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Antimicrobial Agents and Chemotherapy, June 2000, p. 1731-1733, Vol. 44, No. 6
Departments of
Microbiology1 and Infectious Diseases
(Tropical Medicine Unit),2 IDIBAPS (Institut
d'Investigacions Biomèdiques August Pí i Sunyer),
Hospital Clínic, School of Medicine, University of Barcelona,
Barcelona, Spain
Received 30 December 1998/Returned for modification 18 September
1999/Accepted 27 March 2000
Enterotoxigenic Escherichia coli isolates were
identified as a cause of traveler's diarrhea in 82 of 520 (16%)
patients and tested for resistance to seven antimicrobial agents.
Thirty patients (36%) needed antimicrobial therapy: 17 (56%) for
persistence of symptoms and 13 (44%) for severity of symptoms.
Ampicillin, tetracycline, and trimethoprim-sulfamethoxazole resistance
was high. Chloramphenicol showed moderate activity, and amoxicillin
plus clavulanic acid, nalidixic acid, and ciprofloxacin showed very
good activity. Five nalidixic acid-resistant strains were isolated,
four from patients visiting India.
Enterotoxigenic Escherichia
coli (ETEC) is the major cause of traveler's diarrhea in people
from industrialized countries visiting less-developed countries
(6, 7) and is an important cause of dehydrating diarrhea in
infants and children in less-developed countries (13).
Traveler's diarrhea caused by ETEC strains is usually a mild,
self-limited disease, but for severe traveler's diarrhea, early
treatment with loperamide and an antibiotic such as
trimethoprim-sulfamethoxazole, doxycycline, or a fluoroquinolone has
been recommended (4).
Many previous studies of antimicrobial susceptibility of ETEC involved
a small number of isolates from a single geographic location. We,
therefore, performed antimicrobial susceptibility testing of ETEC
isolates causing traveler's diarrhea originating from diverse
geographical locations. Investigation of the mechanisms of acquisition
of quinolone resistance in the nalidixic acid-resistant ETEC strains
was also performed.
During the period from 1994 to 1997, stool specimens from 520 adult
patients with traveler's diarrhea were analyzed. The patients were
recruited from the traveler's clinic of the Tropical Medicine Department of the Hospital Clinic. All patients had diarrhea on arrival
in Spain or within 2 days after their return. The stool specimens were
cultured for E. coli and other bacterial enteropathogens by
conventional methods (11). Single-colony subcultures of all different lactose-fermenting colonial morphotypes growing on MacConkey agar were identified by conventional criteria. The E. coli
isolates were tested by PCR to detect the heat-stable (ST) and
heat-labile (LT) toxin genes (15). ETEC strains were
isolated from 82 patients (16%) with traveler's diarrhea. The
distribution of these strains according to the type of enterotoxin
synthesized was as follows: 58 strains (71%) produced the ST, 11 strains (13%) produced LT, and 13 strains (16%) produced both toxins
(LT/ST). ETEC strains were isolated from stool samples of patients
traveling to different tropical and subtropical areas, except for
Central and South Africa. The range of prevalence was from 7.5% to
31%, with West Africa and the Indian Subcontinent being the two
geographic areas where ETEC strains were more prevalent, at 31 and
22%, respectively. Thirty patients (36%) needed antimicrobial
therapy: 17 patients (56%) because of persistence of symptoms and 13 (44%) because of severity of symptoms. In 14 treated patients, the
cause of diarrhea was ST-producing ETEC (ETEC-ST), representing 24% of the total number of strains of ETEC-ST, whereas in 7 (63%) and 9 (69%) patients, the cause was LT- and LT/ST-producing ETEC, respectively (P < 0.02). All patients were empirically
treated with ciprofloxacin, and in all, the duration of diarrhea was
shortened and the accompanying symptoms such as abdominal discomfort,
flatulence, nausea, and vomiting were relieved. It is noteworthy that
patients with ETEC-LT and ETEC-LT/ST strains required antimicrobial
therapy more frequently than patients with ETEC-ST strains. However,
this finding needs to be confirmed in future studies.
