HEp 2 cell adherence patterns, serotyping, and DNA analysis of Escherichia coli isolates from eight patients with AIDS and chronic diarrhea

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Vol. 35, No. 8

Copyright © 1997, American Society for Microbiology

HEp-2 Cell Adherence Patterns, Serotyping, and DNA Analysis

of Escherichia coli Isolates from Eight Patients with AIDS and

Chronic Diarrhea

Y. POLOTSKY,

_{J. P. NATARO,}

_{D. KOTLER,}

_{T. J. BARRETT,}

_{J. M. ORENSTEIN}

_*

Department of Pathology, George Washington University Medical Center, Washington, DC 20037

_{; Center for Vaccine}

Development, University of Maryland School of Medicine, Baltimore, Maryland 21201

_{; Division of Gastroenterology,}

St. Luke’s-Roosevelt Hospital Center, New York, New York 10025

_{; and Foodborne Diseases Laboratory, Centers for}

Disease Control and Prevention, Atlanta, Georgia 30333

Received 15 October 1996/Returned for modification 19 February 1997/Accepted 24 April 1997

Three morphologic patterns of interaction between bacteria and enterocytes have been observed in colonic

biopsy specimens from AIDS patients with chronic diarrhea in the United States. The DNA encoding virulence

factors and the HEp-2 cell adherence patterns of Escherichia coli strains isolated from the stools of eight

symptomatic AIDS patients were compared with those of five control strains with known adherence patterns.

One clinical isolate from a patient with attaching-and-effacing enteropathy displayed the localized adherence

attaching-and-effacing pattern typical of enteropathogenic E. coli on HEp-2 cells, five isolates displayed the

“stacked-brick” aggregative adherence pattern typical of enteroaggregative E. coli strains, and one isolate

showed the pattern characteristic of diffusely adherent E. coli. One patient’s isolate displayed features of all

three patterns. No clinical isolate hybridized with standard probes for enteropathogenic, enteroaggregative,

diffusely adherent, enterotoxigenic, and enteroinvasive E. coli strains. Thus, isolates from symptomatic AIDS

patients in the United States can display the same interactive patterns with HEp-2 cells as the agents of

pediatric or traveler’s diarrhea, but lack their typical virulence factors.

At least half of all human immunodeficiency virus

(HIV)-infected patients in developed countries and more than 90% of

those in the developing world suffer from chronic diarrhea and

wasting during the course of their HIV disease (1, 18). The

diarrhea is associated with high rates of morbidity and

mortal-ity. Despite significant advances, including the identification of

new intestinal pathogens, the etiology of diarrhea still remains

obscure in a substantial portion of these patients (34).

In the non-HIV-infected population most susceptible to

di-arrhea, infants and young children, the most important

world-wide diarrheagenic bacterial pathogen is Escherichia coli,

al-though Salmonella and Shigella are common in the United

States (26). Six categories of diarrheagenic E. coli are defined

by the presence of specific genes that encode for their

viru-lence traits: enteropathogenic E. coli (EPEC), enteroinvasive

E. coli (EIEC), enterotoxigenic E. coli (ETEC),

enterohemor-rhagic E. coli (EHEC), enteroaggregative E. coli (EAggEC),

and diffusely adherent E. coli (DAEC) (17, 21, 43). In

devel-oping countries, EPEC, ETEC, and EAggEC are particularly

common in young children (43, 48). EPEC, EAggEC, and

DAEC are detected by the HEp-2 assay, and molecular probes

which reveal specific virulence factors are now available (9, 12,

23).

Until recently, outbreaks of diarrheagenic E. coli have

sel-dom been identified in industrialized countries; now EHEC

O157:H7 is recognized as a significant problem (42). However,

since clinical microbiology laboratories do not routinely

at-tempt to isolate and verify lactose-fermenting organisms from

stool specimens, the true prevalence of E. coli diarrhea is

unknown.

