|Year : 2010 | Volume
| Issue : 3 | Page : 503-508
|Phenotypic and molecular characterization of clinically isolated Escherichia coli
Chetana Vaishnavi1, Sukhminderjit Kaur1, Lothar Beutin2, Ulrike Krueger2
1 Department of Gastroenterology, Postgraduate Institute of Medical Education and Research, Chandigarh - 160 012, India
2 National Reference Laboratory for Escherichia coli, Federal Institute for Risk Assessment (BfR), Diedersdorfer Weg 1 D-12277, Berlin, Germany
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|Date of Web Publication||22-Oct-2010|
| Abstract|| |
All diarrheagenic Escherichia coli carry at least one virulence-related property. Stool samples from 244 patients having acute or persistent diarrhea received after the exclusion of routine enteropathogens were investigated. Purely or predominantly isolated E. coli (n = 100) were subjected to serotyping, of which only 25 were typable. They belonged to 14 different O-serogroups comprising 5 O153, 4 O102, 3 O25, 2 each of O130 and O169, and 1 each of O1, O8, O15, O37, O86, O101, O127, O143, and O160. The typable E. coli isolates along with 5 other untypable isolates were investigated for molecular markers, such as intimin (eae), enterohemolysin (EhlyA), a-hemolysin, heat-labile enterotoxins (LT), heat-stable enterotoxins (STa), verotoxins (VT1 and VT2), invasivity (ial), enteroaggregative E. coli (EAEC) gene (EAGG), and enterotoxin (EAST). Two of the isolates (O153 and O86) were positive for enterohemolysin phenotypically and 5 for β-hemolysin both phenotypically and genotypically. Interestingly, 16.6% of the randomly isolated E. coli were O153, a serogroup common in cattle, and 10% belonged to EAEC pathotype of which two-thirds had the EAST gene, which is quite frequent in these strains. Additionally, there was one strain (O153) that was positive for EAST only. Between the two 0130:H6 strains isolated, one belonged to EAEC serogroup. None of the E. coli isolated were positive for verotoxins, eae, LT1, STa, and ial. Data obtained emphasize the need for additional research into the role of eae gene and other putative factors affecting the virulence of diarrheagenic E. coli in India.
Keywords: Clinical isolates, Escherichia coli, molecular characterization
|How to cite this article:|
Vaishnavi C, Kaur S, Beutin L, Krueger U. Phenotypic and molecular characterization of clinically isolated Escherichia coli. Indian J Pathol Microbiol 2010;53:503-8
|How to cite this URL:|
Vaishnavi C, Kaur S, Beutin L, Krueger U. Phenotypic and molecular characterization of clinically isolated Escherichia coli. Indian J Pathol Microbiol [serial online] 2010 [cited 2021 Oct 21];53:503-8. Available from: https://www.ijpmonline.org/text.asp?2010/53/3/503/68298
| Introduction|| |
The pathogenesis of various diarrheagenic Escherichia More Details coli is incompletely understood, although certain characteristic features help to categorize the strains to one or the other pathotypes. Enteropathogenic E. coli (EPEC) are important cause of infantile enteritis, whereas enterotoxigenic E. coli (ETEC), which produce enterotoxins, commonly cause acute watery diarrhea in the tropics, particularly in infants younger than 2 years. Enteroinvasive E. coli (EIEC) isolates have been implicated as a cause of dysentery-like diarrhea with blood and mucus in stools, whereas enterohemorrhagic E. coli (EHEC) are known to cause hemorrhagic colitis. Enteroaggregative E. coli (EAEC), recognizable by their aggregative or "stacked brick" type of adherence is also another important cause of acute and persistent diarrhea in children. Pure isolates of E. coli are frequently obtained from diarrheic stools. However, because serotyping is not routinely done in most Indian laboratories, it is not possible to ascertain the prevalence of E. coli diarrhea. Apart from serotyping, there are several phenotypic and genotypic methods to characterize the pathogenicity of E. coli isolates.
