Phenotypic Characteristics of Enterotoxigenic Escherichia coli Associated with Acute Diarrhea Among Israeli Young Adults

Abstract

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea among infants and children in developing countries, as well as among travelers to these areas. The major virulence factors of ETEC are the colonization factor antigens (CFAs) and a heat-labile enterotoxin (LT) and/or a heat-stable enterotoxin (ST). Among Israeli recruits serving under military field conditions, 107 of all examined isolates expressed LT or ST, and CFAs could be characterized in 68% of the isolates, in which CFAs of the CFA/II group and CS6 were the most prevalent. Additionally, 31% of the 107 ETEC isolates showed resistance to three or more of the antimicrobial agents examined, and the percentage of resistant isolates expressing LT was significantly higher than those expressing ST or LT+ST. These results may be important for development of an effective vaccine and for facilitation of an empirical choice of antibiotic treatment or prophylaxis for traveler's diarrhea in this area.

Introduction

E nterotoxigenic Escherichia coli (ETEC) is a major cause of morbidity and mortality among infants and young children in developing countries (Nataro and Kaper, 1998; Qadri et al., 2005), for example, in Bangladesh (Qadri et al., 2000), Egypt (Abu-Elyazeed et al., 1999; Rao et al., 2003), and South America (Levin et al., 1993; Viboud et al., 1999), and also causes ∼25%–45% of traveler's diarrhea (Nataro and Kaper, 1998; Ericsson, 2003; Qadri et al., 2005). Diarrhea caused by ETEC is also an important medical problem for military personnel stationed in the developing world or under field conditions where sanitation is inadequate. American troops experienced high rates of diarrheal diseases during various military operations in the Middle East (Hyams et al., 1991; Wolf et al., 1993). Diarrheal rates of over 50% were experienced in some of the units under surveillance, making diarrhea one of the most important medical problems encountered during military operations.

Adequate conditions of sanitation, food handling, and hygiene should be sufficient to prevent diarrhea. However, in military populations serving in field units it is frequently difficult, if not impossible, to provide and maintain such conditions. Thus, effective vaccination may be an important means to reduce the attack rate of ETEC-associated diarrhea.

ETEC adhere to the epithelium of the small intestine by means of colonization factor antigens (CFAs) or coli surface antigens (CSs) (Gaastra and Svennerholm, 1996; Qadri et al., 2005). Among human ETEC strains, >22 serologically distinct CFAs have been described (Gaastra and Svennerholm, 1996; Qadri et al., 2005), with most strains expressing either one or two antigenic types on their surfaces. Further, although ETEC bacteria may express more than one CF, CF expression by wild-type ETEC appears to be restricted so that native strains only express CFs in certain combinations. Thus, for example, in the CFA/II group, CS1 and CS2 are generally expressed with CS3, but CS3 can also be expressed alone. Similarly, in the CFA/IV group, CS4 and CS5 are generally expressed together with CS6, but CS6 can also be expressed alone (Gaastra and Svennerholm, 1996; Qadri et al., 2005). Colonizing bacteria are noninvasive but induce fluid and electrolyte secretion into the lumen of the small intestine by the production of heat-labile enterotoxin (LT) or heat-stable enterotoxin (ST) (Sack, 1980; Qadri et al., 2005; Sánchez and Holmgren, 2005), which initiate a cascade of biochemical changes within enterocytes that results in the watery diarrhea characteristic of ETEC disease.

Infections with ETEC are often asymptomatic in people living in endemic areas (Nataro and Kaper, 1998). In developing countries, the incidence of ETEC-associated diarrhea is highest during the first 5 years of life (López-Vidal et al., 1990; Qadri et al., 2005), and children and adults from industrialized areas who travel to these countries are susceptible (Black, 1990; Von Sonnenburg et al., 2000). Data obtained in these and other studies suggest that the immune responses to CFAs contribute to protection (Evans et al., 1988; Qadri et al., 2005; Rao et al., 2005; Tobias et al., 2008).