The immunodeficiency of HIV disease adds a new

dimen-sion to the problem of bacterial enteritis; e.g., EIEC has been

shown to cause recurrent bacteremia (4). Enteric bacterial

infections, including the classic attaching-and-effacing EPEC

type, have recently been observed in colonic biopsy specimens

from AIDS patients with chronic diarrhea in the United States

(35). Bacterial enteropathy was demonstrated in colonic biopsy

specimens from almost 20% of AIDS patients with idiopathic

chronic diarrhea (20). EPEC has been isolated from infants

with diarrhea born to HIV-seropositive mothers in Zaire (36).

Enteroadherent E. coli strains (i.e., EPEC, EAggEC, and

DAEC) were suspected to be the cause of 60% of cases of

diarrhea in AIDS patients studied in Zambia (27).

The aim of this study was to compare the DNA virulence

factors and the light and electron microscopic patterns of

HEp-2 cell adherence of E. coli isolates from eight

symptom-atic patients with AIDS to those of control isolates with known

serotypes.

MATERIALS AND METHODS

Strains and patients.Pure E. coli isolates from the stools of eight AIDS

patients with severe immune depression, weight loss, and chronic diarrhea (three or more loose or watery stools per day for at least 30 days [1, 20, 35]) were evaluated. The stools from seven of the patients (patients A to G) were selected for study because a recent colonic biopsy specimen showed bacteria associated with enterocyte pathology, as reported previously (35). The eighth patient (pa-tient H) was selected because his stool was negative for pathogens that would otherwise explain his diarrhea, and endoscopy was not performed on patient H. Four of the patients’ colonic biopsy specimens had rare cytomegalovirus-infected endothelial cells that did not appear to be associated with epithelial damage. For only two patients was another disease identified in the small bowel: a low level of

Enterocytozoan bieneusi microsporidiosis. Three of the patients (patients A, B,

and H) who were known to have been treated with antibiotic therapy (e.g., ciprofloxacin) responded well (35). Four patients (patients C to F) were from George Washington University Medical Center, and four patients (patients A, B, G, and H) were from the St. Luke’s-Roosevelt Hospital Center. For seven patients CD4 T-cell counts were determined within a few months of the E. coli isolation, and while the counts for two patients (patients E and F) were above 100 cells/mm3_{(patient E, 136 cells/mm}3_{, patient F, 133 cells/mm}3_{), the counts for} the remaining patients were below 50 cells/mm3_.

* Corresponding author. Mailing address: Department of Pathology,

Ross Hall 502, 2300 Eye St., N.W., Washington, DC 20037. Phone:

(202) 994-2943. Fax: (202) 994-2518. E-mail: jmo@gwis2.circ.gwu.edu.

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For E. coli isolation, stool specimens were first streaked onto MacConkey, Hektoen enteric, and MacConkey-sorbitol agar plates. Following overnight in-cubation five suspicious colonies from each plate were tested for motility and indole, ornithine, and citrate utilization. Subcultures of isolated strains were then tested in the API 20E system for final identification of members of the family

Enterobacteriaceae or other gram-negative bacteria. The strains were usually

sensitive to ciprofloxacin.

Bacterial cultures were grown static at 35°C overnight in Trypticase soy broth, and the predominantly growing E. coli strain was isolated and propagated. Nonadherent commensal E. coli I, serotype O9:H14, isolated from a normal stool from a healthy man, served as a negative control. Four E. coli strains from the Center for Vaccine Development, University of Maryland School of Medicine (25, 32), with well-established adherence patterns were used as the standard controls for three adherence patterns: (i) locally adherent (LA) EPEC strain E2348/69, serotype O127:H6, isolated from an infant diarrhea outbreak in En-gland; (ii) aggregatively adherent (AA) EAggEC strain O42, serotype O44:H18, isolated from a child with acute diarrhea in Lima, Peru; (iii) an AA EAggEC hemolytic strain 17-2, serotype O3:H2, isolated from an infant with diarrhea in

Chile; and (iv) diffusely adherent (DA) DAEC strain C1845, serotype O75:NM (nonmotile), isolated in Seattle, Wash., from a child with chronic diarrhea.

