Antibiotic Susceptibility of Staphylococcus aureus and Streptococcus pneumoniae Isolates from the Nasopharynx of Febrile Children under 5 Years in Nanoro, Burkina Faso

(1) Background: nasopharynx colonization by resistant Staphylococcus aureus and Streptococcus pneumoniae can lead to serious diseases. Emerging resistance to antibiotics commonly used to treat infections due to these pathogens poses a serious threat to the health system. The present study aimed to determine the antibiotic susceptibility of S. aureus and S. pneumoniae isolates from the febrile children’s nasopharynx under 5 years in Nanoro (Burkina Faso). (2) Methods: bacterial isolates were identified from nasopharyngeal swabs prospectively collected from 629 febrile children. Antibiotic susceptibility of S. aureus and S. pneumoniae isolates was assessed by Kirby–Bauer method and results were interpreted according to the Clinical and Laboratory Standard Institute guidelines. (3) Results: bacterial colonization was confirmed in 154 (24.5%) of children of whom 96.1% carried S. aureus, 3.2% had S. pneumoniae, and 0.6% carried both bacteria. S. aureus isolates showed alarming resistance to penicillin (96.0%) and S. pneumoniae was highly resistant to tetracycline (100%) and trimethoprim–sulfamethoxazole (83.3%), and moderately resistant to penicillin (50.0%). Furthermore, 4.0% of S. aureus identified were methicillin resistant. (4) Conclusion: this study showed concerning resistance rates to antibiotics to treat suspected bacterial respiratory tract infections. The work highlights the necessity to implement continuous antibiotic resistance surveillance.


Introduction
Bacterial colonization of the nasopharynx in human can lead to the development of invasive and non-invasive disease, caused by common pathogens, such as Staphylococcus aureus and Streptococcus pneumoniae. Although nasopharyngeal carriage with S. pneumoniae and S. aureus is usually asymptomatic, it can lead to serious infections in children, such as pneumonia, sepsis, and otitis [1][2][3]. Moreover, the nasopharynx of healthy individuals is a potential reservoir for transmission of S. pneumoniae and S. aureus to other people in the community or health care setting [2][3][4][5]. Therefore, emerging resistance to commonly used antibiotics to treat infections caused by these bacteria is a serious threat to health systems [5][6][7][8][9]. This situation can lead to treatment failures, extended hospitalization, increased health care costs, and may ultimately lead to increased mortality and morbidity [8,9].
In Burkina Faso, S. pneumoniae became the leading cause of bacterial meningitis after the introduction of the Haemophilus influenzae type b vaccine in 2006 [10] and the serogroup A meningococcal conjugate vaccine (MenAfriVac) in 2010 [11]. However, the introduction of the thirteen-valent pneumococcal conjugate vaccine (PCV13) resulted in a significant decrease of invasive diseases caused by related strains, such as serotypes 6A/6B, 5, 14, 23F, and 18C/18F/B/18A, in children under 5 years of age in the country [12,13]. A reduction of around 50% in absolute number of cases of confirmed pneumococcal meningitis in children under 5 years was observed from the pre-PCV13 period (2011-2013; 478 confirmed pneumococcal meningitis cases) to the post-PCV13 period (2014-2015; 212 confirmed pneumococcal meningitis cases) [12,13]. Similarly, it was reported that the introduction of the pneumococcal conjugate vaccine into routine infant immunization programs substantially decreased invasive pneumococcal diseases in some other African countries [14,15].
Despite this reduction of pneumococci infections, some pneumococci genotypes resistant to antibiotics have emerged worldwide mainly in commensal micro flora [7,16]. In addition, it has been reported that pneumococcal conjugate vaccination might alter the upper respiratory tract flora and subsequently increase the risk of S. aureus colonization and diseases, particularly with methicillin-resistant S. aureus (MRSA) [17]. Although not extensively studied in Burkina Faso, reports from the West Africa region highlight a significant spreading of MRSA strains [18][19][20][21].
Improved insight in emerging bacterial resistance could be obtained when more antimicrobial resistance prevalence studies on nasopharyngeal carriage are conducted in febrile children under 5 years of age. This would improve monitoring and control of these emerging resistant bacteria and would also help to save lives of many children under 5. Bacterial colonization of the nasopharynx could be a proxy to assess bacterial resistance and pneumococcal serotype distribution [22]. Furthermore, the inter-human and environmental transmission of resistant strains are important determinants in the spread of bacteria resistant to antibiotics [23]. Consequently, studying potential pathogens of the nasopharynx can be a substantial add-on value to the antibiotic stewardship and antimicrobial resistance surveillance. Therefore, the present study aimed to determine the antibiotic susceptibility profile of S. aureus and S. pneumoniae isolates from the nasopharynx of febrile children under 5 years in Nanoro, Burkina Faso.

