1997, 35(7):1757.

J. Clin. Microbiol.

T Pal, N A Al-Sweih, M Herpay and T D Chugh

assay.

IpaC-specific enzyme-linked immunosorbent

and Shigella strains in pediatric patients by an

Identification of enteroinvasive Escherichia coli

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J

OURNAL OF

C

LINICAL

M

ICROBIOLOGY

,

0095-1137/97/$04.00

10

July 1997, p. 1757–1760

Vol. 35, No. 7

Copyright © 1997, American Society for Microbiology

Identification of Enteroinvasive Escherichia coli and Shigella

Strains in Pediatric Patients by an IpaC-Specific

Enzyme-Linked Immunosorbent Assay

TIBOR PAL,

1,2

* NOURA AHMED AL-SWEIH,

3

MARIA HERPAY,

4

AND

TULSI D. CHUGH

1

Department of Microbiology, Faculty of Medicine, University of Kuwait,

1

and Mubarak Al-Kabeer Hospital,

3

Kuwait,

and Department of Microbiology, University Medical School, Pecs,

2

and Department of Bacteriology, “Bela Johan”

National Institute of Public Health, Budapest,

4

Hungary

Received 20 November 1996/Returned for modification 4 March 1997/Accepted 27 March 1997

A new method, a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) recognizing a

secreted, invasion plasmid-coded protein antigen (IpaC), was used to identify enteroinvasive Escherichia coli

and Shigella strains among colonies from 859 cultures of fecal samples from children in Kuwait. A total of

33.8% of the samples were diarrheal. By the immunoassay, enteroinvasive E. coli strains were identified from

two diarrheal samples but from none of the samples from children without diarrhea. These strains were fully

virulent and belonged to serogroup O28ac. In addition, 26 Shigella strains were also recognized by the ELISA,

while only 23 were isolated by routine biotyping and serotyping. For two diarrheal patients, Shigella was

identified by culture only. The study showed that the IpaC-specific immunoassay is a simple and useful tool for

identifying enteroinvasive strains. Furthermore, by reporting the first enteroinvasive E. coli isolates from

Kuwait, the study indicates the presence of this group of pathogens as a potential source of diarrhea in the

region.

The microbiological diagnosis of bacillary dysentery is based

on the selective isolation and biochemical identification of the

four Shigella species, S. dysenteriae, S. flexneri, S. boydii, and S.

sonnei. The result is then confirmed and refined by determin-

ing the serotype of the isolate (3). Recently, in order to im-

prove the sensitivity, specificity, and speed of the diagnosis,

new molecular biological methods have been developed. These

techniques are based either on nucleic acid hybridization with

DNA probes specific for various virulence-related genes (2, 25)

or on a PCR technique with primers flanking these sequences

(9). Some target genes are located on the invasion plasmid (IP)

or, like the ipaH gene, on both IP and the chromosomes of

these pathogens (21, 25). These methods were successfully

used in case-control studies (5) or investigations aimed at de-

tecting posttreatment Shigella carriage (21).

A definite advantage of these molecular biological tech-

niques is that they also identify the other, often overlooked

causative agents of bacillary dysentery, enteroinvasive Esche-

richia coli (EIEC) strains carrying the same virulence-related

genes carried by shigellae (11). The enteroinvasive character

had long been recognized in some 10 E. coli serogroups (6);

however, not all members of these serogroups express the

invasive phenotype. Although invasive isolates are often non-

motile, lactose negative, and lysine decarboxylase negative

(22), the detection of these markers, similar to that of the O

antigens, is not specific or sensitive enough for diagnostic pur-

poses (3). An important reason for our rather limited under-

standing of the epidemiology of EIEC infections is the diffi-

culty in identifying these strains by simple laboratory

techniques.

In order to offer a specific and simple technique for the

microbiological diagnosis of enteroinvasive infections, we de-

veloped an enzyme-linked immunosorbent assay (ELISA) that

can be used to identify EIEC and Shigella strains (17). In the

original form of the method, an absorbed rabbit antiserum

recognizing IP-coded antigens (tentatively called virulence

marker antigen [VMA]) was used (17). Although the proce-

dures for the preparation and standardization of the absorbed

sera were cumbersome, the VMA ELISA proved the feasibility

of this approach (24). Recently, the assay was further devel-

oped and simplified by introducing a monoclonal antibody

(MAIC-1) specific for IpaC (8), a component of the tentative

VMA (19). While growing in the microcultures of the wells of

the ELISA plate, enteroinvasive bacteria secrete IpaC (1), thus

sensitizing the plate for antigen detection by the monoclonal

antibody (8). So far, however, the specificity and sensitivity of

this assay have been assessed with a panel of selected invasive

and noninvasive strains only (8), but they have not been eval-

uated with clinical material. Here we report the results of the

first clinical application of the MAIC-1 ELISA to the identi-

fication of EIEC and Shigella strains in pediatric patients.

