2Unité de Formation et de Recherche en Sciences Appliquées et Technologiques (UFR/SAT)/Université de Dédougou, BP 07 Dédougou Burkina Faso
3Centre National de Recherche et de Formation sur le Paludisme (CNRFP), 01 BP 2208 Ouagadougou 01, Burkina Faso
4Institut Des Sciences, 01 BP 1757 Ouagadougou 01, Burkina Faso
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, at north (Gourcy, distance 140 km) and western (Boromo, distance 185 km) of the capital Ouagadougou, Burkina Faso (Figure 1). Stool samples were collected from 275 under five years of age; 228 diarrheal children and 47 healthy children (control group).
Stool samples were taken by trained health staff personnel using a swab transport system (M40 transystemAmies agar gel without charcoal; Copan Italia Spa, Brescia, Italy) and transported to laboratory within 24h of their collection for analysis. Information regarding the age and sex were recorded for each child using a questionnaire.
Selenite broth (Emapol, Pologne) was used for the enrichment of specimens followed by an incubation at 37°C for 18h. Subsequently, samples were cultured on Hecktoen Enteric agar (Liofilchem, Italy) and incubated at 37°C for 24h. 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).
All Salmonella isolates were serotyped by the Salmonella Reference Laboratory. Isolates were serotyped with the somatic O and flagellar H anti-sera according to the Kauffman White scheme .
All Salmonella strains were subjected to antimicrobial susceptibility testing. It was carried out by disc diffusion method on Müller-Hinton agar (Liofilchem, Italy) according to the recommendations of the European Committee on Antimicrobial Susceptibility Testing . Briefly, after depositing the antibiotics, the plates were incubated at +37°C for 18-24h. Nineteen (19) antibiotics belonging to 7 different families were tested : amoxicillin (25 µg), amoxicillin–clavulanic acid (20/10 µg), ceftriaxone (30 µg), cefotaxime (30 µg), cefepime (30 µg), ceﬁxime (10 µg), piperacillin (75 µg), piperacillin–tazobactam (100 +10 µg), imipenem (10 µg), tetracycline (30 µg), chloramphenicol (30 µg), trimethoprim–sulfametoxazole (1.25 ± 23.75 µg), aztreonam (30 µg), colistin sulfate (50 µg), ciproﬂoxacin (5 µg), nalidixic acid (30 µg), gentamycin (15 µg), netilmicin (10 µg), and tobramycin (10 µg) (Bio-Rad, France).
Antibiotyping method involves the simultaneous presence of one or more antibiotic resistance markers. A strain may not wear a resistance marker or wear one or more. When studying the susceptibility of a strain to several antibiotics, its resistance phenotype to antibiotics was determined. If the strain expresses only natural resistances, it is said to belong to the "wild" or sensitive phenotype. If it acquired resistances have changed its sensitivity, it expresses a "resistance phenotype" that can be identified and whose mechanism must be determined. This phenotype is often referred to as initials of antibiotics that have become inactive. A strain is described as multidrug resistant when it is resistant to three antibiotics of different families [17-19].
Strains that were β-lactams resistant were subjected to investigation of extended spectrum ß-lactamase (ESBL) activity according to the recommendations of EUCAST . A disk of amoxicillin-clavulanic acid and two disks of third generation cephalosporins (C3G) (ceftriaxone and cefotaxime) were placed on the bacterial plate separated by a distance of 2 to 3 cm from one another. The presence of ESBL is indicated by a syngergetic effect between the disks, giving rise to an extended halo with the appearance of a “champagne cork” of keyhole.
Data were entried and analyzed using the software package Epi Info 188.8.131.52 (Centers for Disease Control and Prevention [CDC], Atlanta). Multivariable logistic regression was used to estimate odds ratios (ORs) with ninety-five percent confidence intervals (95% CI) also calculated. The statistical significance was evaluated using the Fischer exact 2-tailed p value and a p ≤ 0.05 was considered significant.
Permission to conduct the study was obtained from the hospital authorities of Burkina Faso, and informed verbal consent was obtained from the parents/guardians of every child before sample collection. The National Ethical Committee (s) of Burkina Faso (N° 2009-39) approved the study protocol.
