JAC Advance Access originally published online on March 19, 2008
Journal of Antimicrobial Chemotherapy 2008 61(6):1315-1318; doi:10.1093/jac/dkn108
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Original research |
High rate of resistance to locally used antibiotics among enteric bacteria from children in Northern Ghana
1 Institute of Microbiology and Hygiene, Charité—University Medicine Berlin, Berlin, Germany 2 Institute of Tropical Medicine and International Health, Charité—University Medicine Berlin, Berlin, Germany 3 Department of Infectious Diseases and Tropical Medicine, University of Munich, Munich, Germany 4 Northern Region Malaria Project, NORMAP, Tamale, Ghana 5 Regional Health Administration, Ministry of Health, Tamale, Ghana 6 School of Medicine and Health Sciences, University for Development Studies, Tamale, Ghana 7 Robert Koch Institute, Berlin, Germany
* Correspondence address. Institute of Microbiology and Hygiene, Charité—Campus Benjamin Franklin, Hindenburgdamm 27, 12203 Berlin, Germany. Tel: +49-30-84453620; Fax: +49-30-84453830; E-mail: ralf.ignatius{at}charite.de
Received 7 January 2008; returned 20 January 2008; revised 14 February 2008; accepted 18 February 2008
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Abstract |
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Objectives: Information on antimicrobial susceptibility of bacterial pathogens is scarce in resource-poor settings. We determined the susceptibility of bacterial enteric pathogens and faecal Escherichia coli isolates obtained from children in urban Tamale, Northern Ghana, to antibiotics widely used in the that area [ampicillin or amoxicillin, trimethoprim/sulfamethoxazole (SXT) and chloramphenicol] and to alternative drugs.
Methods: Five Shigella spp., 6 Salmonella spp. and 318 E. coli were isolated from stool specimens obtained from 367 children with or without acute diarrhoea. Isolates were differentiated using standard laboratory procedures and tested using a breakpoint microbroth dilution method for their susceptibility to 18 antimicrobials and by disc diffusion for their susceptibility to chloramphenicol.
Results: Although the salmonellae showed an acceptable resistance pattern, E. coli isolates and the closely related shigellae were highly resistant. About 91% and 81% of E. coli isolates from patients or controls, respectively, were resistant to ampicillin (MICs 
8 mg/L), 88% and 76% to trimethoprim/sulfamethoxazole (MICs 80/4 mg/L) and 46% and 41% to chloramphenicol (inhibition zones 
12 mm). Resistance to β-lactam antibiotics or chloramphenicol was observed more frequently among isolates obtained from infants when compared with older children (1–4 years of age).
Conclusions: Enteric bacteria from children in urban Northern Ghana are highly resistant to antibiotics used in that area. Therefore, new antibiotics should be introduced for the treatment of infections caused by these bacteria. Additionally, the establishment of a surveillance of the prevalence of the main bacterial infectious agents and their antimicrobial resistance is desirable.
Keywords: E. coli , Salmonella , Shigella
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Introduction |
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Increasing antimicrobial resistance of enteric commensals and pathogens constitutes a serious problem in both industrialized and developing countries (reviewed by Okeke et al.1). After we had observed high morbidity and mortality rates due to diarrhoea in a previous study on malaria in children in Tamale, Northern Ghana, we investigated the prevalence of bacterial, viral and parasitic agents of paediatric diarrhoea in this area.2 One additional aim of that study was to collect bacterial pathogens and perform susceptibility testing of those isolates because there is no information on the susceptibility of enteric pathogens—or other bacteria—to the antibiotics most widely used in Northern Ghana. Data acquired in cities more developed than Tamale, such as Accra (the capital), cannot be considered representative for Northern Ghana.
In the preceding study, rotavirus was found to be the main causative agent of acute childhood diarrhoea and only a few enteropathogenic bacteria were isolated.2 To acquire more representative data on the prevalence of antimicrobial resistance of enteric bacteria in Northern Ghana, we isolated Escherichia coli, the main Gram-negative facultative anaerobic species of the colonic flora, as an indicator organism from stool samples. E. coli accounts for a large number of severe infections in neonates and children, e.g. diarrhoea, meningitis, urinary tract infections and sepsis, and has recently been shown to be a major source of community-acquired bacteraemia in infants in rural Kenya.3 E. coli has been used before as source of information on antibiotic resistance of enteric bacteria in surveillance studies.4 Moreover, E. coli constitutes a reservoir of genetic material from which other bacteria can readily acquire genes of antimicrobial resistance.
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Materials and methods |
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Collection of stool samples
The study was conducted between November 2005 and January 2006 in Tamale, the capital of the Northern Region of Ghana. Despite a population of 
350 000, the city has a rather rural character with hamlets and thatched, mud-wall huts scattered over a vast area. Antibiotics are available from numerous licensed and non-licensed sources. At the peri-urban Bulpeila Health Centre, healthcare workers collected faecal specimens from 243 children with acute diarrhoea and from 124 population control children from the same area without acute diarrhoea. The mean age of the children was 13 months (0–11 years); further details of the study population have been published elsewhere.2 The study protocol was approved by the Ethics Committee, University for Development Studies, Tamale, and informed written consent was obtained from the children's guardians.