The MICs for the clinical isolates studied were determined by E-test
according to standard practice. E. coli ATCC 25922 was used
as a reference strain for quality control. The breakpoints considered
to define a resistant strain were those recommended in reference
12. The MICs of antimicrobial agents for ETEC-ST, ETEC-LT, and ETEC-ST/LT strains are shown in Table
1. Randomized, controlled studies have
demonstrated that a 3- to 5-day course of an antibiotic can reduce the
duration of an acute diarrheal episode in travelers. Most studies
done during the 1980s with tetracycline (8) or with
trimethoprim alone (1) or in combination with
sulfamethoxazole (1, 2) showed the effectiveness of these
antimicrobial agents in the treatment of acute diarrhea caused by ETEC,
shortening the duration of excretion of microorganisms, resulting in
earlier termination of illness. However, the emergence and
dissemination of resistance to cotrimoxazole in fecal E. coli were observed during oral administration of this agent
(9). Resistance to cotrimoxazole and tetracycline is now
present in >50% of ETEC strains isolated from tropical and
subtropical countries (Table 1) (10). This fact is due to
the extensive use of these antimicrobial agents in these countries
(10) and precludes their empirical use for the treatment of
traveler's diarrhea. Among the other antimicrobial agents tested in
this study, ampicillin was moderately effective. Chloramphenicol
presented good activity against ETEC, but quinolones such as nalidixic
acid or ciprofloxacin and amoxicillin plus clavulanic acid showed the
best activity against these microorganisms. In the last decade,
fluoroquinolones have been shown to be highly effective in reducing the
duration of traveler's diarrhea (3, 5, 14, 18). However, 5 (6%) of the 82 ETEC strains investigated in our study were resistant to nalidixic acid, and one of them was also resistant to ciprofloxacin (MIC of 32 µg/ml).
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Quinolone Resistance in Enterotoxigenic
Escherichia coli Causing Diarrhea in Travelers to India in
Comparison with Other Geographical Areas
ABSTRACT
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TABLE 1.
MICs of seven antimicrobial agents for 82 ETEC
clinical isolates
The quinolone resistance in E. coli has mainly been
associated with mutations in the gyrA and parC
genes (16, 17). All nalidixic acid-resistant ETEC strains
were analyzed for mutations in the gyrA and/or
parC genes by PCR and DNA sequencing of the quinolone
resistance-determining region as described in references 16 and 17 (Table
2). One ETEC-ST strain for which the MICs were 32 (nalidixic acid) and 0.032 (ciprofloxacin) µg/ml had a substitution in Ser-83 of the GyrA protein to Ala. Three strains (one
each of ST, LT, and ST/LT) for which the MIC of nalidixic acid was 256 µg/ml and that of ciprofloxacin was between 0.19 and 0.25 µg/ml
showed a substitution of Ser-83 by Leu. These results confirm that a
substitution at Ser-83 is sufficient to confer a very high nalidixic
acid resistance level. Finally, one strain for which the MIC of
ciprofloxacin was 32 µg/ml and that of nalidixic acid was >256
µg/ml showed three mutations
two in the gyrA gene, which
generated substitutions of Ser-83 to Leu and Asp-87 to Asn, and the
third in the parC gene, producing a change in Ser-80 to Ile.
In E. coli, a higher quinolone resistance level has been related to the presence of concomitant mutations in the gyrA
and parC genes (16, 17).
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Of the five nalidixic-acid resistant ETEC strains isolated, four (17%) were isolated from patients who had traveled to India. Nalidixic acid was introduced a few years ago as the first-line therapy for shigellosis in some areas of this country, and therefore ETEC strains resistant to nalidixic acid are now emerging.
In summary, ETEC strains, and in particular ETEC-LT strains, are a frequent cause of traveler's diarrhea requiring antimicrobial therapy. The resistance of these microorganisms to the classical agents such as cotrimoxazole, tetracycline, and ampicillin is very high due to the widespread use of these antimicrobial agents in tropical and subtropical countries. Fluoroquinolones are drugs of choice for most adults traveling to high-risk parts of the world. Although the level of resistance to nalidixic acid is generally low, in some geographical areas such as India, it is a matter for concern. Strains for which the MICs of nalidixic acid are high are likely to have at least one mutation of the gyrA gene. The induction of further mutations, e.g., by exposing such strains to fluoroquinolones, is likely to result in a significantly reduced susceptibility to fluoroquinolones. These agents should therefore be used with caution. Furthermore, it is important to continue the surveillance of quinolone resistance levels in these microorganisms.
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ACKNOWLEDGMENTS |
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This work was supported in part by grant SAF97/0091 from the Comisión Interministerial de Ciencia y Tecnología, Plan Nacional I+D.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratori de Microbiologia, Hospital Clínic, Facultat de Medicina, Universitat de Barcelona, Villarroel, 170, 08036 Barcelona, Spain. Phone: 34.3.2275522. Fax: 34.3.2275454. E-mail: vila{at}medicina.ub.es.
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Antimicrobial Agents and Chemotherapy, June 2000, p. 1731-1733, Vol. 44, No. 6
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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