A total of 228 children with diarrhea were included in our study (116 from Boromo Health District and 112 from Gourcy Health District). The age of participants range between 1 and 59 months and mean age was 31 months. From the f all study participants, 39.4% were females while 60.6% were males (Table 1).
|Age (months)||Sexe||Number of patients||Total N (%)|
|Boromo n (%)||Gourcy n (%)|
|0-12||M||30 (13.2)||38 (16.6)||68 (29.8)|
|F||28 (12.3)||22 (9.6)||50 (21.9)|
|13-35||M||29 (12.7)||28 (12.3)||57 (25)|
|F||20 (8.8)||11 (4.8)||31 (13.6)|
|36-59||M||5 (2.3)||8 (3.5)||15 (5.8)|
|F||4 (1.7)||5 (2.2)||9 (3.9)|
|Total N (%)||116 (50.9)||112 (49.1)||228 (100)|
M : Male F : Female n = N = Number
The number of stools loosed per day by the 228 diarrheal children ranged from 1 to 10 stools per 24 hours. Analysis of the clinical data revealed that 183 children (80.3%) loosed 1 to 5 stools per day and only 45 children (19.7%) loosed more than 5 stools per day ie an average of 3 stools per day. These stools were fluid (65.8%), mucous (17.5%) and bloody (16.7%).
|Age (months)||Number of patients (%)||Total (%)|
|Diarrheal group||Control group|
|0-12||1 (14.3)||2 (100)||3 (33.3)|
|13-24||2 (28.6)||0 (0)||2 (22.2)|
|25-59||4 (57.1)||0 (0)||4 (44.5)|
|Total (%)||7 (100)||2 (100)||9 (100)|
Fever, vomiting, dehydration and anemia were associated at diarrhea respectively at 56%, 41%, 17% and 2%. Several symptoms like malnutrition (32/228; 18%) and cough (15%) were also reported in children with diarrhea. Our study reported that 3 (43%) Salmonella in diarrheal children were associated to malnutrition. Our results showed that most of the children were breastfed (58.3%) while 41.7% consumed the family dish. A few cases of herbal teas administered to the children with diarrhea were also reported (1.3%). About water supply sources, nearly half of the participants used well water as a source of drinking (47.8%). The "Office National de l’Eau et de l’Assainissement" (National Water Supply Society) and drilling water were accessible to 27.2% and 22.8% respectively. Mineral water (1.8%) and running water (0.4%) were also drunk by several diarrheal children.
Nine (9) Salmonella sp. were isolated belonging to six different serotypes. The different rates were 22.2% for each serotype S. Typhimurium, S. Poona, S. Virchow and 11.1% for each S. Duisburg, S. Hvittingfoss, S. Ouakam. Sensitivity testing showed that the Salmonella sp strains had different levels of resistance to the antibiotics tested. Strains were resistant mainly to amoxicillin-clavulanic acid (89%), amoxicillin (100%), ceftriaxone (56%) and aztreonam (44%). We also noted resistance to quinolones (22% resistant to nalidixic acid) and fluoroquinolones (11% resistant to ciprofloxacin) (Figure 2).
Legend : AMC = Amoxicillin- clavulanic acid, AMX = Amoxicillin, CTX = Cefotaxime, ATM = Aztreoname, IPM = Imipenem, CRO = Ceftriaxone, FEP = Cefepime, CFM = Cefixime, TET = Tetracycline, CHL = Chloramphenicol, SXT = Trimethoprim-sulfametoxazole CIP = Ciprofloxacine, NAL = nalidixic acid, CST = Colistin sulfate, GMI = Gentamicin, TZP = Piperacillin-tazobactam, PIP = Piperacillin, NTM = Netilmicin, TMN = Tobramycin, I = Intermediate, R = Resistant.