HEp-2 cell adherence assay.HEp-2 cell monolayers (50 to 70% confluence)

were grown on circular 13-mm glass coverslips (BioWhittaker, Walkersville, Md.). Twenty microliters of bacterial cultures (23106_{bacteria) was injected into} each well, and the plates were incubated for 3 h in a humid, 5% CO2atmosphere. By the technique of Cravioto et al. (8) modified by Nataro et al. (32), culture medium was aspirated from the monolayers, which were washed four times with phosphate-buffered saline, fixed in 70% aqueous methanol for 5 min, stained with 10% Giemsa stain (Fisher, Pittsburgh, Pa.), and examined by light micros-copy.

Transmission electron microscopy.Duplicate HEp-2 cell monolayers were

fixed in 2.5% glutaraldehyde in sodium cacodylate buffer (pH 7.25) for at least 1 h. The fixed monolayers were scraped from the glass surface with plastic pipettes, processed into agar blocks, and postfixed for 1 h in 1% OsO4. Cells were washed and dehydrated through graded ethanol and propylene oxide and em-bedded in Spurr’s epoxy. Some 2.5% glutaraldehyde-fixed specimens were also block stained with 1% uranyl acetate during ethanol dehydration.

FIG. 1. Light and electron microscopy of LA of the clinical isolate from patient B (A and B) and control EPEC strain E2348/69 (C and D) on HEp-2 cells. Light microscopy shows clusters of bacteria attached to cell surfaces, while transmission electron microscopy discloses the attaching-and-effacing lesions with villous effacement, cytoskeleton rearrangement, and pedestal formation. (A and C) Giemsa stain. Magnification,3740; (B and D) Transmission electron microscopy. Magnification,330,000.

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DNA probes.DNA probe analysis of the E. coli isolates was performed by a colony blot lift technique (12). DNA probes were labeled with the BIO-PRIME primer extension kit and detected by using the Blue-Gene system (both from Gibco/BRL, Gaithersburg, Md.). Probes specific for ETEC heat-labile toxin (29) and heat-stable toxin (oligonucleotide) (30), EPEC EAF (31) and Eae (16), EIEC pPS2.5 (41), EaggEC pCVD432 (2), DAEC pSLM852 (7), and EHEC pCVD419 (24) were used in this analysis. Probe hybridization was performed overnight at 42°C in a hybridization solution containing 45% formamide. After hybridization, filters were washed in 0.163SSC (13SSC is 0.15 M NaCl plus 0.015 M sodium citrate)–0.01% sodium dodecyl sulfate solution at 50°C as described by Gicquelais et al. (12).

Serotyping.Serotyping was performed at the Centers for Disease Control and

Prevention by using standard procedures and sera.

RESULTS

LA.

The isolate from patient B elicited an LA pattern,

sim-ilar to that of EPEC control strain E2348/69 (O127:H6), when

it was cultured with HEp-2 cells. Bacilli formed compact

at-tachment plaques on the HEp-2 cells, but were not attached to

the glass substrate (Fig. 1A and B). At the transmission

elec-tron microscopy level, the bacilli were observed to be attached

preferentially to the supranuclear plasma membrane of the

HEp-2 cells. Microvilli were effaced by firmly attached bacteria

which were often situated in cupped ends of pedestal-like cell

protrusions (Fig. 1B and D). For the attachment plaques,

rel-atively uniform narrow gaps were found between the bacteria

and the plasma membrane, and subplasmalemmal

concentra-tions of actin-size thin filaments were found in the plaques.

Bacteria were occasionally found within cytoplasmic vacuoles.

Actrich plaques appeared to be maintained throughout

in-ternalization and could completely surround the intravacuolar

bacteria. The strain from patient B was serotyped as O108:NM.

Despite the observation that the isolate from patient B gave

a typical LA, EPEC-type phenotype when it was grown on

HEp-2 cells, it did not hybridize with the eaeA and EAF probes.

AA.