The versatility of E. coli genome is mostly due to virulence-related plasmids, phage or phage-related genes, and the chromosomal pathogenicity islands. All diarrheagenic E. coli carry at least one virulence-related property. The EPEC is characterized by an adhesin called intimin (eae). The ETEC is defined as having at least 1 member of 2 defined groups of enterotoxin-the heat-stable enterotoxin (ST) and the heat-labile enterotoxin (LT). The EIEC is characterized by the ial (marker for invasivity) gene and EHEC by the production of verotoxins (VT) and at times hemolysins (hly) may be present, though not necessarily. The verotoxin-producing E. coli (VTEC) are also known as Shiga toxin-producing E. coli (STEC). The EAEC is characterized by its enteroaggregative E. coli plasmid (EAGG) and by EAST, a genetic marker for ST-associated EAGG.
Schmidt and co-workers  reported the genetic analysis of a new plasmid encoded hemolysin, Ehly, among the VTEC. This hemolysin seemed to be associated with severe clinical diseases in humans.  E. coli produces 2 different hemolysins-the extracellular agent called α-hemolysin and a cell-bound b-hemolysin. Beutin et al studied hemolysin production in a large number of serologically diverse VT+ E. coli strains and found an association between enterohemolysin and verotoxin production in 89% of E. coli strains belonging to 9 different serotypes. These workers propose that enterohemolysin may be useful as an epidemiologic marker for rapid detection of potential VT+ strains of E. coli. The present investigation was carried out to determine the prevalence of different E. coli serogroups in patients presenting themselves with acute or persistent diarrhea in a tertiary care hospital and to characterize the isolates phenotypically and by molecular methods.
| Materials and Methods|| |
Stool samples (n = 244) from patients suffering from acute or persistent diarrhea were investigated. Persistent diarrhea included acute cases having diarrhea lasting for at least 14 days. The subjects comprised 149 males and 95 females of all age groups (1 month to 84 years).
Sample Collection and Exclusion of Other Routine Enteropathogens
Stool samples (watery, mucoid, or bloody) were collected in sterile stool containers for investigation of diarrheagenic E. coli after the initial routine exclusion of other enteropathogens involved in diarrhea. The other enteropathogenic bacteria excluded were Salmonella More Details, Yersinia More Details, Campylobacter, Vibrio cholera, and Clostridium difficile by routine standard culture and biochemical methods. Rotavirus infection was excluded by antigen detection using enzyme immunoassay. Parasitic eggs were excluded by the evaluation of 3 stool specimens obtained on 3 separate days by wet mount examination and iodine staining methods after concentrating the samples.
Preparation of Washed and Unwashed Blood Agar Plates
Washed blood agar plates were prepared as described by Beutin et al.  Briefly, for washed sheep blood agar plate, fresh sheep blood was taken and washed by centrifugation at 3000g for 20 min. The supernatant was pipetted out and replaced by same volume of phosphate buffered saline (PBS) pH 7.2. This procedure was repeated 2 times. Blood agar plates were prepared by adding 5% final concentration of washed sheep blood to molten nutrient agar. Unwashed blood agar was prepared without the washing steps.
Culture, Isolation, and Identification of E. coli
Direct cultures of the stool specimens were done on Xylose lysine deoxycholate agar (XLD), MacConkey agar and MacConkey broth (HiMedia, Mumbai, Maharashtra, India) within 1 h of the receipt of the sample. Both the solid media were incubated at 37°C and examined after 24 h. The MacConkey broth was incubated for 4-5 h till growth occurred and subculture was done on (1) MacConkey agar, (2) washed blood agar, and (3) unwashed blood agar plates.
All inoculated plates were incubated at 37°C. Pure or predominantly obtained lactose fermenting colonies on MacConkey agar were identified by conventional biochemical reactions for E. coli. Serogrouping of E. coli isolates was done using polyvalent and monovalent antisera (M/s Denka Seiken Co. Ltd., Japan). Apart from this, pure or predominant growth of yellow colonies or any red colony on XLD or any nonlactose fermenting colony on MacConkey agar were also identified.