Characteristics of Study Population
The characteristics of the study population are presented in Table 1 In total, 629 nasopharyngeal swabs were obtained from febrile children under 5 years. A significantly higher proportion (5% significance level; 1 degree of freedom) of males (54.1%; 340/629) were recruited. The median age of the enrolled children was 19 months (Interquartile range (IQR): 11.0-32.0). A significantly higher proportion (5% significance level; 1 degree of freedom) of the enrolled children (71.9%; 452/629) were infants (between 1 and 30 months of age) and a portion of this age group (30.1%; 136/452) did not receive pneumococcal vaccination according to the expanded national vaccination (EPI) program of Burkina Faso.
Furthermore, bacterial colonization was not significantly different between gender (p = 0.55) and age groups of vaccinated children (p = 0.08)
The distribution of the antibiotic susceptibility patterns of S. pneumoniae isolates according to the different age groups is also presented in Table 2; no notable differences were observed in terms of resistance between the two age groups.

Discussion
This study aimed to determine the antibiotic susceptibility of Staphylococcus aureus and Streptococcus pneumoniae isolates obtained from the nasopharynx of febrile children under 5 years in Nanoro, a rural area of Burkina Faso. The study provides evidence for concerning high resistance rates to antibiotics that are commonly used to treat suspected respiratory tract infections and septicemia in Burkina Faso. A significant part of these (sometimes serious) infections are often caused by S. aureus and S. pneumoniae that are usually considered to be normal colonizers of the nasopharynx of these children [1][2][3].
The high resistance rates of S. aureus to several commonly used first-line antibiotics are of concern. Particularly, the multidrug resistant S. aureus isolates make many antibiotics clinically inefficient and, thereby, reduces treatment options [8,9]. Moreover, a large proportion of methicillin-resistant S. aureus (MRSA) was also highly resistant to PEN and TET, and this resistance might be due to the β-lactamases produced by S. aureus [5]. In addition, almost all methicillin-sensitive S. aureus (MSSA) were resistant to PEN and TET. These findings are of great concern; this was also mentioned by other studies from Burkina Faso [6] and other African countries [18][19][20][21]24]. In contrast, some antibiotics, including gentamicin (GEN) and vancomycin (VAN), are still effective in our study and could serve as alternative treatment options. A limitation of the current study is that the resistance genes of MRSA have not been characterized and we have not established resistance genes similarities between MRSA and MSSA isolates. The study was restricted to phenotypical resistance assessment of the isolated bacteria. More advanced phenotypic (e.g., automated systems) or genotypic (e.g., polymerase chain reaction) methods to determine antibiotic susceptibility are unfortunately still out of reach for many laboratories in low-and middle-income countries (LMICs), including Burkina Faso.
Relatively few S. pneumoniae isolates were retrieved in the present study. This low colonization rate could be due to the introduction of the 13-valent pneumococcal conjugate vaccine (PCV-13) into the vaccination program of Burkina Faso in 2013 [12,13]. The high efficacy of this vaccine to reduce S. pneumoniae carriage in general was also demonstrated by Kiemde et al. [25], who obtained only three [3] isolates of this species from blood from the same study population. However, high resistance rates to SXT and TET and moderate resistance rate to PEN, which are part of the first-line antibiotics used to treat septicemia and non-severe and severe pneumonia caused by S. pneumoniae [26], were observed. This observation is also in line with other studies from Burkina Faso [7,27] and other African countries [28][29][30]. It should be noted that the majority of colonizing S. pneumoniae isolates were from infants and part of these children are in the process of receiving the full pneumococcal vaccination course [31]. The colonizing of the nasopharynx by resistant S. pneumoniae strains is decreased in older toddlers who have received the full pneumococcal vaccination, confirming the impact of the pneumococcal vaccine after its introduction in the expanded immunization program in Burkina Faso and worldwide [31,32].
Although the introduction of the PCV-13 has decreased pneumococcal meningitis incidence in children under 5 in Burkina Faso, it is important to note that some serotypes, such as serotype 1, 23F, 6A/6B, and 12F/12A/12B/44/46, remain predominant in these children [12,13] including other S. pneumoniae serotypes that the PCV-13 vaccine does not cover. In particular, serotypes 6A and 23F have been reported to develop multi-drug resistance before their inclusion in the PCV-13 [7], and continue to do so even after their inclusion in the vaccine as suggested by some studies [33,34]. Vaccination might facilitate the introduction of new more resistant pneumococcal serotypes that replace the vaccine serotypes [35,36]. Most likely, these serotypes or those that are not covered by PCV13 are responsible for resistance observed in our study, but this cannot be confirmed as serotyping was not performed.
It is relevant to note that all S. pneumoniae strains isolated in this study, albeit, few, were susceptible to ceftriaxone (CRO) and ampicillin (AMP), which are the first-line antibiotics to treat meningitis and suspected septicemia, respectively, in Burkina Faso. Furthermore, the S. aureus isolates were susceptible to vancomycin, which is used to treat infections caused by MRSA [37,38]. In addition, our study outcomes support the use of ampicillin for the treatment of suspected pneumonia. Whenever possible, as a mean to curtail resistance, the use of the narrowest spectrum antibiotics is preferred, which is, in this case, AMP.
The present study showed that a significant proportion of S. aureus and S. pneumoniae isolates from the nasopharynx of young febrile children is resistant to many commonly used antibiotics. This poses a serious problem in the management and treatment of infectious disease. It is, therefore of utmost importance that the spread of (emerging) resistant bacteria in Burkina Faso (and probably the whole West African region) is slowed down and that surveillance structures of antibiotic resistance must be reinforced. In order to manage this resistance problem, more extensive studies on phenotypical and molecular resistance of colonizing bacterial strains from the nasopharynx should be conducted. Knowing that the nasopharynx is a niche propitious to spread resistant strains in communities [2][3][4]21], such studies will provide significant data on the antibiotic resistance and help to refine treatment guidelines at national and global level.

