Antibiotic susceptibility prole of bacterial pathogens isolated from febrile children under 5 years of age in Nanoro, Burkina Faso.

Background: The curative power of antimicrobials is severely threatened due to emerging resistance to rst-line antibiotics worldwide. With a limited reserve of antibiotics, increasing antimicrobial resistance has become a global concern, but there is a paucity of such data in Burkina Faso, and the West African region in general. Therefore, this study aims to determine the antibiotic susceptibility prole of bacterial species isolated from febrile children under 5 years of age in Nanoro (Burkina Faso). Methods: Clinical specimens (blood, stool, and urine) were collected from 1099 febrile children attending the peripheral health facilities and the referral hospital in Nanoro. Bacterial isolates from these clinical specimens were assessed for their susceptibility against commonly used antibiotics by standard disc diffusion procedure and minimal inhibitory concentration method (when appropriate). Results: In total, 141 bacterial strains were recovered from 127 febrile children of which 65 strains were isolated from blood, 65 from the stool, and 11 from urine. Predominant bacterial isolates were Salmonella species (56.7%; 80/141) followed by Escherichia coli (33.3%; 47/141). Antibiotic susceptibility testing revealed Salmonella species were highly resistant to ampicillin (70%; 56/80), trimethoprim-sulfamethoxazole (65%; 52/80), and chloramphenicol (63.8%; 51/80). E. coli isolates were highly resistant to trimethoprim-sulfamethoxazole (100%), ampicillin (100%), ciprooxacin (71.4%; 10/14), amoxicillin-clavulanate (64.3%; 9/14), ceftriaxone (64.3%; 9/14), and gentamycin (50%; 7/14). Moreover, 7 out of 14 E. coli isolates were producers of the ß-lactamase enzyme, suggesting multi-drug resistance against b-lactam as well as non-b-lactam antibiotics. S. pneumoniae isolates were fully resistant to tetracycline and 50% to penicillin G. Multi-drug resistance was observed in 54.6% (59/108) of the isolates of which 56 (54.9%) were Gram-negative bacteria and 3 (50.0%) Gram-positive bacteria. Conclusions: The antibiotic susceptibility proling showed an alarming high resistance to commonly used antibiotics to treat bacterial infections in the study region. The work prompts the need to expand antibiotic resistance surveillance studies in Burkina Faso, and probably the whole region (West Africa). Moreover, it implies the need of a revision of the antibiotic-treatment guidelines by the Ministry of Health in Burkina Faso to avoid further development of resistance. other studies from the same study area (17) and other sub-Sahara African countries who also reported alarming resistance of E. coli and NTS to rst-line antibiotics (32–35). Urinary tract infections suspected to be caused by E. coli or Klebsiella species are treated with trimethoprim-sulfamethoxazole (SXT) or amoxicillin (AMOX) (same antibiotic category as AMP), but these rst-line antibiotics for UTI treatment revealed a resistance rate of 100% in this study. Although low resistance of NTS to CIP (uoroquinolones) was found, the ecacy of this antibiotic must be carefully monitored as it is widely used to treat bacillary dysenteries by children under 5 years in West Africa (18, 36). multi-resistant group β-lactam 39, 40), Gram-negative


Background
The development of antibiotics against bacterial infections has been one of the greatest achievements of modern medicine (1)(2)(3)(4)(5). However, the e cacy of these antibiotics is not endless and this success is now being jeopardized by the increasing occurrence of antibiotic resistance (ABR). Nowadays, ABR is considered as one of the most important threats to public health and one of the biggest health challenges that mankind faces (6)(7)(8)(9)(10)(11). Indeed this is associated with increased risk of infection severity, patient morbidity and mortality rate, prolonged hospitalization time and healthcare costs (12). One of the main obstacles in low-and middle-income countries is the lack of practical tools in the primary healthcare facilities to reliably differentiate bacterial infections from other febrile infections. As a result, antibiotics are systematically prescribed without any evidence-base, thereby signi cantly contributing to increasing ABR (13).
To solve this alarming situation, the World Health Organization (WHO) has developed a global antimicrobial resistance (AMR) action plan that encompasses reinforcing AMR knowledge through surveillance and research (12). A better understanding of local AMR patterns is crucial to rstly guide clinical management of infectious diseases and secondly for the early detection of ABR to rst-line antibiotics used in primary healthcare facilities. However, the information about the true extent of the antibiotic resistance threat in the African region is limited to 6 out of 47 countries where studies on AMR have been performed. The resulting gap in monitoring AMR weakens decision-making on antibiotic resistance policy (14,15).
