC DGC YKT AAC-3′) and dCS20F/CS12R (5′-ATA GTC ATT ACT GCA TTT GCA TCA AC-3′), 10 μl of 5× Green GoTaq Flexi buffer, 2 mM MgCl₂, 100 μM each deoxynucleoside triphosphate, 2.5 U of GoTaq Flexi DNA polymerase (Promega, Madison, WI), and 5 μl of bacterial whole-cell lysate. DNA was denatured at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 44°C (dCS20F and dCS20R) or 54°C (dCS20F and CS12R) for 30 s, and extension of amplified products at 72°C for 1 min, with a final extension time of 7 min at 72°C. Reactions were performed in a GeneAmp PCR system 9700 apparatus (Applied Biosystems, Foster City, CA). Amplified products were resolved by gel electrophoresis through 2.0% agarose (Sigma-Aldrich, Steinheim, Germany) using 1× Tris-acetate-EDTA buffer.

Nucleotide sequence analysis and phylogenetic tree construction.

PCR amplicons were purified using a PCR purification kit (Qiagen, Valencia, CA) according to the manufacturer's specifications. Nucleotide sequences were determined using ABI BigDye Terminator chemistry, and cycle sequence products were purified before they were loaded on an ABI Prism 3100 genetic analyzer (Applied Biosystems) after DyeEx purification (Qiagen). Sequence files were assembled using BioEdit, version 7.0.1, and aligned with Clustal X. Edited nucleotide sequences were used to interrogate GenBank databases (most recent searches were with BLASTn 2.2.21, 1 September 2010, for nucleotides and BLASTx 2.2.21, 28 September 2009, for translated amino acids). Phylogenetic and molecular evolutionary analyses were conducted using the software package MEGA, version 4.0 (33). Phylogenetic trees were constructed using the neighbor-joining method, with genetic distance calculated using the Kimura 2-step algorithm. Bootstrap analysis (7) was performed with 2,000 samplings, and values below 70% were excluded as nonsignificant (12).

PCR phylogenetic grouping.

Determination of the E. coli phylogenetic group was performed using the chuA, yjaA, and tspE genes as previously described by Clermont et al. (3).

MLST.

ETEC isolates generating a PCR amplicon using either combination of degenerate primers were analyzed using the multilocus sequence typing (MLST) analysis scheme of Achtman for E. coli (EcMLST; http://mlst.ucc.ie/mlst/dbs/Ecoli/documents/primersColi_html). New gene sequences were submitted to the curator of the E. coli MLST site, and new allelic and sequence type (ST) numbers were confirmed and assigned by the curator.

Nucleotide sequence accession numbers.

Putative CFAs CS28a and CS28b were submitted to GenBank and can be found under accession numbers HQ203049 and HQ203046.

RESULTS

Development and validation of degenerate PCR primers targeting class 1 b fimbriae.

We examined LT-associated ETEC strains for new CFAs since a large number of these strains from our studies lack a known CFA. We restricted our approach to the genetically related but diverse CFAs found in the class 1b fimbria family as an initial proof of principle. We developed degenerate PCR primers based on the published sequence of the major structural subunit protein from three human CFAs (CS12, CS18, and CS20) and one porcine fimbria (987P) (Fig. 1).

Fig. 1. — Comparison of nucleotide sequences of the degenerate primers used to capture genes encoding published major structural subunits of ETEC expressing class 1b fimbriae.

The two primer pairs were validated for their ability to amplify specific target sequences in the DNA extracted from ETEC reference strains expressing CS12, CS18, CS20, or 987P. Each primer set amplified the specific major structural subunit target gene as expected, yielding anticipated product sizes of 438 bp (fotA), 417 bp (csnA), and 414 (fasA) using primer set 1 (dCS20F/dCS20R) and 414 bp (cswA) using primer set 2 (dCS20F/CS12R). We further demonstrated the specificity of the primer sets for genes encoding the class 1b fimbria major structural subunits by attempting to amplify a product from individual ETEC reference strains expressing other known CFAs (CFA/I, CS1 to CS8, CS10, CS11, CS13, CS15, CS17, CS19, CS21, CS22, and PCFO71). No product was amplified from these nontarget strains (data not shown).

Application of degenerate primers for identification of new putative CFAs.

To determine the utility of the degenerate primer sets for epidemiological studies, we applied them to a collection of phenotypically characterized LT-expressing ETEC strains with no known CFA. Multiplex PCR analysis confirmed the lack of a detectable CFA in all but 13 isolates. We detected the cshE gene (the major structural subunit gene of CS13) in these isolates. An association between CS13 and CS26, a member of class 1b ETEC, has been previously reported (14). The sequence for CS26 was not available for inclusion in the development of the degenerate primer. On the basis of these observations, we included the 13 strains for further analysis. The presence of elt (LT), elt and estA1 (LTSTp), or elt and estA2 to estA4 (LTSTh) genes in the 266 studied ETEC strains was confirmed by mPCR. Using the dCS20 primer sets, a product was detected in 49/266 isolates using both primer sets 1 and 2, indicating that 18.4% of the samples appeared to harbor new putative class 1b fimbria genes. Primer set 1 amplified a product in 16.5% (44/266) of the strains, while primer set 2 detected a product in 1.9% (5/266) of the isolates.

