1 Department of Medical Microbiology, University Hospital Maastricht, Medical Microbiology, P.O. Box 5800, 6202AZ Maastricht; 4 Department of General Practice, University of Maastricht, Maastricht, The Netherlands 2 Department of Biology, Haverford College, Haverford, PA, USA; 3 Kenya Medical Research Institute, Nairobi, Kenya
Received 19 July 2004; returned 10 August 2004; revised 7 September 2004; accepted 8 September 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: Faecal samples of adult volunteers (n=1290) were analysed in one laboratory for the presence of antimicrobial-resistant E. coli using Eosin Methylene Blue agar plates containing, respectively, ampicillin, oxytetracycline, cefazolin, ciprofloxacin, gentamicin, chloramphenicol and trimethoprim at breakpoint concentrations.
Results: The mean age of the volunteers was 35 years; most of them were female. Ciprofloxacin resistance was in the range 1%63%: the highest percentages were found in the urban populations of Asia and South America. In Peru and the Philippines (U and NU), the prevalence of gentamicin resistance was >20%. Cefazolin resistance was the highest in the urban Philippines (25%). Higher prevalences for ampicillin, oxytetracycline and trimethoprim were found for urban areas compared with non-urban ones of Asia, Africa and South America, respectively (P<0.05).
Conclusions: In the populations studied, antibiotic resistance in faecal E. coli from adult volunteers was emerging for cefazolin, gentamicin and ciprofloxacin and was high for the older drugs ampicillin, oxytetracycline, trimethoprim and chloramphenicol.
Keywords: prevalence of antibiotic resistance , emerging resistance , ciprofloxacin
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The faecal flora of the general population represents a potentially large reservoir of antimicrobial-resistant bacteria at sites where resistance genes can be transferred from the commensal flora to potentially pathogenic microorganisms.3,4
In this study, Escherichia coli, the predominant Gram-negative, facultative aerobic organism of the faecal flora, was used as indicator organism to determine the prevalence of antibiotic resistance in faecal samples of adult volunteers in eight developing countries in Africa, Asia and South America.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Faecal samples were randomly collected by medical students of the University of Maastricht in towns and villages of eight developing countries between November 1998July 2002 during door-to-door visits. Participating countries were for the urban (U) areas Mexico, Peru, Kenya and the Philippines, and for the non-urban (NU) regions Ghana, Zimbabwe, Venezuela, Curaçao, Mexico and the Philippines. Classification as urban or non-urban was based on information about the population size of the respective town. The cut-off value for the urban population was 150 000 inhabitants. Adult volunteers were asked to participate in the study by providing a fresh faecal sample (one sample per volunteer) and by filling out a questionnaire on age, gender and prior antibiotic use. Criteria for exclusion were hospitalization and presence of gastrointestinal disease in the past month. At least 90 evaluable volunteers were recruited from each sampling site.
Sample processing
On the day of collection, the faecal samples were diluted 1:10 in sterile saline [0.9% (w/v) NaCl] with 20% (v/v) glycerol at the local laboratory and stored at 20°C until transportation to the Medical Microbiology Laboratory of the University Hospital Maastricht, The Netherlands, in dry ice using shipping services for microbiological analysis. Weekly Eosin Methylene Blue (EMB) agar (Oxoid, CM 69, Basingstoke, UK) plates with and without antibiotics were prepared and stored at 4°C until further use within 1 week. Ampicillin- and oxytetracycline-containing agar plates were prepared daily. The antibiotic concentrations were based on the resistance breakpoints of the National Committee for Clinical Laboratory Standards (NCCLS) guidelines and modified to make comparison with previous studies possible.5 The antibiotics tested and concentrations used were: ampicillin, 25 mg/L; cefazolin, 32 mg/L; ciprofloxacin, 4 mg/L; chloramphenicol, 25 mg/L; gentamicin, 16 mg/L; oxytetracycline, 25 mg/L; and trimethoprim, 8 mg/L. When using trimethoprim, 5% (v/v) lysed horse blood was added to the EMB agar. Faecal samples were 10-fold diluted and 40 µL from each dilution was inoculated on the EMB agar plates with and without antibiotics, using a spiral plater (Eddy Jet; IUL Instruments, Leerdam, The Netherlands). After 1824 h of incubation at 37°C, E. coli appeared as purple colonies with a dark centre and a metallic green sheen. One colony of the dominant flora was randomly picked from each plate for identification using the indole and ß-glucuronidase (ß-glucuronidase discs; Rosco, Denmark) test. When both tests were positive, the colony was considered to be E. coli; if there remained any doubt, further biochemical tests were performed using API 20E strips (bioMérieux, Plainview, NY, USA). The minimum detection level by this method was 300 cfu/g faeces and it has been shown that >95% of the presumptively identified colonies are E. coli.6
The prevalence of resistance to a certain antibiotic was defined as the proportion of faecal samples showing growth of E. coli on agar plates containing that antibiotic compared with the total number of samples tested.