The same applies to Burkina Faso, where studies have revealed the worrying situation of the most commonly prescribed rst-line antibiotics in primary healthcare facilities such as amoxicillin, amoxicillin-clavulanic acid and ampicillin (9,10,(16)(17)(18). These studies highlight that signi cant resistance is recorded for several bacterial species, which have spread into hospitals and communities. It has for example been observed that nurses providing the rst-line care in primary healthcare facilities use the 10 years old national treatment recommendations (18), but this guideline does not contain up to date information about the resistance pro les of different circulating bacterial strains and species. In addition, this guideline is mostly based on ndings in high income countries and does not necessarily re ects the best treatment options for low income countries such as Burkina Faso. Furthermore, this situation is exacerbated due to the fact that the general public has (without a proper prescription) access at local markets and shops to antibiotics, where supply and quality of drugs are not appropriately controlled. This does not only threat the effectiveness of current rst-line antibiotic treatments used in peripheral health facilities, but also the second-and third-line antibiotics (6,19). There are currently no structural mechanisms in place in Burkina Faso to monitor antibiotic use and the susceptibility of bacteria to available antibiotics. The existing sentinel sites for antibiotic resistance surveillance are mainly in tertiary urban hospitals and often not operational.
The present study aims to ll part of the gap in our knowledge on the current effectiveness of antimicrobials by presenting the antibiotic susceptibility pro le of bacteria isolated from various clinical specimens of febrile children less than 5 years of age in the Nanoro health district, Burkina Faso. Among the antibiotics tested in this study, several are recommended as the rst-line antibiotics by the Ministry of Health (MoH) of Burkina Faso to treat various bacterial infections (Table 1). According to this guideline sepsis/suspected bacterial bloodstream infections (bBSIs) and suspected pneumonia are treated with ampicillin (AMP) and gentamycin (GEN). In the cases of suspicion of typhoid fever, it is recommended to treat the infection with the cipro oxacin (CIP).
Furthermore, it is advised to treat suspected simple pneumonia with the trimethoprim-sulfamethoxazole (SXT) (18). For suspected cases of bacterial gastroenteritis (bGE), the rst-line antibiotic is also CIP and for suspected bacterial urinary tract infections (bUTIs) either SXT or amoxicillin (AMOX) are used (18). The rst-line therapy for the treatment of the meningitis infections is chloramphenicol (CL) and AMP; in case CL appears to be ineffective, ceftriaxone (CRO) is used as second line-treatment (18).  (18). For suspected cases of bacterial gastroenteritis CIP is used and for suspected bacterial urinary tract infection either SXT or amoxicillin (AMOX) is used (18). Chloramphenicol (CL) and AMP are mostly used as rst-line therapy for bacterial meningitis and Ceftriaxone (CRO) as second line-treatment (18).

Patients and clinical samples
The present study was conducted in the framework of a larger project that was investigating the aetiologies, diagnoses, and treatment of febrile children in the Health district of Nanoro (20). Brie y, any child under-5 years of age attending the primary healthcare facilities or the referral hospital of Nanoro with documented fever (axillary temperature ≥ 37.5 °C) was invited to participate in the study. Cases were managed by health facility or referral hospital staff according to the Burkinabe national protocol of diseases management based on the Integrated Management of Childhood Illness (IMCI) (21). Furthermore, clinical specimens (blood, stool and urine) were collected at enrolment for microbiological analyses at the laboratory of Microbiology of the Clinical Research Unit of Nanoro (CRUN). In case the children could not produce a urine or stool sample at the time of enrolment, sterile containers were provided to the legal guardian to collect these samples at home and return them as soon as possible to the health facility within 48 hours after inclusion.
Written informed consent was obtained from parents or legal guardians before any data and specimen collection.  (27,28). The diameter of the inhibition zone was measured and recorded in millimetres for each disc and in microgram per millilitre (µg/mL) for each E-test. The results of antibiotic susceptibility testing were interpreted according to the criteria of the CLSI (27,28).
The antibiotic discs (BD Seni-Disc™, Becton Dickinson and Company, B.V., Vianen, The Netherlands) used for antibiotic susceptibility testing as well as the minimal inhibition concentration (MIC; E-tests; Lio lchem S.r.l, Roseto degli Abruzzi(TE), Italy) are reported in Table 1.