MATERIALS AND METHODS

Patients.

A total of 859 fecal samples received from pediatric patients pre-

senting at the Mubarak Al-Kabeer Hospital, Kuwait, were investigated. All fecal

samples received were included in the investigation, no preliminary criteria

concerning the clinical diagnosis or the symptoms of the patients being used. No

special cautions were exercised to exclude from the study samples sent repeat-

edly from the same patients.

Routine bacteriology, biotyping, and serotyping.

The fecal samples were pro-

cessed in the Microbiology Laboratory of Mubarak Al-Kabeer Hospital accord-

ing to the guidelines for public health laboratories in Kuwait (13) for common

bacterial enteric pathogens, i.e., Shigella, Salmonella, Campylobacter, and enter-

opathogenic E. coli. The macroscopic appearance of the specimen was recorded

as formed, loose, or watery, and the presence of blood or mucus was noted.

Loose and watery samples were microscopically examined for the presence of

erythrocytes and leukocytes (RBCs and WBCs, respectively). For the isolation of

Shigella, MacConkey (MC) (Oxoid, Unipath Ltd., Basingstoke, United King-

dom) and salmonella-shigella (SS) (Difco Laboratories, Detroit, Mich.) agar

plates were inoculated before and after Selenite-F broth (Mast Laboratories

Ltd., Herseyside, United Kingdom) enrichment. The plates were investigated by

the technical personnel of the Microbiology Laboratory of Mubarak Al-Kabeer

* Corresponding author. Present address: Department of Microbi-

ology, University Medical School, H-7643 Pecs, Hungary. Phone: 36

(72) 324122. Fax: [phone]. E-mail: [e-mail].

1757

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Hospital. Colonies suspected of being Shigella were isolated on Kligler’s iron

agar slopes (KIA; Mast Laboratories Ltd.) and urea broth. On the next day, KIA

cultures showing reactions typical of Shigella were serotyped with a set of Shigella

typing sera (Murex Diagnostic Ltd., Dartford, England). The species was con-

firmed in the Vitek automatic system by using the Vitek GNI card (bioMerieux,

Marcy l’Etoile, France). Agglutination of eight to nine colonies from the MC

agar plate for samples from patients younger than 2 years of age was done with

Murex diagnostic pooled sera 2, 3, and 4 for pathogenic E. coli. Serotyping of

suspected EIEC isolates identified by MAIC-1 ELISA (see below) were carried

out with diagnostic sera produced at the Department of Bacteriology, “Bela

Johan” National Institute of Public Health, Budapest, Hungary.

MAIC-1 ELISA.

After isolating colonies for biotyping and serotyping, MC and

SS agar plates with primary cultures were used to pick colonies for the MAIC-1

ELISA. A person unaware of the culture results selected three colonies, possibly

with different morphologies and lactose fermentation patterns, from each plate.

The only restriction in choosing various colony types for the immunoassay was

that colonies with signs of H

2

S production on the SS plate were left untouched.

Altogether, six colonies from each patient’s sample were individually inoculated

into 6 wells of a 96-well sterile tissue culture plates (Nunclon Delta; Nunc,

Roskilde, Denmark) (hereafter referred to as ELISA plate) filled with 200

ml of

tryptic soy broth. Simultaneously, these colonies were also inoculated onto a

Congo red (CR) plate (15) as macrocolonies.

No perimeter rows or columns of the 96-well plates were used for the assays.

On each plates three wells were inoculated with a virulent (YSH 6000) and a

nonvirulent (YSH 6200) S. flexneri 2a strain (provided by C. Sasakawa, Tokyo,

Japan), serving as positive and negative controls, respectively, in the immuno-

assay.

