Genotypic Characterization of Egypt Enterotoxigenic Escherichia Coli Isolates Expressing Coli Surface Antigen 6

Introduction: One approach to control enterotoxigenic Escherichia coli (ETEC) infections has been to develop vaccines focused on inducing protective immunity against surface expressed antigenic factors. One such factor is coli surface antigen 6 (CS6); ETEC isolates expressing CS6 may also simultaneously co-express surface antigens CS4 or CS5. However, there is little information regarding the interrelationships of isolates expressing the CS6 antigen alone or in combination with CS4 or CS5. Methodology: A total of 62 CS6-associated ETEC isolates were evaluated for their antimicrobial susceptibility, mechanisms of resistance, toxin genes, colonization factor expression, and XbaI-pulsed-field gel electrophoretic profiles. Results: We observed 46 XbaI profiles; 31 were exclusive to ETEC expressing CS6 alone and 15 among the ETEC co-expressing CS4 or CS5. Nearly half (47%) of these isolates were resistant to ampicillin, a third (37%) of the isolates were resistant to trimethoprim-sulfamethoxazole, and 24% of the isolates were tetracycline-resistant. A bla TEM gene was detected in 24 (83%) ampicillin-resistant isolates. Trimethoprim-sulfamethoxazole-resistant isolates (n = 23) carried either sulI (n = 1, 4%), sulII (n = 8, 35%) or both genes (n = 10, 43%); 4 had no detectable sul gene. Conclusion: Our results show a lack of clonality among Egypt CS6 E. coli isolates and supports the use and the further research on vaccines targeting this cell surface antigen. Copyright © 2013 El-Gendy et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Introduction
Enterotoxigenic Escherichia coli [1] is a significant global cause of diarrhoea in children living in developing countries [2] as well as travellers [3].Diarrhoea results from the secretion of ETEC heatlabile (LT) and/or heat-stable (ST) enterotoxins [4].Prior to toxin secretion, ETEC organisms must colonize the mucosal epithelium; this process utilizes fimbrial and non-fimbrial colonization factors, also referred to as coli surface antigens (CS) [4].Over 21 ETEC CS have been described [5].Individual CS expression may occur alone as is the case with CFA/I, or more than one CS may be co-expressed, such as CS6 together with the CS4 or CS5 fimbriae [6].
To understand the epidemiology and spread of ETEC in an endemic environment it is necessary to characterize the inter-relationship of the isolates.This information can be used to understand the organism's ecology and subsequently control the spread of disease.It may also be possible to use this information to identify regionally important strains and track their dissemination on a global level.Expressed traits such as lipopolysaccharide (O), flagellar (H), and capsular (K) antigens are commonly used to group ETEC into distinct serotypes; toxin-and CS-expression have been used to further differentiate these isolates [8,9,17,18].
Antimicrobial agents such as sulfonamides and beta-lactam-containing compounds can be an effective prophylaxis against traveller's diarrhoea caused by ETEC and in the absence of effective vaccines, treatment with antimicrobials remains the standard of care for many travellers [21].However, concerns over the emergence of antimicrobial resistant strains due to patient self-treatment and abuse of the therapeutic agents discourage this practice [19,20].Genes encoding resistance proteins to commonly used antimicrobial agents in the developing world, such as sulfonamides and beta lactam antimicrobials, are known to reside on horizontally transmissible elements such as conjugative plasmids and integrons, promoting the spread of resistance [60,61].
The present study investigated the diversity of Egypt CS6-expressing ETEC by comparing XbaIpulsetypes and the mechanisms of resistance to selected antimicrobial agents in a collection of previously serotyped ETEC CS6-expressing isolates obtained from children younger than 3 years of age participating in a longitudinal, community-based study [8,22].

Bacterial strains and culture conditions
During a prospective study conducted from 1995-1998, rectal swab specimens were obtained from a cohort of children with diarrhoea residing in the village of Abu Homos, El-Behira Governorate, Egypt.
The Abu Homos field site has been described previously [22].Five individual lactose fermenting colonies with typical morphology of E. coli were isolated from MacConkey-lactose agar medium, cultured overnight in brain heart infusion broth [23], and frozen in 15% glycerol at -70ºC until use.E. coli and other enteric bacterial pathogens (i.e., Campylobacter, Salmonella spp., Shigella spp.) were identified by standard procedures and API20E [24].Expression of ETEC CS was detected using a panel of monoclonal antibodies as previously described [8,9].

