Diarrheal disease is a public health problem worldwide, mostly affecting children in developing countries (Bryce et al., 2005; Vilchez et al., 2009). Diarrhea is one of the leading causes of morbidity and mortality among children under five years in the developing world (Moyo et al., 2011). The aetiological agents include a wide range of viruses, bacteria and parasites (Giordano et al., 2001; Bonkoungou et al., 2011; Nitiema et al., 2011). Salmonella is one of the zoonotic pathogens, which is of great importance in public health science because it is often associated with gastroenteritis (Fashae et al., 2010; Shi et al., 2012). Rural areas are characterized by inadequate hygienic measures, self-medication and poor quality of health care system. Gastroenteritis is associated with about 2500 different serotypes of Salmonella (Matheson et al., 2010) and serotyping is an epidemiological marker for the study of Salmonella spp. infections in a given population and location at a given time (Schutze et al., 1995). Treatment with antimicrobials is crucial for proper management of severe or invasive human salmonellosis (Fashae et al., 2010). Until recently, Salmonella spp. were highly susceptible to the most commonly used antibiotics. However, in the last decade, the emergence of multidrug-resistant strains, including isolates resistant to quinolones, have been reported from some African countries (Gordon et al., 2008; Sire et al., 2008; Hendriksen et al., 2009; Fashae et al., 2010) leading to a major problem associated with the control of diarrhea (Urio et al., 2001). This situation is often associated with bad use of antibiotics in human and animal health. Moreover current information regarding antimicrobial susceptibility pattern of bacteria causing diarrhea in children is limited and thus it is uncertain whether the recommended antibiotics are still effective (Moyo et al., 2011). In Burkina Faso, few data on animal and human Salmonella serotyping and its susceptibility to antibiotics have been reported in urban areas (Bonkoungou et al., 2013; Kagambèga et al., 2013) while there is no information as far as rural areas are concerned. The aim of this study was to investigate the prevalence, serotypes and antibiotic susceptibility patterns of S. enterica in diarrheal children in rural Burkina Faso.

Study design, population and settings

A prospective cross sectional study was conducted to determine the serotypes and antimicrobial susceptibility of Salmonella species among diarrheic children visiting hospitals in rural settings of Burkina Faso. This study was conducted between July 2009 and June 2010 (during one year) in two remote rural areas, in north (Gourcy, distance 140 km) and western (Boromo, distance 185 km) of  the  capital  Ouagadougou, Burkina  Faso  (Figure  1). The main sources of income in these rural settings are subsistence farming, animal husbandry and small scale trade.

Stool samples were collected from 400 children (183 from Gourcy and 217 from Boromo) under five years of age who had acute diarrhea (as described by WHO, 2000) and/or were admitted to medical district hospital of Gourcy and Boromo.

Specimen collection

Stool samples were taken by trained healthcare personnel using a swab transport system (M40 transystemAmies agar gel without charcoal; Copan Italia Spa, Brescia, Italy) and transported to laboratory within 24 h of their collection for analysis. Information regarding the age and sex were recorded for each child using a questionnaire.

Salmonella isolation and identification

Selenite broth (Emapol, Pologne) was used for enrichment of specimens followed by incubation at 37°C for 18 h. Subsequently, samples were cultured on Hecktoen Enteric agar (Liofilchem, Italy) and incubated at 37°C for 24 h.The identity of typical-looking Salmonella colonies on Hektoen was examined by using orthonitrophenyl-ß-D-galactopyranoside (ONPG), citrate, mannitol, lysine decarboxylase tests and the Kliger Hajna medium (Liofilchem, Italy). Finally the isolates were confirmed by API 20E (BioMérieux, Marcy l’Etoile, France) (Kagambèga et al., 2013).

Serotyping

All Salmonella isolates were serotyped by the Salmonella Reference Laboratory. The isolates were serotyped with the somatic O and flagellar H anti-sera according to the Kauffman-White scheme (Popoff et al., 2004).