Five of the clinical isolates (those from patients A, C, D,

E, and G) demonstrated AA to HEp-2 cells and the glass

surface identical to those of the EAggEC O42 (O44:H18, hly

mutant) and 17-2 (O3:H2, hly

_{) control strains (Fig. 2A and}

C). Transmission electron microscopy of the clinical isolates

FIG. 2. Light and electron microscopy of AA of the clinical isolate from patient A (A and B) and control EAggEC strain 042 (C and D) on HEp-2 cells. Light microscopy demonstrates the stacked-brick association of bacteria to cells and glass, and transmission electron microscopy shows the loose association with the cell surfaces. (A and C) Giemsa stain. Magnification,3740. (B and D) Transmission electron microscopy. Magnification,312,500.

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and the control EAggEC strains showed bacteria loosely

asso-ciated with the cell surface (Fig. 2B and D). Neither

attach-ment plaques, pedestals, nor cytoskeleton rearrangeattach-ments

were observed. The strains from patients C to E were

sero-typed as O15:H18, O14:H10, and O153:H19, respectively,

while the nonmotile strain from patient A agglutinated equally

with O8 and O75ac antisera and, therefore, was serotyped as

O8,O75ac:NM. The strain from patient G formed rough, dull

colonies which were devoid of O antigen and therefore could

not be serotyped.

Despite their typical EAggEC phenotype, none of these five

clinical isolates hybridized with the AA probe.

DA.

The isolate from patient F displayed the same DA

pattern to HEp-2 cells as the control DAEC strain, strain

C1845 (Fig. 3A and C). Bacteria were randomly distributed

over the surfaces of the cells and were intercalated into the

FIG. 3. Light and electron microscopy of DA of the clinical isolate from patient F (A and B) and control DAEC strain C1845 (C and D). Light microscopy shows bacteria randomly distributed on the cell surface, and transmission electron microscopy demonstrates that they are intercalated in the microvilli. (A and C) Giemsa stain. Magnification,3740. (B and D) Transmission electron microscopy. Magnification,328,000.

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microvilli (Fig. 3B and D). There were no attachment plaques,

pedestals, or cytoskeletal rearrangements. Occasional bacteria

were found within cytoplasmic vacuoles.

Despite the typical DA phenotype, the strain from patient F

did not hybridize with either the DA or the other probes.

Mixed adherence pattern.

The isolate from patient H

showed a combination of the attaching-and-effacing LA, a

stacked-brick AA pattern, and intercalated DA patterns,

re-spectively (Fig. 4A to D). Bacteria were occasionally found in

cytoplasmic vacuoles.

This strain was serotyped as O8,O75ac:NM, and DNA

hy-bridization studies were negative for this strain.

DISCUSSION

This in vitro study indicates that E. coli strains isolated from

the stools of patients with AIDS and chronic diarrhea in the

United States display the same phenotypic patterns on HEp-2

cells as pathogenic enteric bacteria previously considered to be

predominantly confined to patients with diarrhea in the

devel-oping world. The infection in this immunocompromised

pa-tient population appears to be predominantly of the colon,

especially the right colon (35). Despite the similarities of the

HEp-2 patterns of the clinical isolates to the patterns of known

pathogenic strains, these clinical isolates lack their genotypes

and belong to other serotypes. For example, the strain from

patient B had an O108:NM serotype and elicited the classic

attaching-and-effacing lesions with HEp-2 cells, but it was

found to be eaeA DNA probe negative. This suggests that there

are, as yet, unidentified factors associated with HEp-2 cell and

enterocyte adherence phenotypes that allow bacteria, possibly

nonpathogenic bacteria, even in the developing world, to be

pathogenic for immunocompromised individuals, such as

pa-tients with AIDS.

EPEC has been known as an important enteropathogen for

many years (22, 37, 48), and it is drawing increasing interest

with the emergence of the large immunocompromised

HIV-infected patient population. Autopsies of infants who have

died during the initial stage of disease revealed dense films of

FIG. 4. Light (A) and electron (B to D) microscopy showing mixed adherence pattern of the isolate from patient H on HEp-2 cells. While light microscopy predominantly shows AA, transmission electron microscopy shows LA (B), loose aggregative (C), and DA intercalated (D) patterns of interaction. (A) Giemsa stain. Magnification,31,100. (B to D) Magnification,325,000.