Detection of antimicrobial resistance genes in CS6associated ETEC
The choice of resistance genes studied was based on the high frequency of resistance to AMP and SXT (the most available inexpensive antimicrobial), observed previously [27].CS6-expressing ETEC isolates were screened for the presence of bla TEM1, and two sulfonamides genes (sulI and sulII) by PCR (Table 1).Bacterial DNA extraction was performed as described elsewhere [28].Oligonucleotide primers bla TEM F and bla TEM R (Table1) and the conditions necessary to amplify a 643 bp fragment were previously described [29].Oligonucleotide primer pairs used were sulIF and sulIR or sulIIF and sulIIR, following conditions described previously.Either a 433 bp or a 293 bp internal portion of the sul1 or sulII genes (Table 1), respectively, was amplified [62].
All PCR amplicons were subjected to electrophoresis through a 1% agarose gel (Bio-Rad Laboratories, Richmond, CA, USA) and molecular masses determined by comparison to a 100 bp DNA Ladder (Promega, Madison, WI, USA).E. coli ATCC 25922, a non-ETEC, antimicrobial-susceptible strain, was used as a negative control for each PCR assay.Gels were stained with ethidium bromide and visualized under UV light prior to digital image capture.

XbaI pulsetypes
XbaI macrorestriction profiling using pulsed-field gel electrophoresis of digested chromosomal DNA was used to detect genetic diversity among CS6expressing isolates as described by PulseNet USA [31].PFGE was performed using a CHEF DRII electrophoresis chamber (Bio-Rad); DNA was exposed to an electric current for 22 hours at 6 V/cm at 14ºC in 1% agarose gel by using a linear pulse ramp of 1.79 to 18.66 s in 0.5 X Tris-borate-EDTA electrophoresis buffer.PFGE profiles were compared by automated analysis using the BioNumerics software (version 5.10; Applied Maths, Austin, TX, USA) and confirmed by visual inspection.Similarity between PFGE banding patterns was calculated using the Dice coefficient with a 2% tolerance for the band migration distance and clustering was performed using the complete linkage method.This analysis method is considered a more strict interpretation of banding patterns than UPGMA [32].

Statistical analysis
Results were double-data entered into an Access database; descriptive statistics were completed using Epi Info version 6 (CDC, Atlanta, GA USA and WHO, Geneva, Switzerland).Chi-square or Fisher's exact tests were used to determine statistical significance among discrete or categorical variables.

Pulsetype toxin detection and serological characterization of CS6 isolates
Comparison of the results of PCR amplification of the toxin genes with previous phenotying data revealed six discrepant results (Table 3).Two of the isolates (a serotype O159:H20 isolate expressing CS6 alone and an O15:H18 isolate co-expressing CS6 and CS4) phenotypically expressed ST but were also found to contain the eltB gene encoding LT.Two isolates phenotypically LTST (O39:H11, CS6 alone and O39:H12, co-expressing CS6 and CS5) had no detectable gene encoding ST, and two isolates (rough, CS6 alone; O19:H16, CS6 alone) had no detectable gene encoding LT.Eight O-serotypes had multiple isolates; four O-serotypes (O4, 25, 27 and 39) contained isolates expressing different CS combinations.In each case, the unique CS expressing isolates also possessed different toxin genes with the exception of the CS4, CS6-co-expressing O4 isolate that appeared to share the same STh encoding gene.We also found diversity in toxin genes within isolates that had conserved sero-and CS types (O159, O167 and O169).However, the majority of ETEC isolates expressing CS6-alone (75%; n = 32) contained the estA1 gene, encoding for STp.Similarly, the majority of CS4, CS6 co-expressing isolates harboured estA1 (n = 9/11) in addition to the eltB gene.Only two CS4, CS6 co-expressing isolates were detected that had the estA 2-4 gene encoding for STh toxin.In contrast, the