Detection of Hemolysin
The reference strains used for enterohemolysin detection were (1) E. coli 04: K3-H5 for alpha hemolysis.,(2) E. coli 0157:H- (EHEC-hemolysin positive, enterohemolytic phenotype Shiga-toxin negative), and (3) KK7/1 E. coli 02:Kl:H4 (hemolysin-negative fecal isolate). Hemolysis was detected on blood agar plates containing washed or unwashed sheep blood. Plates incubated were initially checked at 3 h. Hemolysis at this time on unwashed blood agar plates was regarded to be due to α-hemolysin, whereas hemolysis after overnight incubation indicated all types of hemolysis. Lysis on blood agar plates containing washed erythrocytes was regarded to be due to the presence of enterohemolysin. All hemolysin-positive strains along with other nonhemolytic E. coli strains (n = 30) were sent to Germany for molecular characterization.
Serogroup and Motility Factor
All antisera against the E. coli O and H type used for serogrouping confirmation and motility factor study were carried out in Germany. For reference, the O- and H-typing strains from the International Escherichia and Klebsiella Reference Laboratory in Copenhagen were used.  O-typing was done with antisera prepared with boiled bacteria to get rid of the proteinaceous surface compounds and capsules present on living bacteria. To obtain a pure O-typing result, the bacteria were boiled before making an agglutination test either in tubes (or in microtiter plates) with serial 2-fold dilutions of the antiserum and a fixed amount of boiled bacteria added. The endpoint titer of the investigation was taken as that obtained not more than 1 dilution to the titer obtained with the O Reference strain, always included in the assay.  Motility factor was detected by doing the H-typing with antisera made against flagellar antigens H1-H56 by titration of formalin-inactivated motile cultures of bacteria. Nonmotile E. coli strains were investigated for the H-type-specific (flic) genes by polymerase chain reaction (PCR) followed by Hha1 digestion of flic PCR products and evaluation of restriction fragment length polymorphism patterns as described previously. ,
Detection of Virulence Properties and Associated Genes
All strains were investigated for the production of cytotoxins by the Vero cell toxicity test as described previously.  All the strains were also studied for the presence of Shiga toxins, that is, stx genes (stx1 or VT1 and stx2 or VT2) by PCR and restriction endonuclease digestion of PCR products as described previously.  Hemolytic activity was detected by growing bacteria on washed sheep blood agar plates for phenotypic characterization of enterohemolytic and α-hemolytic strains of E. coli. Strains showing hemolytic activity were analyzed for α- and EHEC-hemolysin-specific genes by PCR.  Detection and subtyping of intimin (eae) genes were performed as described by Zhang et al. Genes encoding heat-labile enterotoxin (LT-1) and heat-stable enterotoxin (STa) were detected as described previously.  Enteroaggregative E. coli (EAEC)-specific DNA sequences were detected by dot blot DNA hybridization. A 765-bp PCR product from the EAEC plasmid pCVD432  was obtained with primers pCVD432/Start and pCVD432/Stop  and used as a gene probe. E. coli strains 17-2 and HS were used as positive and negative controls, respectively.  EAST1 gene was detected as described previously by Yamamoto and Echeverria.  Inv plasmid-associated DNA sequences were specifically detected with the EIEC gene probe as described previously  and by ial PCR. 
Hep-2 Cell Adhesion Test
The HEp-2 cell adhesion test was performed as described previously.  Bacteria were allowed to absorb for 3 h or, alternatively, for 6 h before the cells were washed with PBS, fixed with 70% methanol and stained with 10% Giemsa (Merck, Gibbstown, New Jersey, USA). Adhesion tests were performed in the presence and absence of d-mannose (1%, wt/vol) in the growth medium.  The following E. coli strains served as positive controls for different adhesion types: E. coli strains E20513 (O111:H2 eae+ eaf+ LA+),  DH5(pSSS1) for DA,  and 17-2 for enteroaggregative adherence.  Strains E20518 (O128:H2, lacking eae and eaf) and HS served as negative controls for local/diffuse adherence and enteroaggregative adherence, respectively. 