Statistical analysis
Statistical differences in the prevalences of antibiotic resistance between the different populations were determined using the MannWhitney U-test.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In total, 1290 healthy volunteers participated in the study: Venezuela, n=230; Curaçao, n=149; Mexico U, n=99; Mexico NU, n=99; Peru, n=95; Zimbabwe, n=207; Ghana, n=100; Kenya, n=100; the Philippines U, n=105; and the Philippines NU, n=106. The populations studied were derived from four urban (Mexico, Peru, Kenya and the Philippines) and six non-urban regions (Table 1).
|
Prevalence of resistance
Five percent (70/1290) of all volunteers showed no growth of faecal E. coli. Resistance to cefazolin, gentamicin and ciprofloxacin was emerging in most of the populations tested (Table 2). There was considerable variation in the prevalence of ciprofloxacin resistance. It ranged from 2% or lower in the African countries, Curaçao and Venezuela to >30% in Mexico, Peru and the Philippines. Gentamicin resistance was the lowest in the African countries, Curaçao, Venezuela and both Mexican populations. In Peru and both areas in the Philippines, the percentage was 20% and higher.
|
In each population, the highest prevalences of resistance were found for oxytetracycline, ampicillin and trimethoprim. In >30% of the faecal samples, E. coli was resistant to chloramphenicol, with the exception of those from Curaçao and Zimbabwe. Samples from the Philippines had the highest prevalence of resistance for three out of seven antibiotics tested.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To our knowledge, the present study was the first in which the prevalence of antibiotic-resistant faecal E. coli from adult volunteers from eight developing countries on three continents was determined in one laboratory. Although time bias could not be avoided, the methods used for collecting the faecal samples and determining the prevalence of antibiotic-resistant E. coli were similar for the five successive years.
In conclusion, an emerging prevalence of resistance to the newer antibiotics was found among faecal E. coli from adult individuals in developing countries. Moreover, the resistance was more common in urban than in rural areas. The resistance prevalences to the older drugs were, as expected, high. Unfortunately, we could not correlate these resistance prevalences to data on antibiotic use due to the lack of official data on the use of antibiotics and over-the-counter sales.
More surveillance studies are necessary both in developed and developing countries to monitor antibiotic use and antibiotic resistance over time. These data are important as a basis for the implementation of an antibiotic policy. In addition, education of healthcare workers, patients and parents is also important, as is control of antibiotic use and resistance percentages in food animals in order to control antibiotic resistance over time.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Calva, J. J., Sifuentes-Osornio, J. & Ceron, C. (1996). Antimicrobial resistance in fecal flora: longitudinal community-based surveillance of children from urban Mexico. Antimicrobial Agents and Chemotherapy 40, 1699702.[Abstract]
3 . Okeke, I. N. & Edelman, R. (2001). Dissemination of antibiotic-resistant bacteria across geographic borders. Clinical Infectious Diseases 33, 3649.[CrossRef][ISI][Medline]
4 . Amyes, S. G., Tait, S., Thomson, C. J. et al. (1992). The incidence of antibiotic resistance in aerobic faecal flora in south India. Journal of Antimicrobial Chemotherapy 29, 41525.[Abstract]
5 . van de Mortel, H. J., Jansen, E. J., Dinant, G. J. et al. (1998). The prevalence of antibiotic-resistant faecal Escherichia coli in healthy volunteers in Venezuela. Infection 26, 2927.[CrossRef][ISI][Medline]
6 . Nijsten, R., London, N., van den Bogaard, A. et al. (1993). Antibiotic resistance of Enterobacteriaceae isolated from the faecal flora of fattening pigs. Veterinary Quarterly 15, 1527.[ISI][Medline]
7 . World Health Organization. (1998). Use of Quinolones in Food Animals and Potential Impact on Human Health. Report of a WHO meeting in Geneva. Switzerland.
8
.
Hart, C. A. & Kariuki, S. (1998). Antimicrobial resistance in developing countries. British Medical Journal 317, 64750.
9 . Grenet, K. (2004). Antibacterial resistance, wayampis amerindians, French Guyana. Emerging Infectious Diseases 10, 11503.[ISI][Medline]
10 . Gulay, Z., Bicmen, M., Amyes, S. G. et al. (2000). ß-lactamase patterns and betalactam/clavulanic acid resistance in Escherichia coli isolated from fecal samples from healthy volunteers. Journal of Chemotherapy 12, 20815.[ISI][Medline]
|