DNA sequencing and comparative analyses of PCR amplicons generated from the 49 ETEC clinical isolates and 5 reference strains (CS12, CS18, CS20, CS26, and 987P) indicated that five subfamilies with amplicon sizes of 399, 414/417, 434, 438, and 441/444 bp were present. Broadly speaking, each gene subfamily could be linked to one of the major structural subunit genes of the class 1b family: subfamily 1 contained the structural gene for CS26 (data presented below), subfamily 2, fotA (CS18); subfamily 3, csnA (CS20); subfamily 4, fasA (987P); and subfamily 5, cswA (CS12). Within subfamilies 1, 3, and 5, we observed unique branches which appeared to be genetically diverse from the major structural subunit gene from the corresponding reference strain (Fig. 2). However, none of the ETEC isolates included in our study specifically clustered with cswA (CS12), fotA (CS18), or fasA (987P). Sequences representing genes encoding previously undescribed putative major structural subunit proteins representing novel CFAs (in this study, designated CS27a, CS27b, CS28a, and CS28b) were used to interrogate a collection of nucleotide and translated protein sequences archived in GenBank. With the exception of subfamily 3 (CS28a), which showed a maximum identity of 82% with csnA, none of the new putative major structural subunit genes displayed strong similarity to major structural genes of known CFAs at the primary nucleotide level. However, after translation of the nucleotide to amino acid sequences, similarity to class 1b fimbria major subunit proteins was evident. We performed phylogenetic analysis of all sequenced major structural subunits of known ETEC CFAs, including those characterized in this study (Fig. 3). All of the 1b subfamilies identified in this study were more closely related to one another and were located in one discrete cluster distinct from all of the other CFAs.

Fig. 2. — Phylogenetic relationship observed for the newly indentified CFA in respect to known CFAs in the class 1b family (CS12, CS18, CS20, CS26, and 987P). The phylogeny was based on the nucleotide sequences of the whole PCR products obtained from each subfamily. To make the tree readable, branch 1 includes only 2 isolates representative of the 22 isolates whose PCR products obtained using dCS20 primer set were sequenced. The additional ETEC CS26 strains (n = 11) were used to confirm that the identified DNA sequence did represent the major structural subunit of CS26 fimbriae. The numbers at the nodes represent bootstrap confidence values based on 2,000 replicates.

Fig. 3. — Comparison of the phylogenetic relationships between the newly characterized ETEC CFA major structural subunit belonging to the class 1b family with respect to all known ETEC CFAs. The phylogeny was based on the nucleotide sequence of the whole genes encoding the major structural subunits. The numbers at the nodes represent bootstrap confidence values based on 2,000 replicates. O71, putative colonization factor O71.

Characteristics of members of putative class 1b fimbria subfamilies. (i) Subfamily 1.

Subfamily 1 is related to the CS26 major structural subunit gene and consists of two branches, with one containing the recently described CFA, CS26 (PCFO39) (S. B. Khalil et al., submitted for publication). Eleven additional LT-expressing ETEC clinical isolates produced an amplicon of 444 bp, and DNA sequence analysis revealed that these products were indistinguishable from the major structural subunit gene sequenced from CS26 (GenBank accession number HQ203050) (Fig. 2). CS26 expression was detected in each of these 11 strains using a dot blot assay based on in-house MAbs targeting CS26. Interestingly, the coexistence of the cshE gene encoding CS13 was identified in nine of these isolates. Taken together, we believe that the DNA sequence presented in this branch of subfamily 1 is the major structural subunit for CS26, and we have provisionally named it crsH.

The second branch of subfamily 1 consists of two closely related arms (Fig. 2; CS27a and CS27b). Sequence analysis of the 441-bp amplicon showed that nine isolates formed two groups (with three members and six members) differing at only 10 bp (four amino acids). We observed that all three members of cluster 1 (GenBank accession number HQ203047) carried the genes encoding LT and STp, while the six isolates in the second cluster, cluster 2 (GenBank accession number HQ203048), carried genes encoding LT only and one isolate also carried the cshE gene encoding CS13. For simplifying the nomenclature of this discussion (but by no means attempting to officially name these CFAs) and on the basis of the large sequence difference associated with the comparison of the major structural subunit genes of cshA to this putative CFA, we have termed the members of this branch CS27 and suggest the associated gene name of cmaH for the major structural subunit genes.

We used MLST to characterize the genotypes associated with members of subfamily 1. Three STs were recognized among the 11 branch 1 CS26-expressing isolates: ST165, ST226, and ST814. ST165 was the dominant ST, and all isolates carrying the cshE gene except one were of this ST. The other two STs were related to ST165; ST814 is a single-allele (gyrB) variant of ST165, and three alleles (icd, mdh, purA) separate ST226 from ST165. Interestingly, all isolates were phylogenetic group A, with the exception of the sole ST165 and ST814 isolates, which were phylotype B2.