| Results|| |
Fecal samples initially screened and excluded for various other routinely encountered enteropathogens were primarily cultured for E. coli. Of the 244 stool samples cultured, various microorganisms grew in 142 samples. Pure or predominant E. coli grew in 100 of these samples of which only 25 were typable and 2 others were rough strains. Thirty of the E. coli strains inclusive of 25 typable and 5 O-untypable (ONT) were sent to Germany for further characterization. The isolates belonged to 14 different O-serogroups. They were 5 O153, 4 O102, 3 O25, 2 each of O130 and O169, and 1 each of O1, O8, O15, O37, O86, O101, O127, O143, and O160. The demographic and clinical profile of patients (n = 30) whose E. coli were characterized is given in [Table 1]. Diarrhea was present in 100% of them with bloody diarrhea in 30%, and diarrhea with mucus in 7%. Apart from this, fever was recorded in 23% and pain abdomen in 53% of these patients.
α-Hemolysin was detected from 20% of the typable E. coli isolates phenotypically as well as genotypically. Three of these belonged to O153 serotype and 1 each to O130 and O102 serotype. Two of the serotypes (O86:H30 and O153:H4) were also positive phenotypically for enterohemolysin. No VTEC, EPEC, ETEC, or EIEC were found in the present study as characterized by molecular methods for verotoxin, intimin, LT1, STa, and ial even though 7 of the O-typable E. coli serogroups belonged to pathotypes considered to be ETEC, 3 to EPEC, and 1 to EIEC. However, 3 EAEC were isolated from the randomly isolated E. coli. Two of the isolates were positive for the ST associated with EAGG (EAST) gene. In addition to this, there was one O153 isolate that was positive for EAST only. All EAEC strains were found to adhere in the characteristic brick stage manner to the cultivated HEp-2 cells. Between the two 0130:H6 strains isolated, one belonged to EAEC pathotype. The molecular markers of the 30 E. coli isolates are shown in [Table 2].
Non-E. coli isolates that grew in pure or predominant culture were Klebsiella sp. (15/142), Enterobacter and Proteus (5/142) each, Streptococcus (4/142), Serratia (3/142), and Pseudomonas, Staphylococcus, and Candida in one each (1/142). Apart from these, Shigella flexneri was also isolated in 7/142 of the stool samples.
| Discussion|| |
The association of enterohemolysin with verotoxin (VT) is well established (more than 95% of the isolates) in classical EHEC strains. But VT-positive strains may also belong to other serotypes that do not encode and produce the EHEC hemolysin because they do not carry the EHEC virulence plasmid, which encodes the hemolysin and other virulence-associated genes. Thus, E. coli strains may contain EHEC hly and carry the plasmid but remain VT negative. These can be VTEC, which have lost their VT gene. However, non-VT strains of some type might occasionally carry the hly gene. In the present study, all our hemolysin-positive strains were negative for VT gene probably due to loss during subculture. Alternatively, the strains could be VT negative and hly positive. Beutin et al reported that the EHEC hlyA gene was absent in both strains that had an α-hemolytic phenotype (ONT:H10 and O rough:H45), but were positive for hlyA gene. One of our hemolytic strains (ONT:H31) was negative for both hlyA and EHEC hlyA sequences and could possibly be producing a different type of hemolysin. Similarly, 2 of our isolates were positive phenotypically for enterohemolysin but no EHEC hlyA gene was detected in them. However, one of the isolates (O153:H4) was positive for α-hemolysin phenotypically and genotypically. The association between infections with intimin-positive STEC and severe disease in humans was demonstrated previously,  but it was also shown that intimin is not essential for the virulence of certain STEC strains for humans. Beutin et al had previously reported that infections with eae-positive STEC are associated with young age but that eae-negative isolates are more frequently isolated from adult patients. Protective immunity to intimin may be acquired in early childhood due to infections with eae-positive EPEC and STEC strains , and this may explain why these strains are less frequently isolated from adults. Moreover, adults are principally more exposed to STEC strains from nonhuman sources due to occupational contact with animals, food, and the environment, and the majority of STEC strains from these sources are negative for intimin. , Our E. coli isolates were negative for both VT and eae implying the presence of yet another variant of eae gene, which could not be detected in the present study. α-hemolysin, a characteristic trait of porcine STEC strains,  indicate their porcine origin. It is likely that the α-hemolysin isolates detected in the present study could have a porcine origin as pigs are commonly reared in this region.