Stool specimens were processed immediately. Standard agar plates and enrichment media (MacConkey, xylose lysine deoxycholate and thiosulphate citrate bile sucrose agars, selenite broth and peptone water; all Oxoid, Wesel, Germany) were used to detect Salmonella, Shigella and Vibrio species. In addition, selective media (cefsulodin irgasan novobiocin, Ryan and Karmali agars; all Oxoid) were inoculated for the detection of Yersinia, Aeromonas and Campylobacter species. Suspicious bacterial colonies were further isolated and differentiated using routine techniques (e.g. oxidase and motility for Campylobacter spp., agglutination for Salmonella, Shigella and Vibrio) or the API 20E system (bioMérieux, Nürtingen, Germany). All differentiated and all suspicious isolates as well as all Enterobacteriaceae grown were stored at –20°C using a cryobank system (Mast Diagnostica, Germany). Cryobanks were shipped on dry ice to Berlin, Germany, for further analyses. There, cryobanks containing Enterobacteriaceae from children between 0 and 4 years of age were spread on routine agar plates for detection and differentiation of enteric pathogens and E. coli isolates. The latter were identified as lactose- and indole-positive and confirmed using the API 10 S system (bioMérieux).
Susceptibility testing of Enterobacteriaceae to antibiotics except fosfomycin and chloramphenicol was performed by a breakpoint microdilution test in Isosensitest broth (Oxoid). The following ‘Deutsches Institut fuer Normung' 58940-4 MIC interpretive breakpoints (mg/L) for Enterobacteriaceae were used: ampicillin (0.5, 2, 8), ampicillin/sulbactam (0.5/8, 2/8, 8/8), piperacillin (1, 4, 32), piperacillin/tazobactam (1/4, 4/4, 32/4), mezlocillin, ceftriaxone, ceftazidime and amikacin (all 1, 4, 16), cefuroxime axetil, gentamicin, tobramycin and tetracycline (all 0.25, 1, 4), cefotiam (1, 4, 8), cefotaxime (0.5, 2, 8), cefixime and ciprofloxacin (both 0.25, 1, 2) and trimethoprim/sulfamethoxazole (0.25/5, 20/1, 80/4). The susceptibility to fosfomycin (breakpoints: 4, 16, 64 mg/L) was tested in Mueller–Hinton broth supplemented with glucose-6-phosphate (25 mg/L). The inoculum was prepared by making a saline suspension (in 20 mL NaCl/3% Tween® 80) from three single colonies selected from an agar plate after 18–24 h of incubation. The bacterial suspensions were transferred with a replicator to 96-well-microtitre plates. Accuracy and purity of the inoculum (5 x 104–5 x 105 cfu/mL) were verified by subcultures on blood agar plates.
To determine the susceptibility to chloramphenicol, agar diffusion tests were carried out on Mueller–Hinton agar (Oxoid) using a bacterial inoculum equivalent to a 0.5 MacFarland standard and discs containing 30 µg chloramphenicol (Oxoid). Isolates yielding zone sizes of 18 and 12 mm were considered susceptible and resistant, respectively.
As quality controls, two defined E. coli strains (ATCC 25922 and 35218) were included in the testing procedures throughout the study.
Data on resistant isolates from patients and controls were analysed using the 
2 and Fisher's exact tests.
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Results and discussion |
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From children with diarrhoea, we isolated two isolates each of Campylobacter jejuni, Salmonella enterica Group 2, S. enterica serovar Typhimurium, Shigella boydii and S. flexneri as well as one isolate each of S. enterica serovar Colindale and S. enterica serovar Typhi. One isolate of Campylobacter coli and S. boydii each was isolated from control subjects. Yersinia, Aeromonas or Vibrio species were not detected. Susceptibility testing of the six Salmonella and five Shigella isolates was performed in Tamale by disc diffusion and confirmed and extended in Germany using the breakpoint microdilution method. The results revealed substantial antimicrobial resistance of the shigellae, particularly to the drugs most frequently used in Northern Ghana, i.e. amoxicillin/ampicillin, trimethoprim/sulfamethoxazole and chloramphenicol. In contrast, the salmonellae were more susceptible, and only one S. Typhimurium isolate displayed a similar resistance pattern as the shigellae (Table 1). Although the number of isolates tested was small, the data indicate that trimethoprim/sulfamethoxazole and chloramphenicol are most likely not effective anymore in the treatment of childhood shigellosis in Northern Ghana. Similarly, high proportions of multidrug-resistant Shigella isolates in children and adults have been reported from the Accra region of Ghana.5 Studies with more isolates are warranted for Northern Ghana, including Salmonella Typhi isolates, which have been found to be equally multidrug-resistant in Accra.6
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Susceptibility testing of E. coli isolates revealed high resistance to the locally used antibiotics (Table 2), comparable to the closely related shigellae, which underlines the usefulness of E. coli as a surveillance tool. Although we cannot exclude that some E. coli isolates from patients belonged to diarrhoeagenic strains, the majority may represent normal stool flora since bacteria isolated from control children were also highly resistant to the same antibiotics. Moreover, both diarrhoeagenic and commensal, resistant E. coli may constitute a potential reservoir for resistance genes that can be transmitted horizontally to other bacteria including pathogens. The high proportions of resistant bacteria (and particularly the resistance of these to tetracycline, which is generally not used in children) in samples from children <1 year indicate the