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bacilli adhering to damaged ileal and cecal mucosae (10, 21,

22). These strains display the same cytopathology in animal

models (11), small intestinal biopsy specimens from infants

with diarrhea (39, 46), and now, colonic biopsy specimens from

AIDS patients with chronic diarrhea (35). Electron

micro-scopic studies of EPEC strains demonstrate

attaching-and-effacing lesions with pedestals and cytoskeletal rearrangements

(28, 37, 45).

EAggEC, with its enterotoxin and AA pattern, has been

associated with diarrhea in both children and adults and now in

patients with HIV disease (5, 6, 14, 27, 33, 40, 47). EAggEC

manifests the same characteristic stacked-brick AA pattern on

the intestinal mucosa and urothelium that is seen on the cells

and the glass substrate in the HEp-2 cell assay (19, 44, 49). The

EAggEC control isolate and five clinical EAggEC isolates

dis-played the same loose association with the HEp-2 cells and a

lack of cytoskeletal rearrangement, internalization, or

interca-lation. EAggEC AA resembles the so-called loose pattern of

cytopathic enterocyte interaction that was demonstrated in

colonic biopsy specimens from symptomatic AIDS patients,

including three described here (patients A, D, and G) (35).

DAEC is receiving increased attention as a putative cause of

diarrhea (13, 15, 23, 43). The DA HEp-2 cell growth pattern,

characteristic of DAEC C1845, was encountered as the sole

growth pattern for one clinical isolate. The bacteria were

in-tercalated among microvilli and were occasionally internalized.

Similar intercalation of bacilli with microvilli had been

ob-served in colonic biopsy specimens from seven patients with

AIDS and chronic diarrhea (35).

The apparent combination of three patterns (AA, DA, and

LA) seen in the isolate from one patient could have several

explanations. Some researchers have suggested heterogeneity

of DAEC strains from children with diarrhea. Even among the

cell DA-positive and probe-positive DAEC strains,

LA-posi-tive (clustered adherence) bacteria causing cell cytoskeleton

rearrangement and actin accumulation, like typical EPEC

iso-lates, have been found in experiments with HeLa cells (50, 51).

However, EPEC attaching-and-effacing lesions have not been

observed after attachment of the DAEC C1845 strain to the

brush border of polarized monolayers of Caco-2 or T84 cells

(3). The bacteria did cause elongation of microvilli and

vesic-ulation of their tips.

An additional patient with AIDS and chronic diarrhea was

recently found to have attaching-and-effacing bacteria in

bi-opsy specimens showing ileocolitis (38). Initial PCR studies of

the patient’s two E. coli isolates revealed an O111:NM

sero-type, eaeA probe-positive strain, a classic diarrheagenic LA

strain for infants and young children, and a O130:H27

sero-type, eagg probe-positive strain, an unusual EAggEC strain

that is associated with diarrhea and that displays an AA

pat-tern of interaction (38).

The finding of E. coli isolates from the stools of symptomatic

patients with AIDS that attach to HEp-2 cells and the finding

of attaching-and-effacing lesions in biopsy specimens suggest

the possibility that these strains are the cause of the pathology

(35). The fact that such a strain can be negative with the eaeA,

eaf, and EHEC probes suggests the possibility of a novel

at-taching-and-effacing pathogen. At a recent consensus

confer-ence on EPEC (unpublished data), such organisms were

hy-pothesized to exist and given the name atypical EPEC.

ACKNOWLEDGMENTS

We acknowledge the expert technical assistance of Seth Honig,

Marilou Caparas, Faridah Dahlan, Chip Dye III, Yeubin Zhang, and

Marisa Frieder.

REFERENCES

1. Bartlett, J. G., P. C. Belitsos, and C. L. Sears. 1992. AIDS enteropathy. Clin. Infect. Dis. 15:726–735.

2. Baudry, B., S. J. Savarino, P. Vial, et al. 1990. A sensitive and specific DNA probe to identify enteroaggregative Escherichia coli, a recently discovered diarrheal pathogen. J. Infect. Dis. 161:1249–1251.