CS6 XbaI pulsetypes
XbaI digestion of whole genomic DNA extracted from CS6-expressing isolates revealed extensive band diversity (Figure 1A and 1B).There were 46 distinct XbaI-pulsetypes among the 62 CS6-expressing ETEC isolates.No common XbaI PFGE groups were observed when the pulsetypes for the CS6-expressing and the CS6, CS4 or CS5 co-expressing isolates were combined (data not shown).To facilitate comparisons, we analyzed CS6-expressing isolates separately from CS4 or CS5, CS6-co-expressing isolates.
Thirty-one pulsetypes were observed among the 41 CS6-expressing isolates (Figure 1A), although only 20 serotypes were detected in this same collection of isolates.Four broad clusters (I-IV) of isolates were observed among the isolates (33% similarity).Having a common O type was not an adequate indicator of XbaI pattern relatedness: O27 and O4 expressing isolates were located in three separate clusters; O8 and O159 isolates were found in two clusters.An exception to this was O169-expressing isolates; all seven isolates were located in cluster III.Isolates expressing the same O: H type, however, always grouped in the same cluster, with the exception of O4: H NT strains which grouped in separate clusters.The broad clusters consisted of discrete lineages at greater similarity values; these lines were often composed of isolates of the same serotype (e.g.O169: H41, O27: H NT, O27:H7, O27:H20).Isolates of O169: H41 (n = 7), the most common serotype among CS6-expressing ETEC isolates, had six unique pulsetypes.Six of the seven isolates formed a highly related branch in cluster III; the exceptional isolate clustered to a unique arm in cluster III.This isolate also differed from the other O169:H41 isolates in the ST allele expressed; WS6319 harboured an estA2-4 allele whereas the remaining six isolates all had estA1.
Cluster II consisted of all of the O27 isolates, except O27:H20, in addition to the single O8:H4 isolate and one of the two O4:H NT isolates.The five O27:H20 isolates were split into cluster IV (n = 4) and cluster I (n = 1).All of the O27:H7 isolates expressed STp and were sensitive to all antimicrobial tested.The O27:H NT isolates clustered into a unique branch within cluster II, and all expressed STp but differed in their antimicrobial phenotypes.All the O27:H20 isolates expressed STp and had an identical antimicrobial resistance profile (AMP, SXT) and four of the five isolates were genetically indistinguishable (cluster IV).WS6607 was the sole O27:H20 isolate in cluster I, with a genetic background distinct from other O27:H20 isolates.

XbaI pulsetypes of ETEC isolates co-expressing CS6 with CS4 or CS5
We observed 15 XbaI pulsetypes with CS6-ETEC isolates co-expressing CS4 or CS5 (Figure 1B).All of the CS4, CS6 co-expressing isolates grouped together at a similarity level of 60% and were clearly distinct from the CS5, CS6 isolates.Three distinct lineages were observed emerging from within the CS5, CS6 coexpressing isolates.One branch had an O167 isolate; the second branch consisted exclusively of O39expressing isolates; and the last branch was diverse, containing an O167 isolate, two unique serotypes as well as the sole O27:H7 isolate that co-expressed CS5 and CS6.This last isolate also differed from all the other O27 isolates as it expressed STh.All of the CS5, CS6 co-expressing isolates secreted STh.
Among the eleven isolates that co-expressed CS4 and CS6, six had an indistinguishable XbaI pulsetype, expressed LT and ST, and were serotype O25: NT (Figure 1B).Five of the six isolates secreted STp, while one isolate, WS2577, expressed STh. Four of the six isolates were susceptible to all antimicrobials tested in this study and two were SXT resistant.The six XbaI PFGE indistinguishable isolates were all recovered in August 1995 from six different children in two villages in Abu Homos.This cluster might represent an outbreak among the children in Abu Homos; unfortunately, it was not possible to confirm this in our study.The remaining CS4, CS6 coexpressing isolates differed in PFGE profile, antimicrobial susceptibility pattern, and toxin profile.