In subfamily 1, branch 2, all three isolates expressing CS27a were ST10 phylotype A. The CS27b isolates were evenly distributed over ST616 and the newly described ST750. Isolates belonging to ST616 were phylotype B1, whereas ST750 isolates were phylotype A. All three ST types found in this branch represent different clonal lineages; ST616 and ST750 differ at all seven loci used in the MLST scheme. ST616 differs from ST10 at six loci (only purA is conserved), and ST750 differs from ST10 at five loci (only adk and recA are conserved) (Table 1).

Table 1.

Association of ETEC toxin type, multilocus sequence type, and phylotype of clinical ETEC isolates expressing colonization factor antigens belonging to class 1b fimbriae

Gene	Toxin type	CFA	Multilocus sequence type (no. of isolates)	Phylotype
crsH	LT	CS26	ST165 (1)	B2
	LT	CS26	ST226 (1)	A
	LT	CS26 + CS13	ST165 (8)	A
	LT	CS26 + CS13	ST814 (1)	B2
cmaH	LTSTp	CS27a	ST10 (3)	A
	LT	CS27b	ST616 (3)	B1
	LT	CS27b	ST750^a (17)	A
	LT	CS27b + CS13	ST750 (1)	A
csnA	LT	CS20	ST746 (8)	A
	LT	CS20	ST813^a (1)	A
	LTSTp	CS20	ST747 (9)	A
	LTSTp	CS20	ST749 (1)	B1
cnmH	LT	CS28a	ST315 (17)	D
	LT	CS28a	ST731 (17)	A
	LT	CS28a	ST1500^a (1)	A
	LT	CS28b	ST46 (17)	A
	LT	CS28b + CS13	ST46 (17)	A
	LT	CS28b + CS13	ST641 (1)	B1
cswA	LTSTp	CS12	ST10 (3)	A
	LTSTp	CS12	ST189 (3)	A
	LTSTp	CS12	ST209 (1)	A
	LTSTp	CS12	ST748^a (1)	A
	LTSTp	CS12	ST751 (1)	B1

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New sequence types identified in this study.

(ii) Subfamily 2 (CS18 ETEC).

Subfamily 2 is represented by the major structural subunit gene fotA encoding CS18. Using primer set 1 (primers dCS20F and dCS20R), we were able to amplify 438 bp of the fotA gene from the CS18 reference strain (ARG-2). However, none of the clinical isolates tested carried the fotA gene. DNA sequence analysis of the fotA gene places it into a cluster distinct from its most similar neighbor sequence, subfamily 1 (Fig. 2). At the amino acid level, fotA differs from crsH at a minimum of 65 out of 145 (44.8%) amino acids.

(iii) Subfamily 3 (CS20 ETEC).

Subfamily 3 consisted of two branches. Branch 1 contained alleles of the major structural subunit of the csnA gene, encoding the major structural subunit of CS20 (Fig. 2). Nineteen isolates, with an amplicon size of 417 bp, were detected using primer set 1, and sequence analysis placed them into subfamily 3, branch 1. Two types of toxins were associated with this family: LT (n = 9) and LTSTp (n = 10).

Branch 2 in subfamily 3 consisted of two clusters that differ at 51 bp encoding 21 different amino acids. The 399-bp amplicon of subfamily 3, branch 2, cluster 1, isolates (n = 5) was amplified using primer set 1, whereas the 434-bp amplicon of branch 2, cluster 2 isolates (n = 5) was amplified using primer set 2 (dCS20F and CS12R). Interrogating representatives of these alleles against GenBank sequences showed 82% identity with the csnA gene and 70% similarity with the CS20 major structural subunit protein. We have termed these putative CFAs CS28a (GenBank accession number HQ203049) and CS28b (GenBank accession number HQ203046) and suggest the associated gene name of cnmH for the major structural subunit genes. Three out of five ETEC isolates carried the cnmH (CS28b) and cshE (CS13) genes.

MLST analysis of subfamily 3 strains indicated a strong association between coli surface antigen (CS) type, enterotoxin type, and ST. The majority of isolates carrying the elt gene were ST746 (n = 8); a single elt-expressing isolate was ST813. Similarly, most of the isolates carrying both elt and estA1 genes were ST747 (n = 9); a single isolate was ST749. ST746 and ST747 are genetically unique, differing at all seven MLST genes. ST813 was a single-allele variant (adk) from ST746, while ST749 was also unique, differing at all seven alleles compared to the sequence of ST747. All CS20 isolates were phylotype A, except for the sole ST749 isolate, which was phylotype B1.

MLST and phylogenetic analysis of subfamily 3, branch 2, showed that cluster 1 isolates expressing CS28a displayed more genetic variations than cluster 2 isolates expressing CS28b. Three MLSTs were defined in cluster 1, ST315 (n = 2), ST731 (n = 2), and ST1500 (n = 1), and are described as a part of this study. ST1500 was a one-allele variant (gyrB) from ST731, and both STs differed from ST315 at six of seven alleles (only purA is conserved). According to phylogenetic grouping, ST315 was phylotype D, whereas ST731 and ST1500 were phylotype A. Cluster 2 showed a more conservative genetic pattern, as only two MLSTs were detected: ST46 (n = 4) and ST641 (n = 1). ST46 isolates were phylotype A, whereas ST641 isolates were phylotype B1. ST641 differs from ST46 at six of seven alleles (only purA is conserved). Taken together, we believe that the genetic evidence supports the hypothesis that there are three related but distinct alleles derived from csnA in family 3.

