Antimicrobial resistance of diarrheagenic Escherichia coli isolated from children under the age of 5 years from Ifakara, Tanzania.
ABSTRACT Diarrhea caused by multidrug-resistant bacteria is an important public health problem among children in developing countries. The prevalence and antimicrobial susceptibility of diarrheagenic Escherichia coli in 346 children under 5 years of age in Ifakara, Tanzania, were studied. Thirty-eight percent of the cases of diarrhea were due to multiresistant enterotoxigenic E. coli, enteroaggregative E. coli, or enteropathogenic E. coli. Strains of all three E. coli categories showed high-level resistance to ampicillin, tetracycline, co-trimoxazole, and chloramphenicol but were highly susceptible to quinolones. Guidelines for appropriate use of antibiotics in developing countries need updating.
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ABSTRACT: Faeces from urban children < 2 years old with acute diarrhoeal illness and from non-diarrhoeal infants (controls) were examined for Escherichia coli and other enteropathogens. A total of 990 E. coli isolates from 100 patients and 50 controls was tested for enteropathogenic E. coli (EPEC) serotype (O:H), adherence to HEp-2 cells after incubation for 3 and 6 h, fluorescent actin staining (FAS), DNA hybridisation with EAF, eaeA, STh, STp and EAggEC probes and production of heat-labile enterotoxin (LT) and verocytotoxin (VT) with Y1 and Vero cells. EPEC were the most prevalent enteropathogens in patients (32.7%; and 14% in controls). Enteroinvasive E. coli (EIEC) and Vero cytotoxin-producing E. coli (VTEC) were not detected. The rate of isolation of enterotoxigenic E. coli (ETEC) was identical in both groups. Among the EPEC isolates the prevalent serotypes were O111:H2, O55:NM and O119:H6. Localised adherence (LA) was found significantly more frequently in isolates from patients (19.6%) than controls (2.1%). All LA-positive EPEC isolates were FAS+ and eaeA+, but only 75.2% of them hybridised with the EAF probe. Diffusely adhering E. coli (DAEC) and enteroaggregative E. coli (EAggEC) were found with equal frequency in patients and controls. Twenty-seven E. coli isolates were negative for EAF but positive for eaeA and FAS and produced LA in 6-h adherence tests. These EAF-/eaeA+ strains were the only putative enteropathogen identified in seven patients and were not found in controls. The ability of these strains to elicit ultrastructural cell alterations and cell-signalling events was evaluated in Caco-2 cells (human colon carcinoma cell line) by the gentamicin invasion assay and by transmission electron microscopy. The numbers of intracellular bacteria in cell invasion tests varied from 0.4% to 1.6% of the cell-associated bacteria after a 6-h incubation period. Tyrosine phosphorylation of host cell proteins was assessed in HEp-2 cells by immunofluorescence microscopy and all strains gave positive results. EAF-/eaeA+ E. coli strains express most of the virulence properties found among true EPEC strains and can be a relevant cause of infant diarrhoea in developing countries.Journal of Medical Microbiology 10/1998; 47(9):781-90. · 2.30 Impact Factor
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ABSTRACT: Forty-nine children in need of antibacterial treatment for a severe episode of bacterial diarrhoea were consecutively treated with either an oral paediatric suspension of rifaximin (100 mg every six hours for an average of four days: 24 patients), or paromomycin (125 mg every six hours for an average of four days: 25 patients). Stools (number and form), enteritis symptoms and signs, and intolerance manifestations were all monitored on each day of treatment. A stool culture was performed on the first available stool after enrolment and after the end of treatment to monitor the drugs' antibacterial activity. A similar rate of bacteriological cure, with normalisation of stools and elimination of the clinical symptomatology, was attained by the two antibiotics, with statistical significance of changes vs. baseline being apparent on the second treatment day, in both treatment groups. Rifaximin results were quicker (treatment lasted three days in several cases) and on the whole slightly better (though without statistical significance) than those of paromomycin: 21/24 vs. 20/25 children were completely cured, with a failure rate of three and five cases, respectively. Systemic and local tolerance of both treatments were very good in all children.Current Medical Research and Opinion 02/1997; 14(1):39-45. · 2.26 Impact Factor