Furthermore, the extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae were determined by using both ceftazidime (CAZ) (30 µg) and cefotaxime (CTX) (30 µg) discs, alone or in combination with clavulanate (C) (10 µg) discs, as described in CLSI (27,28). A bacteria strain was considered as potential ESBL-producer, when the inhibition zone diameters were ≤ 25 mm for ceftriaxone (CRO) disc, ≤ 22 mm for CAZ disc, and ≤ 27 mm for CTX disc (27,28). An Enterobacteriaceae phenotype is indisputably considered to be an ESBL producing phenotype bacterium if the difference between the inhibition zone diameter for either antibiotic tested in combination (CAZ + C) or (CTX + C) and the inhibition zone diameter of the corresponding antibiotic tested alone (CAZ or CTX) is ≥ 5 mm (27, 28). S. aureus species were considered as MRSA strains when the inhibition zone diameter of cefoxitin disc (FOX; 30 g) on Mueller Hinton (MH) agar plate is ≤ 21 mm after 16-18 hours of incubation, according to CLSI guidelines (27,28).
An isolate was considered to be multi-resistant when it is resistant to at least one antibiotic agent in each of all three antibiotic categories used for therapy or prophylaxis based on Burkina Faso national treatment guidelines.

Data analysis
The inhibition diameters for each antibiotic tested for each investigated bacterium were recorded using Excel 2016. These data were double entered by 2 independent technicians and subsequently validated by the lab-manager. Data analysis was performed using STATA version 13 software. For the interpretation of the resistant rate of strains identi ed in the present study, the following classi cation was used: low (resistance rate < 20%), moderate (resistance from 20 to 50%), high (resistance rate from 50 to 75%), alarming (resistance rate from 75 to 100%) for the antibiotics tested (30, 31).

Results
The characteristics of the study population are presented in Table 2. In total 1099 children participated in the study of whom 55.2% were male and 44.8% female. One hundred and twenty-seven (11.6%) of the febrile children had one (or more) con rmed bacterial infection(s). In total, 1099 blood samples (100%), 757 (68.9%) stool samples and 739 (67.2%) urine samples were collected for microbiology analyses. Among them, a total of 141 bacterial strains were identi ed. Out of these bacterial strains, 65 were con rmed in bacterial bloodstream infections (bBSI), 65 in bacterial gastroenteritis (bGE), and 11 in bacterial urinary tract infections (bUTI) ( Table 2).

Antibiotic Susceptibility testing
Antibiotic susceptibility of Gram-negative bacteria The antibiotic susceptibility testing was performed on 102 Gram-negative bacteria, but not on 33 EPEC isolates from stool. The antibiotic susceptibility results of 102 Gram-negative bacteria isolates recovered from the various clinical specimen are presented in Table 4. The analysis of the susceptibility patterns of predominant Gram-negative bacteria isolates such as non-typhoid Salmonella (NTS) and E. coli revealed a resistance rate varying between high to alarming for several antibiotics tested (Table 4). For example, among NTS isolated from blood and stools, the rate of reported resistance was alarming or moderated to CL, respectively. However, for NTS, the susceptibility testing revealed a low resistance rate for cipro oxacin (CIP) and nalidixic acid (NA); (Table 4). In contrast, for E. coli isolated from urine, an alarming resistance rate was reported to CIP and NA. In addition, 7 strains of E. coli produced β-lactamase of which 6 were isolated from urine, suggesting multi-drug resistance against β-lactam and non-β-lactam antibiotics. For other Gram-negative bacteria, two out of four strains of typhoidal Salmonella (TS) showed high resistance to trimethoprim-sulfamethoxazole (SXT; 50%) and nalidixic acid (NA; 50%). All N. meningitidis strains tested had an alarming resistance to SXT and one was resistant to penicillin (PEN). Less frequently isolated E. agglomerans (0.7%) and H. in uenzae b (0.7%) were found to be sensitive to most of the antibiotics tested, except for trimethoprim-sulfamethoxazole (SXT; 100% resistance) to H. in uenzae b. The single Klebsiella species isolated from urine was fully resistant to SXT (100%) and AMP (100%).   In Table 5 the resistance rates to the rst-line therapies as recommended by MoH of Burkina Faso are presented in more detail. The resistance rates of bacteria associated with bacterial gastroenteritis were in the general low to moderate. However, in the case of bacterial urinary tract infections, E. coli and Klebsiella resistance rate against ampicillin (AMP) and SXT was 100%. AMP is commonly used to treat invasive bacterial infections, but high to alarming resistance rates were found in the present study. In contrast, GEN and CRO seemed to remain effective against NTS.  In addition to rst-line antibiotic tested, all S. pneumoniae isolates showed resistance to TET (100%). As for S. aureus, 1 out of 2 was resistant to clindamycin (CC) and tetracycline (TET). In contrast, CRO that is used as the rst-line antibiotic to treat bacterial meningitis showed to be effective against S. pneumoniae.