(iv) Subfamily 4 (987P ETEC).

We were able to amplify the major structural subunit gene of a porcine CS (987) using primer set 1. Similar to the CS18-expressing strain, the expected 414-bp amplicon was detected only in reference strain 987p. This family is located in a separate branch between subfamilies 3 (CS20 ETEC) and 5 (CS12 ETEC), with 72 and 73 amino acid differences, respectively.

(v) Subfamily 5 (CS12 ETEC).

Sequences obtained from subfamily 5 represent the major structural subunit of the cswA gene, encoding CS12. Derivatives of this gene were not detected in our set of 266 ETEC clinical isolates. Subsequently, we included 10 LTSTp-expressing CS12 isolates that were detected by dot blot assay to complete our genetic analysis. These isolates were detected using primer set 2, amplifying a 414-bp amplicon. The 10 isolates could be subdivided into two highly related clusters, separated by 13-bp differences.

MLST analysis indicated that the two CS12 clusters showed a high degree of similarity in their genetic backgrounds; three STs were detected in each cluster, although they consisted of only a few isolates. cswA cluster 1 consisted of three isolates, each with a unique ST (ST209, ST10, and ST751). ST751 differs from ST10 and ST209 at six of seven alleles (only gyrB is conserved); however, ST10 differs from ST209 at only one allele (recA). cswA branch 2 consisted of three ST types: ST189 (n = 3), ST748 (n = 2), and ST10 (n = 1). The newly described ST748 is a single-allele variant (purA) of ST189. ST10 differs from ST189 and ST748 at five (only adk and purA are conserved) and six (only adk is conserved) alleles, respectively. All isolates belonging to subfamily 5 were phylotype A, except for ST751, which was phylotype B1.

DISCUSSION

To our knowledge, this is the first study to use degenerate primers to detect uncharacterized ETEC CFAs belonging to class 1b fimbriae. Initially, we demonstrated that the designed primer pairs were able to detect the genes encoding the major structural subunit of the known members of the class 1b fimbria family, namely, CS12, CS18, CS20, and 987P. Subsequently, we have shown that CS26 (PCFO39) is also a member of this fimbrial gene family. Phylogenetic analysis of PCR amplicons generated from LT-expressing ETEC strains using the degenerate primer pairs indicated that members of ETEC class 1b fimbriae could be clustered into five different subfamilies. Application of the degenerate primer sets to ETEC isolates recovered from surveillance studies increased the total number of ETEC isolates with a detectable CFA by almost 20%. Finally, using DNA sequence analysis, we have shown that within the class 1b fimbria subfamilies, there are at least three novel genes encoding previously undescribed colonization factors. We also found that there were multiple alleles of several of the genes encoding major structural subunits. In some instances, the genotypes of the strains harboring these novel genes appeared to be highly conserved, but for other CFAs detected in this study, the strains harboring these genes were genetically diverse.

Application of new mPCR methods has greatly improved the assignment of ETEC strains to a CFA class by detection of genes encoding the CFA major structural subunit genes. A recently established mPCR assay targeting genes known to encode the major structural subunit of CFAs reduced the percentage of ETEC isolates with no detectable CFA from 45% to 18% when it was applied previously (19). In this paper, we have shown that our new assay using degenerate primers further increases the yield in the number of strains with an identified CFA. Using the same approach, similar findings in screens for new members of gene families were reported by Park et al. (24). Recent reports of outbreaks of LT-expressing, CFA-nondetectable ETEC-associated diarrhea following the cyclones in Bangladesh (F. Qadri, personal communication) highlight the epidemiological need for increasing the detection capability of our current assays.

In the present study, we targeted a collection of LT-producing ETEC isolates obtained from a surveillance study of severe pediatric diarrhea in Egypt (39). This set of clinical isolates failed to react phenotypically with a panel of 12 MAbs raised against the most commonly detected CFAs. The degenerate PCR primer approach enabled us to amplify a product in almost 20% of the study isolates. The csnA (CS20) and crsH (CS26) genes were detected in this set of clinical isolates, which was confirmed later by the use of specific MAbs not included in our routine ETEC testing, and neither the fotA (CS18) nor the fasA (987P) gene was identified in any of the pediatric clinical isolates included in this study. Interestingly, approximately 7.0% of the ETEC isolates examined carried previously unreported nucleotide sequences falling within class 1b fimbria subfamilies 1 and 3. According to our data, we believe that these new DNA sequences fall into two general sequence subfamilies most closely related to, but distinct from, csnA (CS20) and crsH, the major structural subunit gene of the recently described CFA, CS26. In addition, we found that the genetic diversity present within strains expressing CS12 was greater than expected.