Although several studies have shown data on the clinical characteristics of ETEC-associated diarrhea among children in endemic areas for ETEC (Qadri et al., 2005), such data among adults are limited. Additionally, several studies have shown the distribution of ETEC enterotoxins and CFAs among ETEC isolates; however, these data are mostly based on ETEC strains isolated from subjects in Africa, Asia, and Latin America (Wolf, 1997; Qadri et al., 2005) or among American soldiers and young adults (El-Mohamady et al., 2006; Rockabrand et al., 2006). Limited data are available on the phenotypic characterization of ETEC among young adults in Israel and Middle East, including the prevalence of the CFAs and the enterotoxins.

Israel is a developed country that is highly endemic for enteric diseases, in general, and ETEC-associated diarrhea, in particular. Young children and young soldiers serving under field conditions are at highest risk. The aims of the present study were to characterize the ETEC strains isolated from cases of diarrhea occurring among Israeli soldiers in field training units and to examine the clinical manifestations of ETEC-associated diarrhea among these subjects. The information from the present study is important for both ETEC treatment and prevention policies.

Materials and Methods

Study population

Cohorts of soldiers aged 18–20 years and serving in field units were kept under clinical and laboratory surveillance for periods of 3–6 months during the summer training cycles of the years 1993–1996 and 1999–2001.

In these units, food was prepared in field kitchens and water supplies were chlorinated. Subjects had access to either flushing toilets or nonflyproof trench latrines. They were encouraged to wash their hands after defecation and before eating. The density of the fly population in these field units is often high. We have reported in the past that the flyborne route is an important mode of transmission of Shigella and ETEC in field military units (Cohen et al., 1991).

Subjects presenting at the unit clinic with gastrointestinal complaints were interviewed and examined, and stool specimens were obtained. Diarrhea was defined as passage of at least three loose stools in 24 hours.

Laboratory methods

Representatives of all nonlactose-fermenting colonies on MacConkey or Salmonella–Shigella agar or nonxylose-fermenting colonies on xylose lysine deoxycholate agar were identified by routine morphological and biochemical testing. Isolates resembling Shigella or Salmonella underwent serogrouping and serotyping. After incubation at 37°C for 48 hours in a microaerophilic environment, suggestive colonies on the Campylobacter selective medium (with Preston antimicrobial supplement) were selected for identification on the basis of bacterial morphology and oxidase positivity.

Two to 3 and 5–10 lactose-positive colonies isolated from MacConkey plates of subjects with diarrhea in 1993–1996 and 1999–2001, respectively, were screened by PCR for presence of genes encoding for LT and ST (Yavzori et al., 2000).

During 1993–1996, prescreening of the fecal specimens was performed by direct stool PCR (Yavzori et al., 2000) and two to three lactose-positive colonies were isolated and examined for presence of genes encoding for LT and ST only from specimens found positive by direct stool PCR. During the years 1999–2001, 5–10 colonies from all fecal specimens were tested by PCR. An ETEC isolate was defined as a lactose-positive colony harboring ST- and/or LT-specific DNA sequences as detected by PCR. A subject with diarrhea and from whose stool specimen ETEC was isolated was defined as a case of ETEC-associated diarrhea. One hundred fifty-seven cases of ETEC-associated diarrhea were identified among Israeli soldiers serving in field units between 1993–1996 and 1999–2001. In 145 (92.3%) of these subjects, ETEC was the only enteropathogen detected, whereas in another 12 (7.6%) cases, Shigella species were also identified (7 S. sonnei, 2 S. flexneri, 2 S. boydii, and 1 S. dysenteriae [non-Shiga]) in addition to ETEC.

Characterization of ETEC isolates

The 157 ETEC isolates were sent to the ETEC Reference Laboratory at Gothenburg University in Sweden to examine expression of LT and ST toxins as well as CFAs.

A ganglioside GM1-enzyme-linked immunosorbent assay (ELISA) and an inhibition GM1-ELISA were used for detection of LT and ST expression, respectively, as described previously (Svennerholm et al., 1983, 1986; Sanchez et al., 1990; Sjöling et al., 2007). Briefly, 96 ELISA plates were coated with GM1 (0.3 nmol/mL in phosphate-buffered saline [PBS]), and the ETEC isolates to be tested were grown in 100 μL of LB broth supplemented with lincomycin (45 μg/mL) and glucose (2.5 mg/mL) in GM1-coated microtiter wells at 37°C overnight. The released LT from the tested bacteria bounds to solid-phase GM1 and was detected using an anti-LT MAb and thereafter goat anti-mouse immunoglobulin G horseradish peroxidase, followed by using H₂O₂ and ortho phenylenediamine as substrates to visualize the results. For detection of ST, half of the bacterial suspension (50 μL) grown in the microtiter plate wells, as described earlier, was transferred to a new GM1 plate freshly coated with a covalently linked ST choleratoxin and subunit conjugate. Thereafter, an anti-ST MAb that competitively binds either free ST in the bacterial supernatant or solid-phase-bound ST choleratoxin and subunit was immediately added. Binding of the MAb was detected as described for the LT GM1-ELISA.