Resistance pro ling of co-infections
The resistance pro ling results of bacterial co-infections are presented in Table 6. In total, 11 bacterial isolates (10 NTS and 1 E. coli) were identi ed simultaneously in blood and stool. The resistance rate of NTS strains identi ed from both infection sites was alarming to the rst-line antibiotics (AMP and SXT) tested. Importantly, 2 children had three types of different infections; one child had an E. coli strain responsible for bBSI, bGE, and bUTI, and in another child, 2 NTS strains were responsible for bBSI and bGE, and one E. coli caused bUTI. Overall, all these bacteria identi ed from these 3 sites of infections were fully resistant to AMP and SXT, which are commonly used as rst-line antibiotics to treat these infections cases. Table 6 Resistance rate to recommended rst-line therapy* for the treatment of bacterial co-infections identi ed.
Co-infections type bBSI + bGE (11) bBSI + bUTI (2) bGE + UTI (3) bBSI + bGE + bUTI (2) Isolated bacteria In total, 56/102 (54.9%) of Gram-negative and 3/6 (50%) Gram-positive bacteria were MDR (Table 7). Ten out of fourteen (71.4%) E. coli strains isolated in this study revealed resistance to SXT, AMP and CIP. For Salmonella species, 56.3% (45/80) of the strains isolated were resistant to SXT, AMP and CL. These antibiotics are recommended by national treatment guidelines of Burkina Faso to treat the infections found in this study (Table 5).   (17) and other sub-Sahara African countries who also reported alarming resistance of E. coli and NTS to rst-line antibiotics (32)(33)(34)(35). Urinary tract infections suspected to be caused by E. coli or Klebsiella species are treated with trimethoprim-sulfamethoxazole (SXT) or amoxicillin (AMOX) (same antibiotic category as AMP), but these rst-line antibiotics for UTI treatment revealed a resistance rate of 100% in this study. Although low resistance of NTS to CIP ( uoroquinolones) was found, the e cacy of this antibiotic must be carefully monitored as it is widely used to treat bacillary dysenteries by children under 5 years in West Africa (18,36).
The present work also reported an ambiguity regarding the role that the urinary tract plays on the proliferation of E. coli producing β-lactam enzyme. It was found that 85.7% E. coli isolates from urine were β-lactamase enzyme producers. The children from whom these bacteria were isolated could transmit these resistant E. coli strains to their mother, and subsequently to their family, and even to the community. Furthermore, the strains producing β-lactamase, usually show co-resistance to non-β-lactam antibiotics, such as aminoglycosides and uoroquinolones (37)(38)(39). This explains the high resistance of E. coli isolated from urine to β-lactam and cross-resistance to non-β-lactam antibiotics reported in this study. It was reported that this enzyme is predominately produced by bacteria that are multi-resistant to the group of β-lactam antibiotics (1,37,39,40), which are frequently used to treat infections caused by Gram-negative bacteria like Enterobacteriaceae. This observation, supported by data form another study from Burkina Faso (10) and from other African countries (15,33,41,42) implies that treatment options for bacterial diseases are further reduced. Especially, the treatment of pediatric bUTIs caused by ESBL-producing E. coli is nowadays seriously jeopardized due to antibiotic resistance in sub-Saharan Africa countries.
The observed high resistance of E. coli to 3rd generation antibiotics cephalosporin (CRO) and uoroquinolones (CIP), which are two essential antibiotics largely used in our study area, further pin points the severe threat of antibiotic resistance at the community level. Together these data con rm that the e cacy of many rst-line antibiotics commonly used in Nanoro to treat principal bacterial infections such as E. coli and NTS is at high risk. This is likely to further undermine the precarious health system in place in low-and middle-income countries (LMICs) such as Burkina Faso if nothing is done to stop the spread of resistance. It should be noted that our results are fully in line with other observations that warned for decaying antibiotic effectiveness (17,43). Therefore, actions have to be taken urgently to prevent the inappropriate use of antibiotics, which are still (highly) effective against common pathogenic bacteria encountered at primary health facilities. In order to deal with this threat, it is essential that practical tools or diagnostic algorithms be developed to correctly diagnose bacterial infections that can be easily implemented in the primary health care settings in LMICs. Furthermore, national and regional guidelines for integrated management of childhood illness (IMCI) that recommend syndrome-based management and treatment of bacterial infection need to be reconsidered as it may contribute to the spread of antibiotic resistance. The untargeted, prolonged, and repeated exposure of bacteria to essential antibiotics, which is a consequence of the use of the IMCI guidelines, is largely contributing to emerging resistance and jeopardizes action plans to ght against this emerging antibiotic resistance.