3. Bernet-Camard, M.-F., M.-H. Coconnier, S. Hudault, and A. L. Servin. 1996. Pathogenicity of the diffusely adhering strain Escherichia coli C1845: F1845 adhesdecay accelerating factor interaction, brush border microvillus in-jury, and actin disassembly in cultured human intestinal epithelial cells. Infect. Immun. 64:1918–1928.

4. Bessesen, M. T., E. Wang, P. Echeverria, and M. J. Blaser. 1991. Enteroin-vasive Escherichia coli: a cause of bacteremia in patients with AIDS. J. Clin. Microbiol. 29:2675–2677.

5. Bhan, M. K., P. Raj, M. M. Levine, et al. 1989. Enteroaggregative

Esche-richia coli associated with persistent diarrhea in a cohort of rural children in

India. J. Infect. Dis. 159:1061–1064.

6. Bhatnagar, S., M. K. Bhan, H. Sommerfelt, et al. 1993. Enteroaggregative

Escherichia coli may be a new pathogen causing acute and persistent

diar-rhea. Scand. J. Infect. Dis. 25:579–583.

7. Bilge, S. S., J. M. Apostol, Jr., K. J. Fullner, et al. 1993. Transcriptional organization of the F1845 fimbrial adhesin determinant of Escherichia coli. Mol. Microbiol. 8:993–1006.

8. Cravioto, A., R. J. Gross, S. M. Scotland, et al. 1979. An adhesive factor in strains of Escherichia coli belonging to the traditional infantile enteropatho-genic serotypes. Curr. Microbiol. 3:95–99.

9. Cravioto, A., A. Tello, A. Navarro, et al. 1991. Association of Escherichia coli HEp-2 adherence patterns with type and duration of diarrhoea. Lancet

337:262–264.

10. Drucker, M. M., A. Polliack, R. Yeivin, et al. 1970. Immunofluorescent demonstration of enteropathogenic Escherichia coli in tissues of infants dy-ing with enteritis. Pediatrics 46:855–864.

11. Drucker, M. M., R. Yeivin, and T. G. Sacks. 1967. Pathogenesis of

Esche-richia coli enteritis in the ligated rabbit gut. Isr. J. Med. Sci. 3:445–452.

12. Gicquelais, K. G., M. M. Baldini, J. Martinez, L. Maggi, W. C. Martin, V.

Prado, J. B. Kaper, and M. M. Levine.1990. Practical and economical

method for using biotinylated DNA probes with bacterial colony blots to identify diarrhea-causing Escherichia coli. J. Clin. Microbiol. 28:2485–2490. 13. Giron, J. A., T. Jones, F. Millan-Velasco, et al. 1991. Diffuse-adhering

Esch-erichia coli (DAEC) as a putative cause of diarrhea in Mayan children in

Mexico. J. Infect. Dis. 163:507–513.

14. Gomez, H. F., J. J. Mathewson, P. C. Johnson, and H. L. DuPont. 1995. Intestinal immune response of volunteers ingesting a strain of enteroadher-ent (HEp-2 cell-adherenteroadher-ent) Escherichia coli. Clin. Diagn. Lab. Immunol. 2:10– 13.

15. Jallat, C., V. Livrelli, A. Darfeuille-Michaud, C. Rich, and B. Joly. 1993.

Escherichia coli strains involved in diarrhea in France: high prevalence and

heterogeneity of diffusely adhering strains. J. Clin. Microbiol. 31:2031–2037. 16. Jerse, A. E., K. Gicquelais, and J. B. Kaper. 1991. Plasmid and chromosomal elements involved in the pathogenesis of attaching and effacing Escherichia

coli. Infect. Immun. 59:3869–3875.

17. Jerse, A. E., and D. J. Kopecko. 1995. Molecular approaches to bacterial detection and species/subspecies characterization: the diarrheagenic

Esche-richia coli groups. Curr. Opin. Gastroenterol. 11:89–95.