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ABSTRACT: OBJECTIVE: To determine the prevalence of the different categories of diarrheagenic Escherichia coli, enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), verotoxin-producing E. coli (VTEC), enteroaggregative E. coli (EAggEC), diffusely adherent E. coli (DAEC), and enteropathogenic E. coli (EPEC), associated with travelers' diarrhea. METHODS: Stool specimens from 350 patients with travelers' diarrhea were collected between 1994 and 1996. The virulence factors of the diarrheagenic E. coli isolated were detected by PCR technique, in subcultures of single colonies of all morphotypes of E. coli observed in culture on MacConkey agar. RESULTS: ETEC (15.7%), EAggEC (13.4%) and DAEC (9.14%) are significantly more prevalent than EIEC (3.4%), EPEC (2.86%) and VTEC (0.86%) (p<0.05; z-test). The prevalence of ETEC and EAggEC was similar in all geographic areas visited. CONCLUSIONS: PCR is a rapid and specific technique to use in the identification of the different categories of diarrheagenic E. coli and greatly increases the yield of potential enteropathogens from cases of travelers' diarrhea. Not only ETEC but also EAggEC and DAEC strains play a major role in the etiology of travelers' diarrhea, whereas EIEC, EPEC, and VTEC strains play a minor role, leading to the question of whether it is necessary to search routinely for these three types of E. coli in diagnostic laboratories.Clinical Microbiology and Infection 02/1998; 4(12):682-688. · 4.58 Impact Factor
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY,
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Dec. 1999, p. 3022–3024Vol. 43, No. 12
Antimicrobial Resistance of Diarrheagenic Escherichia coli
Isolated from Children under the Age of 5 Years
from Ifakara, Tanzania
JORDI VILA,1* MARTHA VARGAS,1CLIMENT CASALS,1HONORATO URASSA,2
HASSAN MSHINDA,2DAVID SCHELLEMBERG,2AND JOAQUIM GASCON1
Departament de Microbiologia, Hospital Clinic, IDIBAPS (Institut d’Investigacions Biome `diques August Pı ´ i Sunyer),
Barcelona 08036, Spain,1and Ifakara Health Research and Development Centre, National Institute
for Medical Research, Ifakara, Tanzania2
Received 15 June 1999/Returned for modification 16 August 1999/Accepted 2 October 1999
Diarrhea caused by multidrug-resistant bacteria is an important public health problem among children in
developing countries. The prevalence and antimicrobial susceptibility of diarrheagenic Escherichia coli in 346
children under 5 years of age in Ifakara, Tanzania, were studied. Thirty-eight percent of the cases of diarrhea
were due to multiresistant enterotoxigenic E. coli, enteroaggregative E. coli, or enteropathogenic E. coli. Strains
of all three E. coli categories showed high-level resistance to ampicillin, tetracycline, co-trimoxazole, and
chloramphenicol but were highly susceptible to quinolones. Guidelines for appropriate use of antibiotics in
developing countries need updating.
Diarrhea caused by multidrug-resistant bacteria has been
recognized as an important public health problem among chil-
dren in developing countries and is a research priority of the
diarrheal disease control program of the World Health Orga-
nization. Among these bacteria, strains of the different diar-
rheagenic categories of Escherichia coli, such as enterotoxi-
genic E. coli (ETEC), enterohemorrhagic E. coli (EHEC),
enteropathogenic E. coli (EPEC), enteroinvasive E. coli, and
enteroaggregative E. coli (EAggEC), are among the most im-
portant causes of acute enteritis and subsequent morbidity and
mortality in children in developing countries (4). Knowledge of
local antimicrobial therapy patterns is important in selecting
the appropriate therapy, since even if microbiology laboratory
facilities are available, which is not the case in most of the
developing countries, susceptibilities will generally not be
known until 72 h after the sample is processed. Acute enteritis
is a widespread health problem in these countries and is an
important cause of mortality among infants and young chil-
dren. In Ifakara, Tanzania, children with acute diarrhea are
treated by oral rehydration plus administration of an antibiotic
(currently co-trimoxazole). Therefore, since many patients
with enteritis are treated empirically with antibiotics, it is im-
portant to know the antimicrobial resistance patterns of the
most prevalent bacteria causing this syndrome.