We have presented evidence that supports the establishment of three new CFAs. Screening of the GenBank DNA database for potential nucleotide identities failed to find any significant sequence matches, with the exception of the 80% identity between cnmH and csnA. This observation suggests that at the gene level, the putative colonization factors are unique. After translation of the nucleotide sequence, it was evident that these gene fragments encode major structural subunits of ETEC CFA. No significant amino acid similarity to any other protein was observed.

Using the crsH-csnA subfamily as an example, we have shown additional information that supports the argument that new major structural subunit genes in the fimbrial 1b family have been discovered. We have demonstrated that (i) a MAb specific for CS26 (developed from reference strain WS5874A) recognized all ETEC strains carrying the crsH sequence but failed to recognize markers in CS27a or CS27b strains. (ii) Similarly, a MAb developed to the CS20 fimbriae (15) expressed by ETEC strain WS7179A recognized all strains carrying the csnA sequence but failed to recognize cnmH-carrying strains (16); and (iii) no other colonization factor was detected, either by dot blot analysis or by mPCR, with the exception of the cshA gene encoding CS13 in one subfamily of isolates. It is not uncommon for ETEC CS26 to also express CS13, as previously reported (14). Data obtained may indicate that the variation observed in certain nucleotide sequences of the genes encoding these four new putative CFAs of subfamily 1 (CS27a and CS27b) and of subfamily 3 (CS28a and CS28b) translated into amino acid sequences may be forming an epitope(s) different than the one specific to C26 MAb or CS20 MAb binding sites. The identification of ETEC organisms carrying more than one CFA gene encoding a fimbria major structural subunit is common (19, 37). While we believe that the data that we have presented support the designation of three new CFAs, we recognize that the lack of direct biological information (e.g., immunoelectron microscopy of wild-type and knockout mutant strains) is a limitation of the study. We are actively pursuing a multidisciplinary approach to characterize each of the new putative colonization factors described in this study.

Previous phylogenetic analyses using a triplex PCR have shown that E. coli strains generally fall into four main phylogenetic groups (A, B1, B2, and D) (3). In general, most commensal and diarrheagenic E. coli strains responsible for acute and severe diarrhea and toxin production, such as ETEC, enterohemorrhagic E. coli, and enteroinvasive E. coli strains, belong to groups A and B1 (6, 35). Virulent extraintestinal strains mainly belong to group B2 and, to a lesser extent, group D (6). In this study, the majority of ETEC strains fell in group A or B1, except for a few isolates identified to be in group B2 (LT-CS26 with or without CS13) or group D (LT-CS28a). Due to the limited number of ETEC strains belonging to phylotype B2 or D, we were not able to investigate its association with disease severity.

MLST is a useful tool for characterizing populations of enteric and nonenteric bacterial pathogens (5, 18, 21, 35). We used MLST to characterize the genotypes of the ETEC isolates expressing class 1b fimbriae. We observed in general that different sequence types were associated with different colonization factors. Typing of our isolate collection for ETEC strains expressing class 1b fimbriae revealed 18 sequence types, implying that the genetic background of these ETEC strains is not conserved and is of polyphyletic origin, as previously reported (35). We did observe an enrichment of certain ST types within each ETEC CFA subfamily, with the exception of ST10, which was identified in two subfamilies (1 and 5). Interestingly, strains categorized by this sequence type carried the same ETEC enterotoxin genes (eltB and estA1). Furthermore, we identified nine new sequence types in this study (ST746, ST747, ST748, ST749, ST750, ST751, ST813, ST814, ST1500) that fall within distinct clusters of ETEC expressing different CFAs.

Phylogenetic analyses revealed that the genetic background in ETEC strains expressing CS28b was more conserved (ST46) than that of ETEC strains expressing CS20 or CS28a. Unlike Turner et al. (35), we observed an apparent association between toxin type and MLST that was observed in eltB- and estA1-carrying or in eltB-carrying isolates (35). However, this might be due to the expansion of geographically and temporally successful clones and requires additional study of isolates from wider geographic regions over time. ETEC CS27a harbored eltB and estA1 genes, unlike the majority of this subfamily of strains, which harbor only the eltB gene, and each clade consisted of unique ST groups. ETEC CS12 isolates, members of subfamily 5, include distinct sequence types divided into two clusters, except for ST10, which was also identified in subfamily 1. Overall, we observed a strong correlation between the nucleotide sequence of a novel putative CFA, phylogenetic grouping, and MLST.

In conclusion, this degenerate PCR approach successfully enabled us to detect novel putative colonization factor antigens of genetic similarity. In addition, the coexpression of CS13 is not uncommon among some subfamilies of ETEC expressing class 1b fimbriae. The same approach should be used to test for unknown CFAs of genetic similarity to other ETEC CFA families (e.g., the colonization factor antigen I [CFA/I] family). Further studies are needed at the protein and immunogen levels to validate that the nucleotide sequences discovered in the current study represent major structural subunits of novel CFAs. MLST analysis suggested that for many CFAs, there may be a requirement for certain genetic backgrounds to acquire and maintain the plasmids carrying genes encoding CFAs. According to our data, a high percentage of ETEC LT secretors with phenotypically undetectable CFAs are due to the presence of hitherto unidentified CFAs.

ACKNOWLEDGMENTS

We thank Abdel Fattah Hamad, Salem Awad, and Ireen Kamal for excellent technical assistance.

