Prevalence and antimicrobial susceptibility pattern of Staphylococcus Aureus isolated from clinical specimens at the Mater Hospital Nairobi, Kenya.

Staphylococcus aureus is a leading cause of hospital and community acquired infections globally. Surveillance of prevalence and antibiotic susceptibility patterns is important to ensure appropriate antibiotic prescription. The aim of this study was to determine the prevalence and susceptibility patterns of Staphylococcus aureus isolated from clinical specimens in a tertiary referral and training hospital. A retrospective study was conducted at the Mater Misericordiae Hospital, Nairobi.It involved analyzing records of specimens analyzed between January 2014 and December 2018. Strains phenotypically resistant to <3 non β-lactam antimicrobial categories were dened as non-multidrug-resistant MRSA (nmMRSA) and strains that were resistant to ≥ 3 non-β-lactam antimicrobial groups were dened as multidrug-resistant MRSA (mMRSA). A specimen was categorized as Penicillin susceptible (PSSA) if susceptible to Penicillin and Oxacillin, MSSA if resistant to Penicillin and susceptible to Oxacillin, and MRSA if resistant to Oxacillin and Penicillin. Tests of difference are two-sided and comprised t tests for mean age, chi-squared tests for sex and site of infection.


Conclusion;
The prevalence of MRSA was low compared to previous studies.All strains isolated were non-multidrug resistant.There was no resistance to Vancomycin detected.Penicillins are still a viable option in the treatment of majority of S.aureus infections in our setting.

Background
According to (Daniel J. Diekema et al., 2019), Staphylococcus aureus (S.aureus) is among the most common and devastating human bacterial pathogens, estimated to cause about 20%-30% of bloodstream and surgical site infections globally, as well as up to half of bone and joint infections.
Furthermore, they highlighted that antibiotic resistance is a major problem in the treatment of infections by S. aureus. The emergence of penicillinase-producing S. aureus strains occurred shortly after the introduction of penicillin for clinical use, and by the 1970s majority of S. aureus infections were penicillin resistant. Methicillin resistance among S. aureus was reported in the early 1960s, after the introduction of methicillin following the acquisition of the mecA-containing Staphylococcal cassette chromosome mec(SCCmec) (Kanjilal et al., 2018) .Research has shown that the emergence of Methicillin Resistant S. aureus (MRSA)has complicated treatment of Staphylococcus aureus infections (Daniel J. Diekema et al., 2019),. In addition infections caused by MRSA are associated with signi cant morbidity, mortality and cost. MRSA strains are not only resistant to nearly all beta-lactams, but many have developed resistance to multiple other classes of antimicrobials. (D. J. Diekema et al., 2000) .The epidemiology of MRSA infections has been marked by sequential "waves" of epidemic clones spreading across geographic regions, nations, and continents. According to (Limbago et al., 2014) as MRSA has become endemic, the use of vancomycin for therapy of invasive MRSA infections has increased, along with concerns about development of vancomycin resistance among MRSA. In a study carried out in the United States, it was found that the prevalence of MRSA blood infections in the United States and Europe has dropped in recent years (Sutter et al., 2016). Similarly (Abubakar & Sulaiman, 2018), found MRSA infection rate in Asia is also declining. In contrast, recent data suggests that the prevalence of MRSA in most African countries is rising, although the rate is estimated to be below 50%. (Falagas et al., 2013) and (Abubakar & Sulaiman, 2018) .According to (Kanjilal et al., 2018) the changing trajectory of MRSA infection in developed countries has been attributed to implementation of control interventions. More recent data by (Cheng et al., 2016) further suggests that penicillin susceptibility may be in a period of renaissance.
In Africa, MRSA prevalence intra-country and intercountry has been reported to be heterogeneous.
According to (Wangai et al., 2019) antimicrobial resistance data from Kenya has been variable and inconsistent due to lack of effective and systematic routine surveillance systems .
The objective of our study was to establish the prevalence of S. aureus and MRSA, to identify patterns of susceptibility of S. aureus to commonly used antibiotics and quantify contemporary penicillin resistance among S. aureus in a tertiary center in Nairobi Kenya. The knowledge acquired will be used to guide the choice of antibiotic therapy and serve as a baseline for measuring the impact of interventions.