Antimicrobial susceptibility testing

The susceptibility of strains to 12 antimicrobials was evaluated by the disc diffusion method on Mueller-Hinton agar (Oxoid, England) at 37°C for 24 h and Escherichia coli RHE 6715 (ATCC 25922) was used for validating the antimicrobial test results (CLSI, 2009). The antimicrobials evaluated were ampicillin (10 μg), chloramphenicol (30 μg), streptomycin (10 μg), sulfonamide (3 μg), tetracycline (30 μg), trimethoprim (5 μg), ciprofloxacin (5 μg), nalidixic acid (30 μg), mecillinam (10 μg), imipenem (10 μg), gentamycin (10 μg), and cefotaxime (30 μg). The inhibition zone were measured and tested isolates were classified as “resistant”, “intermediate sensitive” or “sensitive” according to CLSI, 2009.

Ethical considerations

The study protocol was approved by the Ethical Committee of Burkina Faso. An informed verbal consent was obtained from the parents/guardians of every child before taking the stool samples. 

Prevalence of Salmonella

Of the 400 stool specimens analyzed, 24 (6%) Salmonella enterica ssp. enterica were isolated.

Age and sex distribution of patients with Salmonella infection

Our results show that 50% (38% of males and 12% of females) of the Salmonella serotypes were isolated from patients under 12 months of age. Twenty-nine (17% of males and 12% of females) of patients aged 13-24 months of age had Salmonella and 21% (17% males and 4% females) of patients aged 25-59 months reported Salmonella (Table 1). The sex distribution showed 75% of male versus 25% of females for the patients less than 12 months of age, 57% of males versus 43% of females for patients aged 13-24 months and 80% of males versus 20% of females for patients aged 25-59 months.

Diversity of Salmonella serotypes

The 24 isolates belonged to 14 different Salmonella serotypes (Table 2). The most commonly isolated serotype were S. Typhimurium (n = 9; 37.6%), S. Poona (n = 2; 8.1%) and S. Virchow (n = 2; 8.1%). Several uncommon serotypes were among the isolated Salmonella such as S. Kiambou (n = 1; 4.2%), S. Rubislaw (n = 1; 4.2%), S. Muenster (n = 1; 4.2%), S. Gaminara (n = 1; 4.2%), S. Tounouma (n = 1; 4.2%), S. Cubana (n = 1; 4.2%), S. Fresno (n = 1; 4.2%), S. Montevideo (n = 1; 4.2%), S. Duisburg (n = 1; 4.2%), S. Hvittingfoss (n = 1; 4.2%), and S. Ouakam (n = 1; 4.2%) (Table 2). Sixteen (66.7%) of the Salmonella isolated were from Boromo while eight (33.3%) were from Gourcy.

Salmonella antimicrobials susceptibility

The Salmonella serotypes were all susceptible to ciprofloxacin, gentamicin, nalidixic acid, cefotaxime, mecillinam and imipenem (Table 3). Low frequency of resistance to tetracycline was observed with S. Typhimurium (4%). Considering all the 24 Salmonella isolated, a higher frequency of antimicrobial resistance was observed in 33% of each following antibiotic: ampicillin, chloramphenicol, sulfonamide, trimethoprim and streptomycin. Eight of the nine S. Typhimurium isolates were multiresistant to the same antimicrobials (ampicillin, chloramphenicol, streptomycin, sulfonamides and trimethoprim). S. Poona, S. Virchow, S. Tounouma, S. Duisburg, S. Hvittingfoss, S. Ouakam were all intermediate to streptomycin.

Salmonella spp. remains an important cause of diarrheal illness in developing countries. This was the first study in rural Burkina Faso to investigate the prevalence, sero-types and antibiotic susceptibility patterns of S. enterica among diarrheal children. The frequency of isolation of Salmonella comprising several serotypes was 6%, which is lower than in our previous report in urban Burkina Faso (Bonkoungou et al., 2013). This was unexpected since hygiene and sanitation are better in urban than rural areas and thus may reduce Salmonella incidence. However, some practices like street food system which is more developed in Ouagadougou may justify the high prevalence in urban area. Similar data were reported in Malawi, Tanzania, Congo and Ethiopia (Sigauque et al., 2009; Gordon et al., 2010; Moyo et al., 2011; Beyene and Tasew, 2014).