Financial support for this study was provided by MIDRP Step L, Work Unit No. 6000.000.000.E0501.

The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the U.S. Department of the Navy, U.S. Department of Defense, or the United States Government.

This study (protocol 30969) was approved by the Naval Medical Research Unit No. 3 Institutional Review Board, in compliance with all applicable federal regulations governing the protection of human subjects.

John D. Klena is an employee of the U.S. Government. This work was prepared as part of his official duties.

Footnotes

^▿

Published ahead of print on 2 February 2011.

REFERENCES

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19. Nada R. A., et al. 2010. Design and validation of a multiplex polymerase chain reaction for the identification of enterotoxigenic Escherichia coli and associated colonization factor antigens. Diagn. Microbiol. Infect. Dis. 67:134–142 [DOI] [PubMed] [Google Scholar]
20. Nataro J. P., Kaper J. B. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142–201 [DOI] [PMC free article] [PubMed] [Google Scholar]
21. Nicklasson M., et al. 2010. Enterotoxigenic Escherichia coli multilocus sequence types in Guatemala and Mexico. Emerg. Infect. Dis. 16:143–146 [DOI] [PMC free article] [PubMed] [Google Scholar]
22. Nuccio S. P., Baumler A. J. 2007. Evolution of the chaperone/usher assembly pathway: fimbrial classification goes Greek. Microbiol. Mol. Biol. Rev. 71:551–575 [DOI] [PMC free article] [PubMed] [Google Scholar]
23. Oyofo B. A., Mohran Z. S., el-Etr S. H., Wasfy M. O., Peruski L. F., Jr 1996. Detection of enterotoxigenic Escherichia coli, Shigella and Campylobacter spp. by multiplex PCR assay. J. Diarrhoeal Dis. Res. 14:207–210 [PubMed] [Google Scholar]
24. Park J. M., Han N. S., Kim T. J. 2007. Rapid detection and isolation of known and putative alpha-l-arabinofuranosidase genes using degenerate PCR primers. J. Microbiol. Biotechnol. 17:481–489 [PubMed] [Google Scholar]
25. Putnam S. D., et al. 2004. Antimicrobial susceptibility trends among Escherichia coli and Shigella spp. isolated from rural Egyptian paediatric populations with diarrhoea between 1995 and 2000. Clin. Microbiol. Infect. 10:804–810 [DOI] [PubMed] [Google Scholar]
26. Qadri F., et al. 2005. Enterotoxigenic Escherichia coli and Vibrio cholerae diarrhea, Bangladesh, 2004. Emerg. Infect. Dis. 11:1104–1107 [DOI] [PMC free article] [PubMed] [Google Scholar]
27. Sanchez J., Holmgren J., Svennerholm A. M. 1990. Recombinant fusion protein for simple detection of Escherichia coli heat-stable enterotoxin by GM1 enzyme-linked immunosorbent assay. J. Clin. Microbiol. 28:2175–2177 [DOI] [PMC free article] [PubMed] [Google Scholar]
28. Shaheen H. I., et al. 2004. Phenotypic profiles of enterotoxigenic Escherichia coli associated with early childhood diarrhea in rural Egypt. J. Clin. Microbiol. 42:5588–5595 [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Sjoling A., Wiklund G., Savarino S. J., Cohen D. I., Svennerholm A. M. 2007. Comparative analyses of phenotypic and genotypic methods for detection of enterotoxigenic Escherichia coli toxins and colonization factors. J. Clin. Microbiol. 45:3295–3301 [DOI] [PMC free article] [PubMed] [Google Scholar]
30. Steinsland H., et al. 2003. Development and evaluation of genotypic assays for the detection and characterization of enterotoxigenic Escherichia coli. Diagn. Microbiol. Infect. Dis. 45:97–105 [DOI] [PubMed] [Google Scholar]
31. Svennerholm A. M., Steele D. 2004. Microbial-gut interactions in health and disease. Progress in enteric vaccine development. Best Pract. Res. Clin. Gastroenterol. 18:421–445 [DOI] [PubMed] [Google Scholar]
32. Svennerholm A. M., Wiklund G. 1983. Rapid GM1-enzyme-linked immunosorbent assay with visual reading for identification of Escherichia coli heat-labile enterotoxin. J. Clin. Microbiol. 17:596–600 [DOI] [PMC free article] [PubMed] [Google Scholar]
33. Tamura K., Dudley J., Nei M., Kumar S. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24:1596–1599 [DOI] [PubMed] [Google Scholar]
34. Thompson J. D., Gibson T. J., Plewniak F., Jeanmougin F., Higgins D. G. 1997. The Clustal X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25:4876–4882 [DOI] [PMC free article] [PubMed] [Google Scholar]
35. Turner S. M., et al. 2006. Phylogenetic comparisons reveal multiple acquisitions of the toxin genes by enterotoxigenic Escherichia coli strains of different evolutionary lineages. J. Clin. Microbiol. 44:4528–4536 [DOI] [PMC free article] [PubMed] [Google Scholar]
36. Valvatne H., et al. 2004. Identification and molecular characterization of the gene encoding coli surface antigen 20 of enterotoxigenic Escherichia coli. FEMS Microbiol. Lett. 239:131–138 [DOI] [PubMed] [Google Scholar]
37. Valvatne H., Steinsland H., Sommerfelt H. 2002. Clonal clustering and colonization factors among thermolabile and porcine thermostable enterotoxin-producing Escherichia coli. APMIS 110:665–672 [DOI] [PubMed] [Google Scholar]
38. Wenneras C., Erling V. 2004. Prevalence of enterotoxigenic Escherichia coli-associated diarrhoea and carrier state in the developing world. J. Health Popul. Nutr. 22:370–382 [PubMed] [Google Scholar]
39. Wierzba T. F., et al. 2006. Clinic-based surveillance for bacterial- and rotavirus-associated diarrhea in Egyptian children. Am. J. Trop. Med. Hyg. 74:148–153 [PubMed] [Google Scholar]