Methods
This study was carried out in the microbiological laboratory in Mater hospital, a tertiary referral hospital and training center in Nairobi, Kenya. It serves a large and diverse low and middle income population comprising of patients of African and Asian descent and few Caucasians. The Mater hospital microbiological laboratory is ISO 2012 certi ed. This study was well powered due to the large number of records reviewed. One study by (Gitau et al., 2018) analyzed 944 specimens of S.aureus while (Omuse et al., 2015) looked at 731 non duplicate specimens S.aureus so we felt our study population of 659 non duplicate S.aureus specimens was su cient.

Data and sample collection
This was a retrospective study based on electronic laboratory records from the Mater microbiological laboratory. The following demographic information was extracted from the hospital administrative database about each patient-in vs. outpatient status, age and gender, and antimicrobial susceptibility pro les of S. aureus. Data from 2014 to 2018 was retrieved from the hospital medical records and for 2018 from Vitek 2 (bioMérieux, 2018) antibiotic susceptibility system imported to (LIFELINE, 2014) database.
Standard operating procedures were followed in collection of samples in the ve-year period.
Clinical samples from various anatomical sites of study participants were cultured on blood agar and mannitol salt agar and identi ed to be S. aureus by using catalase and coagulase tests. Tests were performed in vitro, and measured the growth response of an isolated organism to a particular drug or drugs. These tests were performed under standardized conditions so that the results were reproducible. The raw data was either in the form of a zone size or MIC.
The most common method employed by the Mater hospital diagnostic laboratory between 2014 and 2018 was a simpler agar disk diffusion test (Kirby-Bauer method).
After incubation, the diameters of the complete growth inhibition zones around each disk were measured.
Pre-speci ed breakpoints were used to interpret the zone sizes and classify them as susceptible(S), intermediate (I), or resistant(R). Clinical Laboratory Standards Institute (CLSI) M100-S19 breakpoints were used for interpretation. Isolates that were "intermediate" by CLSI breakpoints were grouped with resistant isolates for all analyses. Methicillin resistance was determined using Oxacillin. This is similar to a study by (Udobi et al., 2013) where analysis was done according to the NCCLS (2002) guidelines using oxacillin in agar screen test. Isolates from a solution adjusted to 0.5 McFarland standard were spot inoculated unto Mueller Hinton agar supplemented with 6 micrograms/mL oxacillin and 4% sodium chloride. The plates were incubated at 35°C for 24 hours. The isolates that survived showing more than one colony were considered Methicillin resistant. Approval for this retrospective study was obtained from the Ethics committee of the Mater hospital. All patient information was anonymized preceding the analysis.

Data Analysis
The data collected was analyzed using (IBM-SPSS, 2018). We categorized a specimen a priori, as Penicillin Susceptible Staphylococci aureus(PSSA) if it was susceptible to Penicillin and Oxacillin, Methicillin Susceptible Staphylococci aureus (MSSA) if it was resistant to Penicillin and susceptible to Oxacillin, and MRSA if it was resistant to Oxacillin and Penicillin. (Kanjilal et al., 2018) The ndings were presented in tables, graphs and charts. Bivariate analysis was done by use of Chi square to assess for association between categorical variables such as susceptibility of antibiotics and infections location (Gitau et al., 2018). Multivariate logistic regression was used to determine the association between the presence of S. aureus isolates and the source of the clinical specimen (in vs. outpatient), age and gender. A p value<0.05 was considered statistically signi cant.

Results
Prevalence; A total of 659 specimens with S. aureus isolates were included in the study. Initially we had 879 specimen collection forms however 220 were eliminated due to incomplete data.
The annual prevalence of MRSA uctuated between 0-1% during the ve-year study period.
Demographics 62.37 %( 411) of all specimens were collected from outpatients as compared to 37.63 %( 248) from admitted patients. All MRSA isolates were cultured from specimens from inpatients (general medical wards (50%, 2/4) as compared to the intensive care unit (25%, ¼)) The average age of all patients with S. aureus infections (N=659) was 29.66 MRSA cultured was predominantly from specimens obtained from patients between the age of 0-19(2/4, 50%).