Study Design and Participants
The present observational study was embedded in a large research project implemented in the Health District of Nanoro (central-west Burkina Faso) and performed from 2014 to 2018 that investigated the etiology, diagnosis, and treatment of fever episodes in children under 5 years [25]. Written informed consent was obtained from the parent or legal guardian prior to enrolment of a child in the study. The study protocol was approved by the National Ethical Committee in Health Research, Burkina Faso (Deliberation N • 2014-11-130).
Febrile children under the age of 5 years with an axillary temperature ≥37.5 • C presenting at one of the study health facilities were diagnosed according to the International Classification of Diseases in Childhood [39]. They were treated according to the national guidelines for the management of childhood diseases based on the world health organization (WHO) guidelines for the Integrated Management of Childhood Illness [40]. The children were not further follow-up to determine treatment outcome in the context of this study. From each recruited child, different samples were collected, regardless of the potential cause of fever. The clinical specimens were transported to the Microbiology Laboratory of the Clinical Research Unit of Nanoro (CRUN) for microbiology analysis according to the standard operating procedures (SOPs). A standard case record form (CRF) was used to collect details of clinical examinations, diagnosis, and antibiotics prescription.

Laboratory Procedures
Nasopharyngeal samples were collected with sterile cotton swabs from each participant by trained study nurses. After collection, the nasopharyngeal swabs were inoculated in skim milk, tryptone, glucose, and glycerin broth, and transported to the laboratory where they were processed immediately. Each sample was vortexed and 200 µl was subsequently transferred to 10 mL of Todd-Hewitt broth for enrichment and incubated at 35 ± 2 • C for 24 h. Next, each broth was sub-cultured onto sheep blood agar, and mannitol salt agar (MSA). The sheep blood agar plates were incubated under 5% CO 2 , and the MSA plates under aerobic conditions, at 35 ± 2 • C for 24 h.
Bacterial isolates were identified using standard microbiology methods [41][42][43]. S. pneumoniae were identified by their ability to produce alpha hemolysis on sheep blood agar, and their inability to produce catalase [42]. S. aureus was identified as small to large yellowish colonies (ability to ferment mannitol) on MSA plates, and by positive reaction to the catalase and coagulase tests [42].