Discussion
Antimicrobial-resistant ETEC are a growing public health concern.While treatment of diarrhoea with antimicrobial agents is generally restricted to severe cases or the immunocompromised, at least one travel clinic has reported that 35% of returning travellers have been treated with an antimicrobial due to disease severity or duration [57].Antimicrobial resistance profiles of 3,627 isolates of ETEC and nonenterotoxigenic E. coli recovered during 1995-2000 from infants and children residing in the Abu Homos district, Egypt, have previously been examined [27].The percentage of antimicrobial resistant ETEC isolates was 64.6% for ampicillin, 53.8% for   trimethoprim-sulfamethoxazole, and 41.5% for tetracycline.Overall, the ETEC isolates examined in this study were sensitive to the majority of the antimicrobials tested.Resistance to ampicillin was observed in nearly half of the isolates, and in most cases, this resistance could be explained by acquisition of a member of the bla TEM1 family.Unlike previous studies conducted in Egypt, observed resistance in this study is consistent with ETEC resistance reported in India [57] and Vietnam [58], but less than that observed in Thailand [59].Our data also revealed the presence of resistance genes sul1 (n = 1, 4%) sulII (n = 8, 35%) and both genes (n = 10, 43%) among trimethoprim-sulfamethoxazole resistant isolates.We observed that sulI and sulII resistance tended to be associated more often with CS6-expressors (n = 9, 69%), whereas sulII alone was more commonly associated with CS4 or CS5, CS6 expressing isolates (n = 6, 60%).
Correlation of observed antimicrobial resistance to expressed colonization factors has not previously been performed in Egypt and it is possible that ETEC expressing colonization factors other than CS6 are more resistant to ampicillin and trimethoprimsulfamethoxazole.
Detection of antimicrobial resistance mechanisms for ampicillin and sulfa drug resistance should provide a reference point for studies of emerging resistance in ETEC isolated from Egypt.
Diversity in toxin type and serotype of ETEC isolates expressing CS6 has been previously reported.Our results also suggest that CS6 antigen is expressed in a wide representation of phenotypically and genetically dissimilar ETEC isolates, supporting the use of CS6 antigen as a vaccine candidate to control ETEC infections.In this study 16 of 17 (94%) O27expressing isolates expressed only CS6.McConnell et al. [42] also reported that 94% and 86% of O25-and O27-expressing isolates were found to express CS6 alone.McConnell et al. were the first to suggest that CS6 might be able to function as a colonization factor, independent of an association with either CS4 or CS5.
The majority of the O25 isolates described in the present study secreted LT and ST and co-expressed CS4 with CS6 (6/7; 86%); this observation is in contrast to other studies where O25 isolates were LTonly.Serological analysis of 108 CS6-associated ETEC isolates (79 expressing CS6-only and 29 coexpressing either CS4 or CS5) revealed 33 distinct O: H serotypes: 20 distinct O: H serotypes among the ETEC isolates expressing CS6 alone and 13 O: H serotypes within the CS4 or CS5 co-expressing isolates [8].The most common serologic feature among CS6-expressing isolates was detection of the O27 lipopolysaccharide [O27: H7 (n = 6), O27: H20 (n = 5) or O27: H NT (n = 5)], while the most common serotype detected was O169: H41 (n = 7).However, because genetically unrelated strains may express the same phenotypic markers [43] and genetically related strains may express traits differently, phenotypic testing has a restricted ability to discriminate between strains.More recently, several groups have reported nucleotide heterogeneity in the operon containing the genes encoding CS6 [23,44], supporting the concept of genetic diversity within CS6 expressing isolates.Since genes that code for toxins and CS usually are located in plasmids, these markers are not likely reliable markers of clonality of ETEC [18].
DNA-based techniques such as PFGE have been used for the characterization of diarrhoeal pathogens, including ETEC [45,46].PFGE is recognized as a useful tool for comparative analyses of bacterial isolates, but genomic inferences may be difficult to support without additional restriction enzyme analyses [47].
Multilocus sequence analysis (MLST) of a global collection of ETEC isolates, including some strains from Egypt, indicate that there are at least 42 lineages [51].Consistent with our findings, strains of CS6 populate 15 of these lineages, indicating widespread genomic heterogeneity.PFGE has also been shown to be useful for outbreak [52,53] and nonoutbreak/surveillance of E. coli [54,55].In this study we selected PFGE as the method for phylogenetic analysis, in part to create a database of ETEC isolates that would form the basis of the PulseNet Middle East E. coli system.Noller et al. [56] have previously shown that strain diversity in one serotype of E. coli may be underestimated using MLST.Current studies are underway evaluating the diversity of our CS6expressing ETEC isolates using both methods.
In conclusion this study has used PFGE to investigate the diversity of CS6 expressing ETEC in Egypt.Our observations, based on PFGE analysis, suggest a high degree of heterogeneity among CS6 strains in this region, indicating a non-clonal origin and revealing intra-serotype variation.However, CS6 isolates that express the same O: H serotype, such as O27: H7 and O27: H20, appear clonally related.Implementation of standardized protocols of DNAbased typing methods will yield a PFGE database of ETEC strains that could improve the analysis of data generated at different locations and could contribute to a better understanding of the genetic structure of ETEC strains.

Figure 1B .
Figure 1B.Dendrogram and characteristics of ETEC expressing CS6 co-expressing either CS4 or CS5 based on XbaI-digested genomic DNA

Figure 1A .
Figure 1A.Dendrogram and characteristics of ETEC expressing CS6 alone based on XbaIdigested genomic DNA.

Table 1 .
Characteristics of CS6-expressing ETEC isolates used in this study

ETEC-CF type No of Isolates ETEC-CF type Toxin (gene) Toxin type O group H group CS6 isolates
Abbreviations: a NT: non-typeable; b R: rough colonies

Table 2 .
Antibiotic resistance of CS6 isolates

Table 3 .
Carriage of β-lactam and sulfonamide resistance genes among CS6 isolates