Serotyping is an important data for understanding the epidemiology of salmonellosis in a geographical area and measuring trends in serovars over time can provide information about emerging serotypes (Fernandes et al., 2006). The relative high proportion of S. Typhimurium isolates in this study is similar to results of previous studies from Burkina Faso (Bonkoungou et al., 2013), other African countries such as Tanzania (Moyo et al., 2011), Botswana (Urio et al., 2001) from Australia (Doyle et al., 2009), Colorado (Berg, 2008) and Denmark (Ethelberg et al., 2008). In contrast, S. Enteritidis, which is the most common serotype reported from human isolates globally (Galanis et al., 2006), was not found in this study. This result may be due to increased awareness of the community about food and environmental hygiene from the continuous interventions made by the health care agents recently because the main reservoir of S. enteritidis is food. In addition, some of Salmonella serotypes (S. Kiambou, S. Rubislaw, S. Gaminara, S. Tounouma, S. Cubana, S. Montevideo, S. Duisburg, S. Hvittingfoss) were never detected in human stool in Burkina Faso up to this study. Some of those serotypes have been isolated in food and animal products and thus it could be suggested that humans have been contaminated from food or animal reservoirs. In fact, animals husbandry (chickens particularly) is well developed both in Boromo and Gourcy. Sure enough, besides through food, direct transmission from chicken to humans (children) could easily happen in Burkina Faso, since chickens roam free scattering their feces anywhere in the house yards (Kagambèga et al., 2013). However, the following serotypes (S. Typhimurium, S. Muenster, S. Fresno, S. Ouakam, S. Poona and S. Virchow) were isolated in Burkina Faso from poultry (Kagambèga et al., 2013) and the close association between humans and domestic animals could play an important role in human infection. In general, the primary sources of salmonellosis are considered to be food-producing animals such as cattle, poultry and swine (Thorns, 2000). It is interesting to note that the majority of the S. Typhimurium was isolated from Boromo whereas very few found from Gourcy. This result might be due to location of Boromo as crossroads between Ouagadougou, the capital city, and Bobo-Dioulasso, the second largest city in Burkina Faso. Street-foods are produced and sold there which may explain this higher rate of Salmonella in comparison to Gourcy. Children and young adults are the most affected Salmonella infection. The children under 12 months group was the most infected followed by 13-24 months of age group and 25-59 age group. Our results are consistent with those reported in southwest Ethiopia (3.9% for children under 12 months and 5.4% for 13-60 months) (Beyene and Tasew, 2014). The anti-microbial susceptibility tests showed that all Salmonella strains except S. Typhimurium were susceptible to all the tested antibiotics, suggesting the efficacy of old antimi-crobials for treatment of these strains of Salmonella. Most of S. Typhimurium were resistant to ampicillin, chloram-phenicol, streptomycin, sulfamethoxazol, tetracycline and trimethoprim. It has been reported that resistance to antimicrobials may increases the difficulty and cost of treating seriously ill patients (Doyle et al., 2009). This is likely due to the uncontrolled overuse of these antimi-crobials for various sicknesses leading to the emergence of resistant strains. In many ways, the S. Typhimurium isolates in this study match the resistance profile of the multidrug-resistant invasive S. Typhimurium ST313 clone found in sub-Saharan Africa (Kingsley et al., 2009) but further investigation by MLST typing is needed before concluding. No resistance was detected to the newer generation antimicrobials (ciprofloxacin, gentamycin, nalidixic acid, cefotaxime, mecillinam, Imipenem) in our study these could thus be used in the future for empiric treatment.

Diarrhea caused by bacterial pathogens, especially Salmonella, remains one of the major causes of morbidity and mortality among infants and young children in Burkina Faso. Our results show that part of diarrhea in rural Burkina Faso is caused by antimicrobial-resistant Salmonella. Resistance of Salmonella spp. has shown a progressive increase over the course of time in many areas of the world.

Approaches to minimize the development of antimicro-bial resistance and optimize therapy include educational interventions for physicians and parents, appropriate use of antimicrobial agents, reduction in the use of growth promoters in animal feed. 

The authors have not declared any conflict of interests.

This study was supported by the “Union Economique et Monétaire Ouest-Africaine (UEMOA)” and the “Centre de Recherches en Sciences Biologiques, Alimentaires et Nutritionnelles (CRSBAN)”.