Inoculated ELISA and CR plates were incubated overnight at 37°C. On the

next day the MAIC-1 ELISA was run as described previously (8). Briefly, the

microcultures in the wells were discarded and the free binding sites were blocked

for 1 h at room temperature with 1% bovine serum albumin (BSA) in phosphate-

buffered saline (PBS; pH 7.2). The plates were washed three times with PBS

containing 0.05% Tween 20 (PBS-Tw), and then the IpaC-specific monoclonal

antibody was added as a serum-free supernatant of the cell culture medium

(RPMI 1640; Gibco BRL, Life Technologies Ltd., Paisley, England) of the

hybridoma cell line MAIC-1 (8), which was diluted 1:250 in PBS-Tw containing

0.1% BSA (PBS-Tw-BSA). The plates were incubated for 1 h at 37°C, washed,

and reacted with anti-mouse immunoglobulin–horseradish peroxidase (P260;

Dakopatts, Copenhagen, Denmark). Reactions were developed with 1,2-phe-

nylenediamine substrate (Dakopatts), and the optical density (OD) at 490 nm

was measured on a Dynatech MR 5000 ELISA reader.

Previously (8), it was shown that a cutoff value for the positive reaction in the

ELISA, defined as the mean

1 3 standard deviations for nonvirulent isolates,

differentiated between enteroinvasive and nonenteroinvasive strains. In prelim-

inary experiments with a set of virulent and nonvirulent Shigella and EIEC strains

from our strain collection, this value was determined for the purpose of the

present study to be 0.150. In case of a positive reaction (i.e., OD

$ 0.150) the

corresponding colony taken from the CR plate was retested by the MAIC-1

ELISA. In order to recognize isolates binding to the enzyme-conjugated anti-

mouse antibodies (i.e., false-positive results), the assay was also carried out with

the anti-mouse–horseradish peroxidase conjugate only, replacing the IpaC-spe-

cific antibodies with PBS-Tw-BSA.

Clones that were saved on the CR plate and that showed a positive reaction in

the ELISA and all six selected clones from samples from which an enteroinvasive

pathogen was isolated in the hospital laboratory were also subjected to biochem-

ical and serological identification, as described above.

Confirmatory assays for enteroinvasiveness.

All Shigella and E. coli isolates

recognized by the MAIC-1 ELISA were tested for their capacity to invade HEp-2

cells (10). After Giemsa staining of the infected tissue cultures, the invasive

potentials of the strains were calculated as the number of infected cells of 200

cells examined and was expressed as the percentage of that for the positive

control strain, strain YSH 6000. The E. coli isolates exhibiting a positive reaction

in the immunoassay were also investigated for the expression of the characteristic

pattern of invasion plasmid-coded proteins detected by sera from convalescent-

phase dysenteric patients (16) received from Alf A. Lindberg, Stockholm, Swe-

den. The extraction of bacteria, polyacrylamide gel electrophoresis, and Western

blotting (immunoblotting) were carried out as described previously (19). As

ultimate proof of the enteroinvasive character, E. coli isolates positive by the

assays described above were subjected to the Sereny test (i.e., the guinea pig

keratoconjunctivitis assay) (20).

RESULTS

Altogether, 859 samples were included in the study. On the

basis of macroscopic observations, 33.8% of the samples were

scored as loose or watery, of which 8.2% contained macro-

scopic blood. Microscopically, RBCs were detected in 23.4% of

the diarrheal samples and WBCs were detected in 27.5% of the

diarrheal samples. Sixty-eight nontyphoid Salmonella and 12

Campylobacter jejuni strains were isolated. One patient ex-

creted both Salmonella enteritidis and C. jejuni. From the pa-

tients younger than 2 years of age, 11 E. coli strains belonging

to enteropathogenic E. coli serogroups were isolated.

The results of the isolation and identification of enteroinva-

sive bacteria are presented in Table 1. Shigella species were

identified either by culture or by ELISA in 28 samples, all from

different patients. All 28 samples were marked as loose or

watery (3.25% of the total and 9.62% of the diarrheal sam-

ples). Twenty-six of these samples contained RBCs and WBCs,

and 8 of the samples were macroscopically bloody. By bio-

chemical and serological assays, Shigella organisms were iden-

tified, all from the primary culture plates, in 23 samples, and no

further strains were found after enrichment in Selenite-F

broth. By MAIC-1 ELISA, members of the genus were de-

tected among colonies from 26 samples. In four of the five

samples in which Shigella strains were missed by routine cul-

ture, the pathogen grew as a few colonies of non-lactose fer-

menters (NLF) on the MC agar plate only in a heavy lactose

fermenter background. In the fifth case a few NLF colonies of

S. dysenteriae were also missed on the SS plate in a similar

background.