Enterotoxin-positive ETEC isolates were also assayed for surface expression of CFAs, that is, CFA/I, CS1-CS8, CS12, CS14, and CS17, by dot blot. The isolates were grown overnight on CFA agar with or without bile salts for optimal expression of CFAs (Evans et al., 1979). Bacterial colonies were assayed for CFA expression by dot blots, as described previously (Binsztein et al., 1991). Briefly, appropriately cultured cells were harvested by centrifugation and washed once in PBS. Washed cells were resuspended in PBS to a concentration of 10⁹ cfu/mL. Two microliters aliquots of the resulting bacterial suspension was applied to nitrocellulose filter papers, blocked with bovine serum albumin, and incubated with anti-CFA MAb, followed by goat anti-mouse IgG conjugated to horseradish peroxidase, for 1.5 hours each. The final development was performed using 4-chloro-1-naphtol-H₂O₂ in Tris-buffered saline for up to 15 minutes. Development was stopped by washing the filters with water.

Antimicrobial susceptibility

Susceptibility of the ETEC isolates to amoxicillin–clavulanate (Amc), ampicillin (Amp), chloramphenicol (Chl), ciprofloxacin (Cip), ceftriaxone (Cro), cefuroxime (Cxm), gentamicin (Gent), nalidixic acid (Nal Ac), sulfamethoxazole–trimethoprim (Sxt), and tetracycline (Tet) was determined by the Kirby–Bauer disk diffusion method (Bauer et al. 1966). The tests were carried out on Muller–Hinton medium using Oxoid antimicrobial susceptibility disks (Oxoid, Hampshire, England). Multidrug resistance was defined as resistance to three or more antimicrobials agents.

Statistical analysis

Fisher exact and χ ² tests were used to determine the significance of differences in the rates of symptoms or signs of disease, and antibiotic susceptibility related to infection caused by the different ETEC toxin phenotypes.

Results

Clinical symptoms of subjects, and enterotoxin and CFA distribution among ETEC isolates

LT, ST, and LT+ST toxin genes were identified by PCR in 77 (49.0%), 49 (31.2%), and 31 (19.7%) of the 157 ETEC cases, respectively. Among the cases of ETEC-associated diarrhea (with only ETEC isolated in the stool specimens), 96% suffered from abdominal pain, 35% vomited, and 29% reported five or more liquid stools per 24 hours. Seven percent of the patients had fever (38°C or higher), and in 50% diarrhea lasted for >4 days (Table 1). There was no significant difference in the rate of signs or symptoms of disease among patients according to the toxin of the ETEC isolate (Table 1). Among the 12 cases of ETEC-Shigella mixed infection, all suffered from abdominal pain, 25% vomited, 50% reported five or more liquid stools per 24 hours, and 1 (8.3%) had fever (38°C or higher). In 33%, diarrhea lasted for >4 days.

Table 1.

Clinical Characteristics Among Patients with Enterotoxigenic Escherichia coli–Associated Diarrhea

Signs or symptoms		LT	LT+ST	ST	Total ETEC	p-Value
Abdominal pain	No	1	2	3	6	0.72
	Yes	35 (97.2%)	28 (93.3%)	72 (96%)	135 (95.7%)
Vomiting	No	25	17	43	85	0.63
	Yes	10 (28.5%)	10 (37%)	26 (37.6%)	46 (35.1%)
Nausea	No	9	10	8	27	0.18
	Yes	13 (59%)	6 (37.5%)	16 (66.6%)	35 (56.4%)
Max. no. of stools per 24 hours	≤4	22	19	53	94	0.84
	5 or more	12 (35.2%)	10 (34.4%)	23 (30.2%)	25 (21.0%)
No. of stools per last 24 hours	≤4	29	18	55	102	0.24
	5 or more	8 (21.6%)	12 (40%)	21 (27.6%)	41 (28.7%)
Fever	<38°C	26	27	63	116	0.77
	≥38°C	2 (7%)	1 (3.5%)	5 (7.3%)	8 (6.9%)
Duration of diarrhea	≤3 days	10	11	19	40	0.23
	4 days^a or more	13 (56.5%)	5 (31.2%)	22 (70.9%)	40 (50%)

Median, 7 (range, 4–30).