[B1] 1. Anantha R. P., et al. 2004. Evolutionary and functional relationships of colonization factor antigen i and other class 5 adhesive fimbriae of enterotoxigenic Escherichia coli. Infect. Immun. 72:7190–7201 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2. Binsztein N., et al. 1991. Colonization factors of enterotoxigenic Escherichia coli isolated from children with diarrhea in Argentina. J. Clin. Microbiol. 29:1893–1898 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3. Clermont O., Bonacorsi S., Bingen E. 2000. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66:4555–4558 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4. Contrepois M., Bertin Y., Pohl P., Picard B., Girardeau J. P. 1998. A study of relationships among F17 a producing enterotoxigenic and non-enterotoxigenic Escherichia coli strains isolated from diarrheic calves. Vet. Microbiol. 64:75–81 [DOI] [PubMed] [Google Scholar]

[B5] 5. Dingle K. E., Colles F. M., Falush D., Maiden M. C. 2005. Sequence typing and comparison of population biology of Campylobacter coli and Campylobacter jejuni. J. Clin. Microbiol. 43:340–347 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6. Escobar-Paramo P., et al. 2004. A specific genetic background is required for acquisition and expression of virulence factors in Escherichia coli. Mol. Biol. Evol. 21:1085–1094 [DOI] [PubMed] [Google Scholar]

[B7] 7. Felsenstein J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791 [DOI] [PubMed] [Google Scholar]

[B8] 8. Gaastra W., Svennerholm A. M. 1996. Colonization factors of human enterotoxigenic Escherichia coli (ETEC). Trends Microbiol. 4:444–452 [DOI] [PubMed] [Google Scholar]

[B9] 9. Gomez-Duarte O. G., et al. 2007. Genetic diversity of the gene cluster encoding longus, a type IV pilus of enterotoxigenic Escherichia coli. J. Bacteriol. 189:9145–9149 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10. Gupta S. K., et al. 2008. Part III. Analysis of data gaps pertaining to enterotoxigenic Escherichia coli infections in low and medium human development index countries, 1984-2005. Epidemiol. Infect. 136:721–738 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11. Gutierrez-Cazarez Z., Qadri F., Albert M. J., Giron J. A. 2000. Identification of enterotoxigenic Escherichia coli harboring longus type IV pilus gene by DNA amplification. J. Clin. Microbiol. 38:1767–1771 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12. Hall B. G., Barlow M. 2006. Phylogenetic analysis as a tool in molecular epidemiology of infectious diseases. Ann. Epidemiol. 16:157–169 [DOI] [PubMed] [Google Scholar]

[B13] 13. Hall T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41:95–98 [Google Scholar]

[B14] 14. Khalil S. B., et al. 1999. PCFO39: a novel colonization factor identified in enterotoxigenic Escherichia coli strains from Northern Africa, abstr. B/D-58. Abstr. 99th Gen. Meet. Am. Soc. Microbiol American Society for Microbiology, Washington, DC [Google Scholar]

[B15] 15. Khalil S. B., et al. 2007. Development and evaluation of monoclonal antibodies against new ETEC colonization factors and incorporation into field studies, abstr. B-113. Abstr. 107th Gen. Meet. Am. Soc. Microbiol American Society for Microbiology, Washington, DC [Google Scholar]

[B16] 16. Koprowski H., II, et al. 2000. Attenuated Shigella flexneri 2a vaccine strain CVD 1204 expressing colonization factor antigen I and mutant heat-labile enterotoxin of enterotoxigenic Escherichia coli. Infect. Immun. 68:4884–4892 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B17] 17. Lopez-Vidal Y., Svennerholm A. M. 1990. Monoclonal antibodies against the different subcomponents of colonization factor antigen II of enterotoxigenic Escherichia coli. J. Clin. Microbiol. 28:1906–1912 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B18] 18. Maiden M. C., et al. 1998. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Natl. Acad. Sci. U. S. A. 95:3140–3145 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19. Nada R. A., et al. 2010. Design and validation of a multiplex polymerase chain reaction for the identification of enterotoxigenic Escherichia coli and associated colonization factor antigens. Diagn. Microbiol. Infect. Dis. 67:134–142 [DOI] [PubMed] [Google Scholar]