Antimicrobial Susceptibility Testing (AST)
Antimicrobial susceptibility was tested by disk diffusion (Kirby-Bauer) and Epsilometer (E-test) methods, and interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines [44]. Antibiotics used for susceptibility testing are listed in Table 3. Mueller-Hinton agar with 5% of sheep blood was used for AST of S. pneumoniae while Mueller-Hinton agar was used for S. aureus. The agar plates were inoculated aseptically with bacterial suspension at McFarland 0.5 (measured by BD PhoenixSpec, Nephelometer Becton Dickinson and Company, Sparks, Maryland, USA) and incubated under either atmospheric condition or 5% of CO 2 for 18-24 h for S. aureus and S. pneumoniae, respectively. According to CLSI guidelines, the minimal inhibitory concentration (MIC) as determined by Epsilometer (E-test) was used for some antibiotics (see Table 3) [44]. Furthermore, methicillin-resistant Staphylococcus aureus (MRSA) strains were phenotypically identified when the diameter of the cefoxitin disc (30 µg) was ≤21 mm [44]. Inducible resistance for both S. aureus and S. pneumoniae to Clindamycin (CC) was determined by D-testing.

Quality Control
Standard bacteriological procedures were followed in accordance with the local microbiology standard operating procedures (SOPs) to ensure the reliability of the laboratory results. In addition, all the laboratory processes (culture media, reagents, AST disks and equipment) were quality controlled using American Type Culture Collection (ATCC ® ) standard reference strains. Furthermore, CRUN microbiology laboratory is enrolled to the external quality assessment program of the National Institute for Communicable Diseases (NICD, Johannesburg, South Africa), supported by the World Health Organization (WHO) Africa.

Data Analysis
Data were entered into Microsoft Excel version 2016, checked by two independent technicians, and subsequently validated by the laboratory manager. The data were then analyzed using STATA ® statistical software version 13 StataCorp LLC, College Station, TX, USA. Categorical variables were summarized as proportions and Pearson's chi-square test were performed. The median was used for continuous variables. A p value of <0.05 was considered significant.
Children were stratified in age groups following the expanded national vaccination program of Burkina Faso Ministry of Health (MoH) [31]. The following age categories were made:
Infants: ≥1-<30 months of age, in progress of receiving the full course of pneumococcal vaccination; 3.
Older toddlers: ≥30-<60 months of age, completed the full course of pneumococcal vaccination (expected to be fully immunized).
A bacterial isolate was considered MDR when it was resistant to at least one antibiotic agent in three antibiotic categories [45].

Conclusions
This study revealed high resistance rates to antibiotics that are commonly used to treat suspected bacterial respiratory tract infections. Children who received the 13-valent pneumococcal conjugate vaccine carried the highest proportion of resistant bacteria. The research highlights the necessity to perform frequent and extensive antibiotic-resistance studies, including molecular assessment, to ensure that the shrinking arsenal of effective antimicrobials remains effective.

Ethics Approval and Consent to Participate
A child was only enrolled in the study after obtaining signed written informed consent of her/his parent or legal guardian. The study protocol was reviewed and approved by the National Ethical Committee in Health Research, Burkina Faso (Deliberation No. 2014-11-130).  Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper.

Data Availability Statement:
Data is contained within the article. Datasets used and/or analyzed in this study are available from the corresponding author upon reasonable request. WHO Africa is thanked for supporting the external quality assessment of the laboratory. Standard reference strains Staphylococcus aureus ATCC 25923, Streptococcus pneumoniae ATCC 49619, Staphylococcus epidermidis ATCC 14990, Escherichia coli ATCC-25922, Streptococcus pyogenes ATCC 19615, Enterococcus faecalis ATCC 29212 were obtained from The American Type Culture Collection.

Acknowledgments:
We would like to acknowledge the study staff of the rural health facilities (Nanoro Health District) and the reference hospital CMA Saint Camille de Nanoro for their valuable support to the work. We are very grateful to all the children who participated in the study. We also acknowledge the staff of the Microbiology Department of Clinical Research Unit of Nanoro for their enormous help in analyzing the clinical samples and data.

Conflicts of Interest:
The authors declare no conflict of interest.