The main objective of this study was to analyze the preva-
lence of diarrheagenic E. coli as a cause of diarrhea in children
under the age of 5 years in Ifakara, Tanzania, as well as to
study the susceptibilities of these isolates to six antimicrobial
Stool specimens from 346 children under 5 years of age from
Ifakara, Tanzania, who presented with acute diarrhea were
cultured for E. coli and other enteropathogens by conventional
methods in the microbiology laboratory of the Ifakara Center
(from April to June 1998). The identified strains were kept on
conservation agar. Detection of the virulence factors of E. coli
strains and susceptibility testing were performed in the clinical
microbiology laboratory of the Hospital Clinic, Barcelona,
Spain. DNA from each E. coli isolate was subjected to PCR
under the conditions described in reference 9 to determine the
correct diarrheagenic category. The MICs of six antimicrobial
agents for the diarrheagenic E. coli isolates were determined
by the E-test method. Colonies from 24-h McConkey agar
cultures were homogenized in 0.85 saline, and the turbidity was
adjusted to that of a 0.5 McFarland standard. The inoculum
suspension was spread on a Mueller-Hinton agar plate surface
with a swab, and after that plates had sat on the bench 15 min,
E-test strips were applied. The inoculated medium was incu-
bated for 20 h at 37°C, and the MICs were read. The National
Committee for Clinical Laboratory Standards breakpoints
were used to differentiate between susceptible and resistant
isolates (6). E. coli ATCC 25922 was used as a reference strain
for quality control purposes. ETEC strains were isolated from
44 children (12.7%). The distribution of these strains accord-
ing to the type of enterotoxin synthesized was as follows: 33
strains (75%) produced the heat-stable toxin (ST), 6 strains
(14%) synthesized the heat-labile toxin (LT), and 5 strains
(11%) produced both toxins. EAggEC strains were isolated
from 82 (23%) children, and verotoxin-2-producing E. coli was
detected in one child; no enteroinvasive E. coli strains were
isolated. Seventeen of these 82 EAggEC strains also produced
LT and/or ST. Of these 17 strains, 15 synthesized ST, one
strain produced LT, and one strain synthesized both toxins.
Finally, EPEC strains were isolated in 21 cases (6%).
In this study, three PCR techniques, amplifying the bfp gene,
the eaeA gene, or the EPEC adherence factor (EAF), were
used to detect EPEC strains. Twenty-one strains were found to
have at least one of these genes; 13 of the 21 strains had only
the eaeA gene, 1 strain had only the bfp gene, 5 strains were eae
and bfp positive, 1 strain was positive for both eae and EAF,
and 1 strain was positive for eae, bfp, and EAF. Therefore, it
seems that the presence of the eae gene is more highly linked
to EPEC than is the presence of either the bfp gene or EAF, as
has been previously suggested (7).
In this study, ETEC was found to be the category of diar-
rheagenic E. coli which most frequently causes diarrhea in
children under 5 years of age, as has been reported for many
* Corresponding author. Mailing address: Laboratori de Microbio-
logia, Hospital Clinic, Villarroel 170, 08036 Barcelona, Spain. Phone:
34.93.2275522. Fax: 34.93.2275454. E-mail: firstname.lastname@example.org.
studies in developing countries (4). Several studies have also
shown the importance of EAggEC and EPEC as a cause of
diarrhea in children (5). In our study, only one EHEC strain,
producing verotoxin-2, was isolated. This result is in agreement
with previous studies in which EHEC strains were not found
(4). Overall, 38% of diarrhea cases in children under 5 years of
age are due to multiresistant diarrheagenic E. coli, with ETEC,
EAggEC, and EPEC strains being the most prevalent.