18. Keusch, G. T., D. M. Thea, M. Kamenga, et al. 1992. Persistent diarrhea associated with AIDS. Acta. Pediatr. Suppl. 381:45–48.

19. Knutton, S., R. K. Shaw, M. K. Bhan, H. R. Smith, M. M. McConnell, T.

Cheasty, P. H. Williams, and T. J. Baldwin.1992. Ability of

enteroaggrega-tive Escherichia coli strains to adhere in vitro to human intestinal mucosa. Infect. Immun. 60:2083–2091.

20. Kotler, D. P., T. T. Giang, M. Thiim, et al. 1995. Chronic bacterial enterop-athy in patients with AIDS. J. Infect. Dis. 171:552–558.

21. Levine, M. M. 1987. Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. J. Infect. Dis. 155:377–389.

22. Levine, M. M., and R. Edelman. 1984. Enteropathogenic Escherichia coli of classic serotypes associated with infant diarrhea: epidemiology and patho-genesis. Epidemiol. Rev. 6:31–51.

23. Levine, M. M., C. Ferreccio, V. Prado, et al. 1993. Epidemiologic studies of

Escherichia coli diarrheal infections in a low socioeconomic level periurban

community in Santiago, Chile. Am. J. Epidemiol. 138:849–869.

24. Levine, M. M., J. Xu, J. B. Kaper, et al. 1987. A DNA probe to identify enterohemorrhagic Escherichia coli of O157:H7 and other serotypes that cause hemorrhagic colitis and hemolytic uremic syndrome. J. Infect. Dis.

156:175–182.

25. Levine, M. M., V. Prado, R. M. Robins-Browne, et al. 1988. DNA probes and HEp-2 cell adherence assay to detect diarrheagenic Escherichia coli. J. In-fect. Dis. 158:224–228.

26. Losonsky, G. 1992. Diarrhea and gastroenteritis. Curr. Opin. Infect. Dis.

5:576–581.

27. Mathewson, J. J., Z. D. Jiang, A. Zumla, et al. 1995. HEp-2 cell-adherent

Escherichia coli in patients with human immunodeficiency virus-associated

V

. 35, 1997

E. COLI ADHERENCE IN AIDS PATIENTS WITH DIARRHEA

1957

on May 15, 2020 by guest

http://jcm.asm.org/

diarrhea. J. Infect. Dis. 171:1636–1639.

28. Moon, H. W., S. C. Whipp, R. A. Argenzio, M. M. Levine, and R. A.

Gian-nella.1983. Attaching and effacing activities of rabbit and human

entero-pathogenic Escherichia coli in pig and rabbit intestine. Infect. Immun. 41: 1340–1351.

29. Mosely, S. L., P. Echeverria, J. Seriwatana, et al. 1982. Identification of enterotoxigenic Escherichia coli by colony hybridization using three entero-toxin gene probes. J. Infect. Dis. 145:863–869.

30. Murray, B. E., J. J. Mathewson, H. L. DuPont, et al. 1987. Utility of oli-godeoxyribonucleotide probes for detecting enterotoxigenic Escherichia coli. J. Infect. Dis. 155:809–811.

31. Nataro, J. P., M. M. Baldini, J. B. Kaper, et al. 1985. Detection of an adherence factor of enteropathogenic Escherichia coli with a DNA probe. J. Infect. Dis. 152:560–565.

32. Nataro, J. P., J. B. Kaper, R. Robins-Browne, et al. 1987. Patterns of adher-ence of diarrheagenic Escherichia coli to HEp-2 cells. Pediatr. Infect. Dis. J.

6:829–831.

33. Nataro, J. P., D. Yikang, S. Cookson, et al. 1995. Heterogeneity of entero-aggregative Escherichia coli: virulence demonstrated in volunteers. J. Infect. Dis. 171:465–468.

34. Orenstein, J. M. 1995. Diagnosis of gastrointestinal diseases in AIDS. Adv. Pathol. Lab. Med. 8:453–477.

35. Orenstein, J. M., and D. P. Kotler. 1995. Diarrheagenic bacterial enteritis in acquired immune deficiency syndrome: a light and electron microscopy study of 52 cases. Hum. Pathol. 26:481–492.