The MAIC-1 ELISA did not detect one S. sonnei isolate and

one S. flexneri isolate from diarrheal patients in whom the

pathogen was identified by culture (Table 1). By microscopy,

RBCs and WBCs were detected in both mucoid samples, while

no macroscopic blood was seen. Reinvestigating the colonies

tested by ELISA from the S. sonnei-infected patient revealed

that two of the six colonies (both taken from the MC agar

plate) were biochemically S. sonnei, indeed, agglutinating in

the phase I and II typing sera. However, both clones were CR

negative and lost their invasive capacity, as tested on tissue

culture cells, indicating the loss of the IP coding for the target

antigen of the immunoassay (data not shown). No phase I,

CR-positive, invasive, ELISA-positive colonies were found on

reinvestigations of 20 S. sonnei colonies from the MC and SS

agar plates. In the sample from the other patient excreting

Shigella but in which Shigella was not identified by the immu-

noassay, two of the six colonies tested proved to be S. flexneri.

The colonies were taken from MC and SS agar plates, respec-

tively. Similar to the S. sonnei isolate, these colonies did not

bind to CR and they were noninvasive (data not shown). How-

ever, after reinvestigating the SS and MC agar plates contain-

ing the sample from this patient, 5 ELISA-positive, invasive

colonies of 20 colonies tested were found.

By the MAIC-1 ELISA we could identify two E. coli strains

secreting the IpaC antigen. They expressed the O28ac cell wall

TABLE 1. Identification of enteroinvasive bacteria among colonies

isolated from 859 fecal samples

Species

No. of strains identified

By ELISA

By biotyping

and serotyping

Total

a

S. dysenteriae

1

0

1

S. flexneri

13

12

14

S. boydii

0

0

0

S. sonnei

12

11

13

All Shigella

26

23

28

EIEC

2

0

2

Total

28

23

30

a

Total number of strains identified by ELISA and/or biotyping and serotyping.

1758

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antigen and fermented lactose, but they were unable to decar-

boxylate lysine. Both isolates were nonmotile. The isolates

formed red colonies on CR plates, and they were invasive in

the tissue culture invasion assay, showing 15.2 and 12.3% in-

vasive capacities compared to that of the positive control

strain. They expressed the characteristic protein pattern (i.e.,

VirG and Ipa A, B, C and D) (16) of the virulent strains when

tested with reconvalescent-phase human serum by polyacryl-

amide gel electrophoresis-Western blotting and were positive

by the Sereny test (data not shown). The two EIEC strains

were isolated from independent patients presenting with diar-

rhea at the casualty section of the hospital. The stool samples

were loose but contained no macroscopic blood. They showed

moderate numbers of RBCs and WBCs (i.e., less than 10/high-

power field).

The OD values for the Shigella strains identified by ELISA

varied between 1.405 and 2.000, while those for the two EIEC

isolates were 0.962 to 1.111. Of the total of 5,154 colonies from

the 859 samples tested by ELISA, 3 colonies from three inde-

pendent patients gave false-positive, low OD readings (0.205 to

0.322). All three isolates were shown to bind to the conjugate

in the ELISA without the monoclonal antibody, they were not

invasive, and they did not express the VirG-Ipa proteins by

Western blotting. They were identified as Serratia spp. by bio-

typing.

DISCUSSION

In some developing countries EIEC strains can be respon-

sible for 1 to 6% of cases of diarrhea or dysentery (4, 7, 23),

and an age-related isolation rate of as high as 7% was reported

for 2- to 4-year-old Thai children with diarrhea (5). EIEC

strains also can cause food- or waterborne outbreaks (14). Our

knowledge of the incidence and epidemiology of EIEC infec-

tions is mostly based on limited-scale pilot studies instead of

regular surveillance by clinical microbiology laboratories. This

is due to the fact that neither biotyping nor serotyping is

specific and sensitive enough to identify EIEC strains, and the

confirmatory assays for virulence, i.e., the guinea pig kerato-

conjunctivitis test (20), the epithelial cell invasion assay (10), or

the detection of the IP-coded proteins by Western blotting

(16), are research tools rather than techniques suited for clin-

ical laboratories. The introduction of molecular biological

techniques that detect virulence-specific plasmid or chromo-

somal genes of Shigella and EIEC (2, 3, 9, 25), while highly

sensitive and specific, may still not offer a solution for routine

work, particularly in developing countries. Recently, we pro-

posed that a simple ELISA recognizing an IP-coded antigen

(i.e., IpaC) of these pathogens could provide a highly specific

alternative for the detection of EIEC (8).