acquisition of resistant bacteria by the children rather than resistance induced through antimicrobial treatment. This is supported by a recent population-based study, which suggests that children acquire resistant E. coli isolates from household contacts.7 Resistance to β-lactam antibiotics or chloramphenicol was observed more frequently among isolates obtained from infants when compared with older children. Although this association was statistically significant only for control children, this group may be more representative for the overall harbouring of resistant enteric bacteria by children in Northern Ghana because their faecal flora was not disturbed by acute diarrhoea. This tended to be reverse for the resistance to aminoglycosides; however, here we detected only small percentages of resistant isolates in either group, and larger studies may yield more accurate results. These data suggest that infants may acquire the commensal enteric flora from their parents who have likely been more exposed to antibiotics than older children. Notably, a high prevalence of resistant E. coli has been shown for adults from other parts of Ghana, i.e. Accra and the Brong Ahafo region in West Ghana.8,9 With increasing age, the children may lose some of the resistant isolates transmitted originally. Moreover, the prevalence of resistance was significantly higher among E. coli isolates from patients than in strains obtained from controls, which may suggest unknown demographic differences between these two groups.
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Our data also indicate that the commonly used drugs cannot be considered any longer as first-line treatment for E. coli infections in children. Reassuringly, we did not find isolates producing extended-spectrum β-lactamases, which have recently been reported for Cameroon.10 Although around one-third of the isolates were resistant to the second-generation cephalosporin cefuroxime axetil (used for oral therapy), none of the isolates showed resistance (MIC
8 mg/L) to the second-generation cephalosporin cefotiam (for systemic application) or the third-generation cephalosporins tested (cefotaxime, MIC 8 mg/L; ceftriaxone, MIC 16 mg/L; ceftazidime, MIC 16 mg/L; cefixime, MIC 2 mg/L). Similarly, all isolates were susceptible to fosfomycin (MIC 64 mg/L). Therefore, these drugs could be introduced in Northern Ghana. Moreover, quinolones could be used in adults since the prevalence of antimicrobial resistance of enteric bacteria in this population may be similar to that found in children. In conclusion, the antibiotics used most widely in Northern Ghana are not appropriate for the treatment of infections caused by E. coli or Shigella and new effective drugs need to be introduced. The data underline the importance of regular antimicrobial testing in resource-poor settings. Consequently, laboratory infrastructure and protocols for the surveillance of antimicrobial resistance must be established and sustained.
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Funding |
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The study was supported by funding from the German Rotary Volunteer Doctors, Bayer Vital GmbH, the Charité and the Robert Koch Institute.
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Transparency declarations |
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None to declare.
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Acknowledgements |
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We thank the members of the Northern Region Malaria Project (NORMAP) for patient recruitment, Jutta Imlau and Ursula Rüschendorf for expert technical assistance and the German Rotary Volunteer Doctors and Bayer Vital GmbH for financial support.
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References |
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2 Reither K, Ignatius R, Weitzel T, et al. Acute childhood diarrhoea in northern Ghana: epidemiological, clinical and microbiological characteristics. BMC Infect Dis (2007) 7:104.[CrossRef][Medline]
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Berkley JA, Lowe BS, Mwangi I, et al. Bacteremia among children admitted to a rural hospital in Kenya. N Engl J Med (2005) 352:39–47.
4 Erb A, Sturmer T, Marre R, et al. Prevalence of antibiotic resistance in Escherichia coli: overview of geographical, temporal, and methodological variations. Eur J Clin Microbiol Infect Dis (2007) 26:83–90.[CrossRef][Web of Science][Medline]
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6 Mills-Robertson F, Addy ME, Mensah P, et al. Molecular characterization of antibiotic resistance in clinical Salmonella typhi isolated in Ghana. FEMS Microbiol Lett (2002) 215:249–53.[CrossRef][Web of Science][Medline]
7 Lietzau S, Raum E, von Baum H, et al. Household contacts were key factor for children's colonization with resistant Escherichia coli in community setting. J Clin Epidemiol (2007) 60:1149–55.[CrossRef][Web of Science][Medline]
8 Newman MJ, Seidu A. Carriage of antimicrobial resistant Escherichia coli in adult intestinal flora. West Afr J Med (2002) 21:48–50.[Medline]
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Nys S, Okeke IN, Kariuki S, et al. Antibiotic resistance of faecal Escherichia coli from healthy volunteers from eight developing countries. J Antimicrob Chemother (2004) 54:952–5.
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Gangoue-Pieboji J, Bedenic B, Koulla-Shiro S, et al. Extended-spectrum-β-lactamase-producing Enterobacteriaceae in Yaounde, Cameroon. J Clin Microbiol (2005) 43:3273–7.
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