[B20] 20. Nataro J. P., Kaper J. B. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:142–201 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B21] 21. Nicklasson M., et al. 2010. Enterotoxigenic Escherichia coli multilocus sequence types in Guatemala and Mexico. Emerg. Infect. Dis. 16:143–146 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22. Nuccio S. P., Baumler A. J. 2007. Evolution of the chaperone/usher assembly pathway: fimbrial classification goes Greek. Microbiol. Mol. Biol. Rev. 71:551–575 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B23] 23. Oyofo B. A., Mohran Z. S., el-Etr S. H., Wasfy M. O., Peruski L. F., Jr 1996. Detection of enterotoxigenic Escherichia coli, Shigella and Campylobacter spp. by multiplex PCR assay. J. Diarrhoeal Dis. Res. 14:207–210 [PubMed] [Google Scholar]

[B24] 24. Park J. M., Han N. S., Kim T. J. 2007. Rapid detection and isolation of known and putative alpha-l-arabinofuranosidase genes using degenerate PCR primers. J. Microbiol. Biotechnol. 17:481–489 [PubMed] [Google Scholar]

[B25] 25. Putnam S. D., et al. 2004. Antimicrobial susceptibility trends among Escherichia coli and Shigella spp. isolated from rural Egyptian paediatric populations with diarrhoea between 1995 and 2000. Clin. Microbiol. Infect. 10:804–810 [DOI] [PubMed] [Google Scholar]

[B26] 26. Qadri F., et al. 2005. Enterotoxigenic Escherichia coli and Vibrio cholerae diarrhea, Bangladesh, 2004. Emerg. Infect. Dis. 11:1104–1107 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27. Sanchez J., Holmgren J., Svennerholm A. M. 1990. Recombinant fusion protein for simple detection of Escherichia coli heat-stable enterotoxin by GM1 enzyme-linked immunosorbent assay. J. Clin. Microbiol. 28:2175–2177 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B28] 28. Shaheen H. I., et al. 2004. Phenotypic profiles of enterotoxigenic Escherichia coli associated with early childhood diarrhea in rural Egypt. J. Clin. Microbiol. 42:5588–5595 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29. Sjoling A., Wiklund G., Savarino S. J., Cohen D. I., Svennerholm A. M. 2007. Comparative analyses of phenotypic and genotypic methods for detection of enterotoxigenic Escherichia coli toxins and colonization factors. J. Clin. Microbiol. 45:3295–3301 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30. Steinsland H., et al. 2003. Development and evaluation of genotypic assays for the detection and characterization of enterotoxigenic Escherichia coli. Diagn. Microbiol. Infect. Dis. 45:97–105 [DOI] [PubMed] [Google Scholar]

[B31] 31. Svennerholm A. M., Steele D. 2004. Microbial-gut interactions in health and disease. Progress in enteric vaccine development. Best Pract. Res. Clin. Gastroenterol. 18:421–445 [DOI] [PubMed] [Google Scholar]

[B32] 32. Svennerholm A. M., Wiklund G. 1983. Rapid GM1-enzyme-linked immunosorbent assay with visual reading for identification of Escherichia coli heat-labile enterotoxin. J. Clin. Microbiol. 17:596–600 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33. Tamura K., Dudley J., Nei M., Kumar S. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24:1596–1599 [DOI] [PubMed] [Google Scholar]

[B34] 34. Thompson J. D., Gibson T. J., Plewniak F., Jeanmougin F., Higgins D. G. 1997. The Clustal X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25:4876–4882 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B35] 35. Turner S. M., et al. 2006. Phylogenetic comparisons reveal multiple acquisitions of the toxin genes by enterotoxigenic Escherichia coli strains of different evolutionary lineages. J. Clin. Microbiol. 44:4528–4536 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B36] 36. Valvatne H., et al. 2004. Identification and molecular characterization of the gene encoding coli surface antigen 20 of enterotoxigenic Escherichia coli. FEMS Microbiol. Lett. 239:131–138 [DOI] [PubMed] [Google Scholar]

[B37] 37. Valvatne H., Steinsland H., Sommerfelt H. 2002. Clonal clustering and colonization factors among thermolabile and porcine thermostable enterotoxin-producing Escherichia coli. APMIS 110:665–672 [DOI] [PubMed] [Google Scholar]

[B38] 38. Wenneras C., Erling V. 2004. Prevalence of enterotoxigenic Escherichia coli-associated diarrhoea and carrier state in the developing world. J. Health Popul. Nutr. 22:370–382 [PubMed] [Google Scholar]

[B39] 39. Wierzba T. F., et al. 2006. Clinic-based surveillance for bacterial- and rotavirus-associated diarrhea in Egyptian children. Am. J. Trop. Med. Hyg. 74:148–153 [PubMed] [Google Scholar]

PERMALINK

Discovery and Phylogenetic Analysis of Novel Members of Class b Enterotoxigenic Escherichia coli Adhesive Fimbriae▿

Rania A Nada

Hind I Shaheen

Sami B Khalil

Adel Mansour

Nasr El-Sayed

Iman Touni

Matthew Weiner

Adam W Armstrong

John D Klena