ETEC, enterotoxigenic Escherichia coli; LT, heat-labile enterotoxin; ST, heat-stable enterotoxin.

Of the 157 ETEC isolates sent to the ETEC Reference Laboratory at Gothenburg University in Sweden, 107 isolates expressed LT or ST when examined by GM1-ELISA, and in 68 ETEC of the 107 isolates a CFA was characterized, as listed in Table 2. LT, ST, and LT+ST toxins were expressed in 38 (35.5%), 40 (37.4%), and 29 (27.1%) of the 107 isolates, respectively. Twenty-two percent of the isolates expressed the CFA/II group, containing CS1, CS2, and CS3. CS6 alone or in combination with other CFAs was the most prevalent CFA among the ETEC isolates, followed by CFAs from the CFA/II family (Table 2). In 3 of the 12 ETEC strains isolated in patients with ETEC-Shigella mixed infections, a CFA was characterized (1 CS1–CS3, 1 CS12, and 1 CS20). The ETEC isolates were resistant to Amc (20.5%), Amp (45.5%), Chl (4.9%), Sxt (37.5%), and Tet (21.5%). All the 107 isolates were sensitive to Cip, Cro, Cxm, Gent, or Nal Ac. Thirty three (31%) of the 107 ETEC isolates were resistant to three or more of the antimicrobial agents examined. Of the 12 ETEC isolated in patients with ETEC-Shigella mixed infections, 50%, 75%, 8.3%, 75%, and 66.7% were resistant to Amc, Amp, Chl, Sxt, and Tet, respectively. All were sensitive to Cip, Cro, Cxm, Gent, or Nal Ac.

Table 2.

CF Distribution Among Israeli Enterotoxigenic Escherichia coli Isolates

	Number (%) of ETEC isolates with defined toxin and CF phenotypes
	LT	LT+ST	ST	All ETEC
CF	n = 38	n = 29	n = 40	n = 107
CFA/I		1 (3.5)	3 (7.5)	4 (4)
CFA/II		22 (75.5)	1 (2.5)	23 (21.5)
CS1+CS3		10		10
CS2+CS3		11	1	12
CS3		1		1
CFA/IV			10 (25)	10 (9)
CS6			10	10 (9)
CS6+CS8	8 (21)		0	8 (7.5)
CFA/I+CS21			1 (2.5)	1 (1)
CFA/I+CS6+CS21			1 (2.5)	1 (1)
CS1+CS3+C21		1 (3.5)		1 (1)
CS2+CS3+CS6		1 (3.5)		1 (1)
CS5+CS6+CS7			10 (25)	10 (9)
CS12	1 (3)	2 (7)	1 (2.5)	4 (4)
CS17+CS19	4 (10)			4 (4)
CS20			1 (2.5)	1 (1)
No CF detected	25 (66)	2 (7)	12 (30)	39 (36)

CFA, colonization factor antigen; CS, coli surface antigen.

We also determined the resistance rate of the ETEC isolates in relation to enterotoxin expression. As shown in Table 3, except the case of Chl in which the number of the resistant isolates was low, the rate of resistance was significantly higher among isolates with LT phenotype than among isolates expressing ST or LT+ST toxins.

Table 3.

Number and Percentage of LT or ST/LT+ST Enterotoxigenic Escherichia coli Isolates Resistant to Various Antibiotics

	Number (%) of resistant ETEC isolates
Type of expressed toxin	Amc	Amp	Sxt	Chl	Tet
LT	16 (43.2)	28 (75.7)	23 (62.2)	1 (2.7)	13 (35.1)
ST/LT+ST	6 (9.4)	18 (28.1)	16 (25.0)	4 (6.3)	10 (15.6)
p-Value	<0.01	<0.01	<0.01	0.65	0.029

Amc, amoxicillin–clavulanate; Amp, ampicillin; Chl, chloramphenicol; Sxt, sulfamethoxazole–trimethoprim; tet, tetracycline.