It may be possible that in India VTEC may be rare though not ETEC and EPEC  quite in contrast to Germany, where EPEC and ETEC are a few to absent, but VTEC is present in 2-3% of diarrheal cases. From the epidemiologic point of view, the most important non-O157 VTEC serotypes are O26:H11/HNM, O103:H2, O111:HNM, and O145:HNM. Vaz et al identified 29/2607 VTEC belonging mainly to O111 nonmotile, O111:H8 and O26:H11 in Brazil. In earlier studies, Vaishnavi and co-workers reported the isolation of O26 and O111  as well as O153 from pediatric patients,  although no H-typing and molecular study were carried out.
In the present study of the 5 O153 isolated, 3 were positive for α-hemolysin and 1 for enterohemolysin. Thus, sometimes nonhemolytic O153 strains may also be detected because hemolysin might not be a stable marker associated with all the O153 strains. It is unclear whether the O153 strains isolated from humans in the present study were closely associated with cattle even though transmission from animals to humans has been reported.  Wolk et al studied endemic occurrence of E. coli infections and found that 83% of isolates of E. coli belonged to 1 of 4 predominant serotypes (O153:H31, O101:H-, O2:H42, and O102:H6) in hospitalized patients. In the present study, 3 of the same serogroups (O153, O101, and O102) were detected, however, with different H types except for O102 all of which had the same H types (H6) and one of them was also positive for α-hemolysin phenotypically and genotypically.
ETEC O15 strains are mainly represented by serotypes O15:H11 and O15:H45.  This serogroup shows a high diversity in both their flagellar antigens and their virulence attributes. However, our lone O15:H15 isolate was found to be negative for all the virulence characters.
Enteroaggregative E. coli has been implicated as an emerging cause of persistent and acute diarrhea in developing and developed countries. , It has recently been shown that EAEC can induce growth impairment and malnutrition among children regardless of diarrhea.  Bhan and co-workers [34,35] reported significantly higher occurrence of EAEC in persistent diarrhea in a cohort of rural children and in hospital-based case-control study in India. Pai et al reported the occurrence of a diarrheal outbreak in South India due to EAEC contaminating drinking water source. EAEC has been reported to cause traveller's diarrhea and persistent diarrhea in children in developing countries with 19% reported in Goa and 33% in Guadalajara.  EAEC is an important etiologic agent of acute diarrhea among infants in and around Kolkata.  It is noteworthy that 10% of the randomly isolated E. coli strains in the present study were found to have EAEC of which two thirds had the EAST gene, which is frequent in these strains. In addition, only one strain of O153 was EAST positive. Other interesting results obtained were that of two other strains belonged to serotype 0130:H6. One was EAEC positive and the other was not. It can be speculated that the other had lost the EAEC plasmid and therefore was negative during EAEC gene probe. Similar could be the case with 1 0153:H6 serotype showing EAEC gene positivity and another showing EAST positivity. Kahali et al reported 90.1% of the EAEC strains positive for EAST and adherence.
Dutta et al isolated EAEC belonging to several O serogroups from 9% of diarrhea cases. Our randomly isolated EAEC belonged to 3 different serogroups, namely, 0127, 0130, and 0153. Study by Czeczulin et al revealed that EAEC strains are heterogenous with respect to chromosomal and plasmidborne genes but that the majority harbor a member of a conserved family of virulence plasmids. In our study, two thirds of the EAEC had the EAST gene as observed by PCR studies. Fernandez-Prada et al reported that α-hemolysin in EAEC appears to be critical for both oncosis and apoptosis in cell cultures. We have 1 EAEC with α-hemolysin detected both by phenotypic and genotypic methods.
The absence of VTEC in our isolates in the present study could be due to selection criteria or these pathogens may in fact not be present in our patient group. However, we have reported the presence of both EPEC and ETEC in our earlier studies. , The data obtained in the present study emphasize the need for additional research into the role of the eae gene or other putative factors affecting the virulence of VTEC strains. The finding of ONT strains in this study suggests that further O types need to be designated.
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Department of Gastroenterology, PGIMER, Chandigarh
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]
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