ANTIMICROBIAL RESISTANCE IN STAPHYLOCOCCUS AUREUS ISOLATES FROM INPATIENTS AND OUTPATIENTS IN OUAGADOUGOU, BURKINA FASO

1. Laboratoire de Biochimie et Immunologie Appliquées (LABIA), Ecole Doctorale Sciences et Technologies, Université Joseph KI-ZERBO, 03 BP 7021 Ouagadougou 03 Burkina Faso. 2. Service de Bactériologie-Virologie du Centre Hospitalier Universitaire Yalgado Ouédraogo (CHU-YO). 3. Laboratoire National de Santé Publique, Ouagadougou, Burkina Faso. 4. Département Technologie Alimentaire (DTA), CNRST/IRSAT, 03 BP 7047 Ouagadougou 03, Burkina Faso. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 20 January 2020 Final Accepted: 22 February 2020 Published: March 2020

843 medical costs, prolonged hospital stays, and increased morbidity and mortality (Founou et al., 2017). This antimicrobial resistance is putting the gains of the Millennium Development Goals at risk and endangers achievement of the Sustainable Development Goals and requires action across all (WHO, 2018). Staphylococcus aureus, a bacterium commonly found on the skin and in the nose of about 30% of individuals has become one of the major disease-causing organisms because of AMR. The infections list ranges from benign cutaneous lesions (furuncles, paronychia) to fatal sepsis and toxic shock syndrome (Tong et al., 2015;Hale et al., 2018). Staphylococcus aureus has been responsible for almost 17.5% of sepsis in children in hospitals (Odutola et al., 2019). The emergency of Methicillin-resistant Staphylococcus aureus (MRSA) strains or multidrug-resistant S. aureus strains, in the last decade has resulted rapidly progressive, potential fatal diseases including life-threatening pneumonia, necrotizing fasciitis, endocarditis, osteomyelitis, severe sepsis, and toxinoses (Monecke et al., 2011). MRSA is associated with significant mortality and morbidity and imposes a serious economic burden on scarce healthcare resources worldwide. According to the World Health Organization (WHO), methicillin-resistant Staphylococcus aureus increases the death rate of infected patients by 64%. The use of different types of antibiotics over the years has led to the emerging of multi-resistant Staphylococcus aureus strains. The MRSA prevalence varies according to the regions and the living conditions of the populations (Schaumburg et al., 2015).

Microbiological analyses:
S. aureus strains have been identified based on morphological characteristics and ability to ferment mannitol to Chapman mannitol media. The colonies of golden yellow that fermented mannitol were subjected to Gram staining and biochemical tests. Biochemical tests used to confirm S. aureus strains included tests of catalase, coagulase, and Dnase. Subsequently, the DNase agar plates were flooded with HCl (1N) (Oxoid Limited, UK). Isolates that showed the ability to hydrolyze DNA were identified as S. aureus.

Antimicrobial susceptibility testing:
Antibiotics susceptibility testing was carried out on Mueller Hinton Agar (MHA) (HIMEDIA, India, REFM1084-500G) using the Kirby-Bauer disc diffusion technique. Screening of MRSA was done using cefoxitin disc (30μg) according to the Clinical and Laboratory Standards Institute (CLSI) recommended disc diffusion method. A reference strain of Staphylococcus aureus (ATCC 25923) was used as a control strain. The antibiotics discs used for identification of antibiotic sensitivity pattern of MRSA isolates were: erythromycin (15 μg), tetracycline (30 g), gentamicin (10 μg), clindamycin (2 μg), ciprofloxacin (5 μg), levofloxacin (30 μg), tobramycin (30 μg), kanamycin (30 μg), cotrimoxazole (1.25 + 23.75 μg), fusidic acid (10 μg), chloramphenicol (30μg) (HIMEDIA, India, REFM1084-500G). Each test isolate was emulsified in peptone water to create a suspension with turbidity similar to 0.5 McFarland standards. The swab was dipped into the suspension and pressed against the interior walls of the container to drain excess fluid. The dried surface of a MHA plate was inoculated by streaked the swab over the entire agar surface. After an application of antibiotic discs to inoculated agar plates, the plates were inverted and incubated at 37 o C for 18-24 h. The diameter of the zones of inhibitions around the antimicrobial discs were measured and interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST/CA-SFM, 2018).
The multi-resistant (MRSA) is defined as the resistance to at least of three different antibiotics family.