Discussion

ETEC is one of the most common types of diarrheagenic E. coli causing diarrhea in infants and children in developing countries and in travelers to these areas. The main purpose of the study was to characterize the distribution of the different CFAs of clinical ETEC strains that were isolated from Israeli army recruits, in association with enterotoxin production and antimicrobial resistance. The clinical features of the young adult patients from whom ETEC strains were isolated were characterized.

Young recruits changing abruptly their home environment living conditions to the worse ones in field units can be in some regard analogous to adults from countries of low diarrheal disease endemicity traveling to countries highly endemic for diarrheal diseases.

The CFA/II group, consisting of CS1, CS2, and CS3, is the most prevalent CFA among the tested isolates, followed by CS6. Several studies carried out to determine the prevalence of CFAs in ETEC strains isolated from infants and children (Qadri et al., 2005) showed that CFA/I is the most prevalent CFA, followed by CFA/II, CS5+CS6, or CS6. However, the number of such studies conducted among adults, especially in the Middle East, is very low (Qadri et al., 2005; Rao et al., 2005). Our results are in agreement with those reported by Rockabrand et al. (2006), which showed that CS6 and the CFA/II group were the most prevalent CFAs expressed by ETEC clinical isolates from American soldiers during the Operation Bright Star exercise in Egypt. These findings may indicate that certain CFA-expressing ETEC strains causing diarrhea are particularly prevalent in the Middle East and/or among young adults.

Although no significant differences were found in the rates of signs or symptoms of disease among patients according to ETEC toxin phenotype, a trend to more intense symptoms among subjects infected with ST-expressing ETEC was observed. It has been shown that among children who were infected with ETEC expressing ST and LT+ST, the severity of disease was greater than in those infected with ETEC expressing only LT (Qadri et al., 2000).

The antimicrobial susceptibility of the ETEC isolates was determined and the highest rate of resistance found was to Amp and Sxt, with lower resistance rates found to tetracycline and Amc. Almost a third of the isolates showed multidrug resistance. A similar extent and pattern of resistance of ETEC isolates has been previously reported (Shaheen et al., 2004; Al-Gallas et al., 2007).

ETEC isolates expressing LT displayed a significantly higher resistance in vitro to various antimicrobial drugs when compared with isolates expressing ST or LT+ST. These results are in agreement with data previously reported by Turner et al. (1998), although in the present study, in addition to Amc and Amp, significant differences in resistance rates also exist to Sxt and tetracycline. A possible explanation for this finding could be that ETEC isolates expressing only LT have a lower pathogenicity index when compared with ETEC ST or LT+ST with LT being similarly present among cases of diarrhea and asymptomatic controls as shown in several case–control studies in highly endemic areas (Qadri et al., 2005; Gupta et al., 2008). As a more prevalent commensal organism, ETEC LT has a greater chance of being exposed to antimicrobial drugs, whereas ST-expressing ETEC isolates, which are more virulent and more rapidly cured from the body, may have a lesser chance of acquiring resistance to antimicrobials.

The collection of samples and clinical and epidemiological data were conducted in the mid to late 1990s till 2001 and this might be a limitation of this report. However, we consider that the presented data such as clinical manifestation of ETEC-associated diarrhea in young adults and the association of LT expression but not ST with increased antibiotic resistance are of current interest. We cannot rule out the possibility that the overall antibiotic antimicrobial susceptibility patterns and the relative distribution of ETEC isolates expressing various CFAs may change from time to time, and therefore, additional studies are needed.

Conclusions

This study reports the distribution of different CFAs of ETEC strains isolated in Israeli young adults, with CFA/II group as being the most prevalent. These data may influence the design of an effective broadly protective vaccine incorporating multiple CFAs as protective antigens. Moreover, this study shows a significantly higher antibiotic resistance of ETEC isolates expressing LT, but not ST or LT+ST. The data are useful for the empirical choice of antibiotic treatment for ETEC-associated diarrhea when indicated.

Footnotes

Disclosure Statement

No competing financial interests exist.

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