In the present study we showed that the IpaC-specific im-

munoassay can indeed successfully be used to recognize EIEC

and Shigella strains among colonies grown from stool samples

from pediatric patients. By the ELISA technique, two E. coli

strains belonging to the recognized EIEC serogroup O28ac

were detected from patients in whom no enteric pathogens

were isolated by biotyping and serotyping. The enteroinvasive

character of the isolates was confirmed in the epithelial cell

invasion assay (10) as well as in the Sereny test (20). Both

strains expressed the characteristic IP-related protein profile

(16) on Western blots. Similar to other invasive isolates of the

same serogroup (3, 22), the two strains did not decarboxylate

lysine, but fermented lactose, which makes their identification

by biotyping difficult. These two EIEC strains are the first ones

ever reported from Kuwait. Together with the recent detection

of similar strains among U.S. troops stationed in the northern

region of Saudi Arabia during Operation Desert Shield (12),

this finding indicates the presence of these pathogens as a

potential cause of diarrhea in this part of the world.

More Shigella isolates were detected by the immunoassay

than by the traditional culture techniques (26 versus 23). How-

ever, in case of this genus, the immunoassay, similar to colony

hybridization with DNA probes, should not necessarily offer an

advantage. Once suspected colonies were carefully spotted on

the plates, the biological and serological identifications should

be straightforward. It should be noted that in the present study

Shigella colonies were seen only on the MC agar plates con-

taining samples from four of five patients in whom shigellae

were identified by the immunoassay only but not by culture. It

is known that for some Shigella strains, media with selective

power better than that of MC agar could be inhibitory (3). If no

NLF colonies were seen on the highly selective plates (e.g.,

agar plates), the culture could mistakenly be declared negative.

The colonies of the two Shigella strains selected for but not

identified as IpaC secretors by ELISA had lost their IpaC-

producing capacity as well as invasive capacity due to the

probable loss of or deletion from the IP. We surmise, however,

that encountering noninvasive colonies only from clinical ma-

terial should be a relatively infrequent phenomenon (one S.

sonnei isolate from the 30 patients excreting either Shigella or

EIEC in this study) due to the counterselection of the non-

virulent clones in the host. In the ELISA the problem of

plasmid instability may partially be overcome by testing larger

numbers of colonies from individual patients. This was found

to be the case for the S. flexneri-infected patient not identified

by the immunoassay, for whom, upon reinvestigation, 5 of 20 S.

flexneri colonies were still found to secrete IpaC. It should be

noted that the loss of or deletion from the IP may result in a

negative reaction with the DNA probes or by PCR as well

unless the deletion leaves the corresponding sequences intact

(18) or genes also present on the chromosome are targeted

(25).

The present study did not aim to compare the diagnostic

performances of ELISA to that of the molecular biological

techniques; therefore, no screening assays other than the

ELISA were used to identify EIEC. However, by using the

polyclonal antibody- and monoclonal antibody-based versions

of this assay, it has previously been found that of the total of ca.

5,500 E. coli strains from various strain collections tested so

far, not a single virulent EIEC strain was missed by the method

(8, 24). Therefore, we believe that among the total of 5,154

colonies tested in this study, none but those identified as Shi-

gella or EIEC expressed the IpaC antigen, i.e., were enteroin-

vasive.

Weak false-positive reactions in this assay are known to

occur rarely (8). In the present study 3 of 5,154 colonies tested

(0.058%) fell in this category, but due to their low frequency

and to the fact that they could easily be identified in repeated

or parallel assays run without the IpaC-specific antibodies, they

hardly indicate a serious diagnostic problem.

The labor requirement of this immunoassay is relatively low,

and it could provide a positive identification in 24 h. Since the

ELISA technique has been used for various purposes in many

laboratories in developing countries, the method could easily

fit into the diagnostic armory, even in clinical laboratories with

relatively low levels of technical sophistication. One could en-

vision the use of the assay as a screening method, as was done

in this study, or it could be applied only to isolates recovered

from diarrheal samples in which no other enteric pathogens

were identified.

On the basis of these results we believe that the IpaC-

specific MAIC-1 ELISA offers a specific and relatively inex-

V

OL

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ELISA FOR EIEC AND SHIGELLAE

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pensive technique for the identification of enteroinvasive

strains. This is particularly true for the members of the EIEC

group, for which no other simple alternatives are available.

ACKNOWLEDGMENTS

This work was supported by grants MI O92 from Kuwait University

and T 016190 from the National Scientific Research Foundation

(OTKA), Hungary.

The skillful technical assistance of Akbar P. Kalandath is highly

appreciated.

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