Statistical analysis:
The data obtained were analysed with SPSS software version 21 using simple descriptive statistics. Tests for significance for associations computed for categorical variables were based on the chi-square. P-values less than 0.05 (p<0.05) were regarded as statistically significant.

Results:-Bacterial Strains:
A total of 150 isolates were collected during the study period (103 strains were from outpatients and 47 from hospitalized patients over 2 days). Most of isolates were isolated in adults' patients (20-59 years' group) ( Table 1). Staphylococcus aureus strains have been isolated from different pathological products as shown in Table 2. It can be noticed that most of these strains were from pus samples (90/150), followed by those obtained from urine samples (30/150) and from vaginal swabs (24/150). Only six (6) S. aureus strains were from blood specimens. The antibiotic sensitivity pattern of MRSA isolates are given in figure 1. Staphylococcus aureus strains were resistant to 5 antibiotics. A higher frequency of antimicrobial resistance was observed to tetracycline (58.7%), followed by levofloxacin (56.25%), ciprofloxacin (44.12%), kanamycin (36%) and erythromycin with (30%).
Most of the strains (61.7%) of the MRSA were multidrug-resistant (Table 4). Seventy-one percent (71.43%) of MRSA isolated in inpatient had resistance to several antibiotics, compared to MRSA strains isolated from outpatient (42.4%) ( Table 5). Most MRSA that had multi-antibiotic resistance were isolated from patients who had spent more than two days in hospital care.
845   More than 30% of MRSA isolates were resistant to tetracycline (58.7%), followed by levofloxacin (56.25%), ciprofloxacin (44.12%), kanamycin (36%) and erythromycin (30%). The high resistance of MRSA isolates to antibiotics could be due to the widespread use of these antibiotics for the treatment of staphylococcal infections in healthcare, because high consumption of antibiotics leads to the emergence of antibiotic-resistant Staphylococcus aureus. Multidrug resistance to these old and commonly used antimicrobials is a public health problem and could facilitate usage of fluoroquinolones and third-generation cephalosporins for empiric treatment (Somda et al., 2017).
Resistance of MRSA to tetracycline was found at 58% in the study, which is correlated with previous findings that MRSA isolates were 53.2% and 42% (Rajesh et Rashmi et al. (2017) have showed that all MRSA isolates in their study showed resistance to erythromycin.In our study, it was noted the emergence of strains resistant to ciprofloxacin, levofloxacin and kanamycin. This situation might be explained by the fact that in developing countries, populations frequently practice self-medication and use proscribed drugs sold on the street because of their low economic power and their ignorance of the danger of these practices. These practices increase the phenomenon of bacteria resistance (Somda et al., 2017). These observations suggest the necessity to undertake the regulation of antibiotics usage in Burkina Faso to avoid or control the spread of resistance to antimicrobials.

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The high frequency of multiple resistance of MRSA to antibiotics in hospital care units could be related to inappropriate use of antibiotics, self-medication, and practices for preventing and controlling non-standard infections.

Conclusion:-
This study documents the strong involvement of MRSA in community and hospital infections in Ouagadougou. Among the risk factors associated with the acquisition of MRSA are the older age, prolonged hospitalization, previous antibiotics. The increase in the number of methicillin-resistant strains and their multiresistance to antibiotics is a problem in the management of bacterial infections. Establishing measures to control antibiotic infections and management could help reduce the emergence and spread of these MRSA.

Conflicts of interest:
The authors declare that they have no competing interest.

Authors' contributions:
TR carried out the sampling and strains isolation and their antibiotics susceptibility and drafted the manuscript; ZC, KDS, SNS, TY, LS, SA and TY supervised the sampling and strains isolation, antibiotics susceptibility and participated in writing the manuscript. All authors read and approved the final version of the manuscript.