Results for the antimicrobial susceptibility testing of the
different categories of diarrheagenic E. coli strains are shown
in Table 1. For all three categories of diarrheagenic E. coli, the
MICs of ampicillin, tetracycline, and co-trimoxazole at which
50% of the isolates tested were inhibited (MIC50s) were ?256
?g/ml. Chloramphenicol showed moderate activity, with resis-
tance ranging from 25 to 57%; the MIC50s of chloramphenicol
were 4 ?g/ml for ETEC and EPEC and ?256 ?g/ml for
EAggEC. Nalidixic acid and ciprofloxacin had very good activ-
ity against these microorganisms; however, one EAggEC strain
and one ST-producing ETEC strain showed resistance to na-
lidixic acid, with MICs of 32 and 256 ?g/ml, respectively. Re-
cently, Sang et al. (8) described four cases of diarrhea caused
by multidrug-resistant EAggEC in Kenyan children. It was
therefore reasonable to predict that this multiresistance
showed by the different categories of diarrheagenic E. coli
might emerge in other developing countries where these clas-
sical antibiotics (ampicillin, tetracycline, and trimethoprim-sul-
famethoxazole) have been widely used. It has been shown that
the treatment of diarrhea caused by ETEC with antibiotics,
specifically co-trimoxazole, decreases the duration and inten-
sity of the diarrhea (1). However, in our study, ETEC exhibited
high-level resistance to this antimicrobial agent. According to
the antibiogram, different resistance patterns were defined
(Table 2) in the three categories of diarrheagenic E. coli, with
being the two most prevalent multiresistance patterns.
Acute or chronic enteritis due to the different categories of
E. coli, mainly ETEC and EAggEC, is an emerging problem in
many parts of the world (4). It has been estimated that 9.2
million deaths in the developing world have been caused by
infectious diseases, and diarrheal diseases are the fourth most
prevalent cause (3). Most mild diarrhea cases are successfully
managed with oral rehydration therapy. Only for more severe
or persistent diarrhea cases should antimicrobial treatment be
added. Ampicillin and cotrimoxazole have been recommended
by the World Health Organization. Local information about
antimicrobial resistance should be used in clinical manage-
ment, and treatment guidelines should be updated (10). In this
sense, ampicillin and co-trimoxazole should be excluded. One
alternative for enteritis treatment is the use of quinolones.
However, they are not recommended for children. Moreover,
it should be taken into consideration that in spite of the min-
imal use of quinolones in Tanzania, we have detected nalidixic
acid-resistant E. coli strains. If quinolones are used as a first-
line treatment for enteritis in these countries, where the use of
antibiotics is not regulated, a rapid emergence of quinolone
resistance will likely occur. Rifaximin is a nonabsorbable anti-
microbial agent which has been shown to be effective as a
treatment for severe episodes of bacterial diarrhea in children
in developed countries (2). However, studies to assess the role
of this antimicrobial agent in the treatment of ETEC or
EAggEC infections in children from developing countries are
This work was supported in part by grant SAF97/0091 from Plan
Nacional I?D, Spain, and by the Spanish Agency for International
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Infect. Dis. 4:450–455.
2. Frisari, L., V. Viggiano, and M. Pelagalli. 1997. An open controlled study of
two non-absorbable antibiotics for the oral treatment of paediatric infectious
TABLE 2. Resistance patterns of the diarrheagenic E. coli isolates
No. of strains
with pattern (%)
EPEC (21) AmpsClsTcsSxtsNalsCips
an, total number of strains analyzed.
TABLE 1. Antimicrobial susceptibilities of different diarrheagenic E. coli isolates from children under 5 years of age in Ifakara, Tanzania
MICs for and antimicrobial susceptibilities of E. coli isolatesa
EAggEC (n ? 65) ETEC (n ? 44) EPEC (n ? 21)
No. of resistant
No. of resistant
No. of resistant
an, total number of clinical isolates tested.
VOL. 43, 1999NOTES3023
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3024 NOTESANTIMICROB. AGENTS CHEMOTHER.