Despite the rare cases of N. meningitidis (2 cases) and H. in uenza b (1 case) reported in the present study, it is relevant to note that these bacteria were fully susceptible to the CL and CRO. This is important as these antibiotics are used to treat meningitis as recommended by MoH of Burkina Faso (the country is located in Lapeyssonnie's belt). Moreover, GEN used in combination with AMP as a rst-line antibiotic showed to be effective against most of the pathogens isolated in this study, except for E. coli, which showed moderate resistance. In addition, low resistance of NTS isolated from blood to this antibiotic was found in the present study, and this is worrying as this combination was always highly effective against Enterobacteriaceae in Burkina Faso and this might be an indication for upcoming resistance against this antibiotic.
The study also reported a high prevalence of MDR bacteria. This emergence of MDR is a serious public health problem and a threat to effectively treating bacterial infections. The emergence of speci c MDR bacteria is closely linked to the use of broad-spectrum antibiotics for both presumptive and de nitive therapy. The occurrence of community spread of MDR bacteria leads to the large increase of the population at risk and increases the number of infections caused by MDR bacteria.
A limitation of the study is that the work did not include respiratory tract infections, as these infections are often (presumptively) treated with antibiotics, irrespective of the cause of infection (being bacterial or viral). Often this treatment practice leads to signi cant resistance (19,44). In the case of suspected simple pneumonia, it is for example advised to treat with the trimethoprim-sulfamethoxazole (SXT). In the present study, it was found that this antibiotic was ineffective to many of the bacterial infections studied and it would be valuable to determine its effectiveness against bacterial infections causing pneumonia.
Another possible restriction of the study is the low number of Gram positive bacteria isolated from the clinical specimens studied. The low prevalence of S. pneumoniae is likely a positive effect of the introduction of the pneumococcal conjugate vaccine in the expanded program of immunization (EPI) in October 2013 (45,46). However, it remains a concern that the few isolates recovered in the present study (from blood) showed moderate to high resistance against the rst-line antibiotics recommended in our study area (6,18).
Finally, another limitation of our study is the fact that the recruited children were not followed up post-treatment in the framework of the study. Consequently, it remains unknown whether the treatments installed actually failed or were successful in vivo. However, based on the evidence provided by the susceptibility testing it is likely that several treatments have failed thereby jeopardizing the health of the children. Therefore, we propose to update the current national antibiotic treatment guidelines in order to use effective drugs to treat the infections.
The study demonstrated that various rst-line antibiotics are no longer effective to treat common bacterial infections and a revision of the current treatment guidelines in Burkina Faso and probably the whole West-Africa region is needed. Based on our study outcomes we recommend the following revision (Table 8): when sepsis or a simple (uncomplicated) bBSI is suspected; the proposed treatment would be with a single 3rd generation cephalosporin (CRO). In the case of a severe sepsis or severe bBSI, the proposed treatment would be a combination of a 3rd generation cephalosporin such as CRO combined with an aminoglycoside, like Gentamycin (GEN). In the case of a suspected bUTI, we suggest distinguishing between hospitalized and non-hospitalized cases, because the route of administration of GEN may have a health safety risk for the outpatient as it needs to be administered intravenously. For a hospitalized patient with bUTI the proposed treatment would be with an aminoglycoside (GEN). However, for a non-hospitalized case, we propose to use amoxicillinclavulanate (AMC) which is a combination of a β-lactamase inhibitor, Clavulanic acid (C), together with another antibiotic agent, Amoxicillin, which can be administered orally. For the treatment of bGE we propose to use a uoroquinolone (CIP), but it is important to monitor resistance to this antibiotic too as it is very frequently used even without proper laboratory examinations and/or prescriptions.

Conclusions
In conclusion, this study showed an alarming high resistance to many rst-line antibiotics used to treat common bacterial infections in Burkina Faso and a revision of the current treatment guidelines is needed. The work prompts the need to expand antibiotic resistance surveillance studies in Burkina Faso, and probably the whole region (West Africa). Abbreviations