36. Pavia, A. T., E. G. Long, R. W. Ryder, et al. 1992. Diarrhea among African children born to HIV-1-infected mothers: clinical, microbiologic and epide-miologic features. Pediatr. Infect. Dis. J. 11:996–1003.

37. Polotsky, Y., E. Dragunsky, and T. Khavkin. 1994. Morphologic evaluation of the pathogenesis of bacterial enteric infections. Crit. Rev. Microbiol.

20:161–208.

38. Polotsky, Y., E. Henrikson, T. Barrett, et al. 1996. Enteropathogenic and enteroaggregative E. coli isolated from stool of an AIDS patient with diar-rhea. Modern. Pathol. 9:131A (abstr. 762) and Lab. Invest. 74:131A (abstr. 762).

39. Rothbaum, R. J., A. J. McAdams, R. A. Giannella, and J. C. Partin. 1982. A clinico-pathologic study of enterocyte-adherent Escherichia coli: a cause of

protracted diarrhea in infants. Gastroenterology 83:441–454.

40. Savarino, S. J., A. Fasano, D. C. Robertson, et al. 1991. Enteroaggregative

Escherichia coli elaborate a heat-stable enterotoxin demonstrable in an in

vitro rabbit intestinal model. J. Clin. Invest. 87:1450–1455.

41. Small, P. L., and S. Falkow. 1986. Development of a DNA probe for the virulence plasmid of Shigella spp. and enteroinvasive Escherichia coli, p. 121–124. In L. Lieve, P. F. Bonventre, J. A. Morello, and S. D. Silver, Microbiology—1986. American Society for Microbiology, Washington, D.C. 42. Tarr, P. I. 1995. Escherichia coli O157:H7: clinical, diagnostic, and

epidemi-ological aspects of human infection. Clin. Infect. Dis. 20:1–10.

43. Thielman, N. M. 1994. Enteric Escherichia coli infections. Curr. Opin. Infect. Dis. 7:582–591.

44. Tzipori, S., J. Montanaro, R. Robins-Browne, P. Vial, R. Gibson, and M. M.

Levine.1992. Studies with enteroaggregative Escherichia coli in the

gnoto-biotic piglet gastroenteritis model. Infect. Immun. 60:5302–5306. 45. Tzipori, S., R. M. Robins-Browne, G. Gonis, et al. 1985. Enteropathogenic

Escherichia coli enteritis: evaluation of the gnotobiotic piglet as a model of

human infection. Gut 26:570–578.

46. Ulshen, M. H., and J. L. Rollo. 1980. Pathogenesis of Escherichia coli gas-troenteritis in man—another mechanism. N. Engl. J. Med. 302:99–101. 47. Vial, P. A., R. M. Robins-Browne, H. Lior, et al. 1988. Characterization of

enteroadherent-aggregative Escherichia coli, a putative agent of diarrheal disease. J. Infect. Dis. 158:70–79.

48. Wanke, C. A. 1994. Enteropathogenic and enteroaggregative strains of

Esch-erichia coli: clinical features of infection, epidemiology, and pathogenesis.

Curr. Clin. Top. Infect. Dis. 14:230–252.

49. Yamamoto, T., S. Endo, T. Yokota, and P. Echeverria. 1991. Characteristics of adherence of enteroaggregative Escherichia coli to human and animal mucosa. Infect. Immun. 59:3722–3739.

50. Yamamoto, T., M. Kaneko, S. Changchawalit, O. Serichantalergs, S. Ijuin,

and P. Echeverria.1994. Actin accumulation associated with clustered and

localized adherence in Escherichia coli isolated from patients with diarrhea. Infect. Immun. 62:2917–2929.

51. Yamamoto, T., Y. Koyama, M. Matsumoto, et al. 1992. Localized, aggrega-tive, and diffuse adherence to HeLa cells, plastic, and human small intestines by Escherichia coli isolated from patients with diarrhea. J. Infect. Dis. 166: 1295–1310.

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