Among the nine (9) Salmonella sp. strains, the most resistant phenotypes were Extended Spectrum β-lactamases (ESBL) phenotype (n = 4; 44.5%), Low Level Penicillinases (LLP) phenotype (n = 3; 33.3%). We also reported Low Level Penicillinases/quinolones cross-resistance phenotype (LLP/QCR) (n = 1; 11.1%) and Low Level Penicillinases/fluoroquinolones cross-resistance phenotype (LLP/FQCR) (n = 1; 11.1%) (Table 3). Multiple drug resistance (MDR) has been reported for most of the Salmonella serotypes (7/9 :78%) (Table 4).
|Antibiotic resistance phenotypes||Resistance (I+R) N (%)|
|Age groups (years)||Total N (%)|
|LLP||1 (33.3)||1 (33.3)||1 (33.4)||0 (0)||3 (100)|
|ESBL||2 (50)||1 (25)||1 (25)||0 (0)||4 (100)|
|QCR + LLP||0 (0)||0 (0)||1 (100)||0 (0)||1 (100)|
|FQCR + LLP||1 (100)||0 (0)||0 (0)||0 (0)||1 (100)|
Legend: LLP = Low Level Penicillinases, ESBL = Extended Spectrum β-lactamases, QCR = Quinolones Cross-Resistance phenotype, FQCR = Fluoroquinolones Cross-Ressistance phenotype, N = number
|Salmonella code||Serovar (Formula)||Phenotype of antibiotic resistance(a)||MDR Salmonella(b)|
|084B||Duisburg (4, 12:d:e,n,z15)||AMC, AMX, CTX, PIP, ATM, CRO, FEP, CFM, TET, SXT, CST, GMI, TMN, ESBL||Yes|
|057B||Poona (13, 22:z:1,6)||AMC, AMX, FEP, TET, CHL, CST, GMI, LLP||Yes|
|066B||Typhimurium (4,5, 12:i:1,2)||AMC, AMX, TET, SXT, CIP, NAL, GMI, LLP, FQCR||Yes|
|068B||Typhimurium (4,5, 12:i:1,2)||AMX, CTX, ATM, CRO, FEP, CFM, SXT, CST, ESBL||Yes|
|078B||Ouakam (9, 46:z29:-)||AMC, AMX, TET, CST, TMN, LLP||Yes|
|063G||Hvittingfoss (16:b:e,n,x)||AMC, AMX, CTX, ATM, CRO, FEP, PIP, CFM, CHL, ESBL||No|
|087G||Poona (13, 22:z:1,6)||AMC, AMX, CTX, ATM, PIP, CRO, FEP, CFM, TET, CHL, CST, TMN, ESBL||Yes|
|112G1||Virchow (6, 7:r:1,2)||AMC, AMX, CTX, CRO, FEP, CFM, TET, SXT, NAL, CHL, LLP, QCR||Yes|
|112G2||Virchow (6, 7:r:1,2)||AMC, AMX, CFM, LLP||No|
(a)Antibiotic resistance patterns of Salmonella. AMC, Amoxicillin- clavulanic acid ; AMX, Amoxicillin ; CTX, Cefotaxime ; ATM, Aztreoname ; CRO, Ceftriaxone ; FEP, Cefepime ; CFM, Cefixime ; TET, Tetracycline ; CHL, Chloramphenicol ; SXT, Trimethoprim-sulfametoxazole ; CIP, Ciprofloxacine ; NAL, nalidixic acid ; CST, Colistin sulfate ; GMI, Gentamicin ; PIP, Piperacillin ; TMN, Tobramycin ; LLP, Low Level Penicillinases; ESBL, Extended Spectrum β-lactamases; QCR, Quinolones Cross-Resistance phenotype; FQCR, Fluoroquinolones Cross-Resistance phenotype.
(b)MDR : Multidrug resistance
The authors gratefully thank the "Laboratoire de Biologie Moléculaire, d’Épidémiologie et de Surveillance des Bactéries et Virus Transmissibles par les Aliments (LaBESTA)", the "Centre National de Recherche et de Formation sur le Paludisme (CNRFP)" for their technical support. We also thank the parents and guardians of children as well as the authorities of the "CMA de Boromo" and "CMA de Gourcy" for their kind cooperation.
The authors declare that they have no conflict interests.
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