Discussion
The current study demonstrates that there has been a high prevalence of PSSA (60.85%) and a low prevalence of MRSA (0.61%) and MSSA (38.54%). A similar prevalence has been reported in previous studies by (Omuse et al., 2015).This could be explained by low community transmission of MRSA and MSSA (Aiken et al., 2014).
MSSA and PSSA were predominantly isolated in outpatients unlike MRSA which was more in admitted patients. This has similarly been observed by (Nishijima et al., 1992)in a study in Japan where MRSA was more prevalent in admitted patients . Even though this study was not designed to identify risk factors for MRSA acquisition, risk factors that have previously been associated with acquisition of MRSA in hospitals such as broad-spectrum antimicrobial therapy, admission to an intensive care unit and proximity to other patients with MRSA could play a major role in our study site (Dilnessa & Bitew, 2016).
Majority of S. aureus isolates were from specimens cultured from patients between the ages of 0-19. However, there were no signi cant differences in age between patients with MRSA and MSSA. In a study carried out in Australia (Agostino et al., 2017), MRSA infections were found to occur more often in younger patients (under 40).This transmission could be explained by the fact younger people may have close skin-to-skin contact more often during participation in sports or when working.
There were statistically signi cant differences in gender between patients who grew MRSA and PSSA.
Similarly, there were signi cant differences in gender between patients with MSSA and PSSA however there were no signi cant differences in gender between patients whose cultures were positive for MRSA and MSSA. Most S. aureus isolates were from female patients. This difference could have arisen from the fact that female patients constituted majority of the study population (76.33%) and this could further be explained by the differences in health seeking behavior across different genders (Thompson et al., 2016).
MRSA infections were highest in male patients unlike MSSA and PSSA. Several studies have found the isolation, carriage and infection of MRSA to be higher in males (Humphreys et al., 2015). This could be because hand-hygiene behavior varies according to gender. Males are less compliant, which in turn may predispose them to higher colonization and infection rates.
MSSA, PSSA and MRSA were all predominantly isolated from pus. This nding is similar to what was found by Dilnessa et al in Ethiopia (Dilnessa & Bitew, 2016). MRSA was also isolated from a tracheal aspirate specimen and cerebrospinal uid. The three clinical situations that predispose to the development of S aureus meningitis are neurosurgical intervention, contiguous infection and S aureus bacteremia. Until recently, most cases have been caused by methicillin-susceptible S aureus strains.
There are now an increasing number of reports of severe infections attributed to methicillin-resistant strains of S aureus (Pereira et al., 2015).
Methicillin Resistant S. aureus cultured in our setting was sensitive to Linezolid, Vancomycin, Tigecycline, Rifampicin, Fusidic acid, Teicoplanin and Tetracycline. These antibiotics have proved effective for treatment of MRSA infections in other parts of the world (Liu et al., 2011), (Khalili et al., 2010).On the contrary, all the isolates cultured in our setting were resistant to Clindamycin which is considered an option in MRSA treatment in some parts of the United States of America(USA).
Notably, in a sub analysis we found that resistance of MSSA and PSSA to Amoxicillin-Clavulanic acid and Penicillin G declined gradually over the past ve years however, resistance to Amoxicillin rose. This could be explained by the overuse of Amoxicillin which is more readily available and cheaper than amoxicillin-clavulanic acid and a decline in use of Penicillin G over the years. Furthermore, recent publications have reported a rise of Penicillin-Sensitive Staphylococcus aureus. This trend has now been observed on several continents and has been shown to involve bloodstream infections and other serious infections (Cheng et al., 2016). In the United states, this increase in the incidence of PSSA infections has also been mirrored by a decrease in /methicillin-resistant S. aureus infection rates (Butler-Laporte et al., 2018) .
In our study 50%of MRSA were susceptible to trimethoprim-sulfamethoxazole. (Pappa et al., 2009) reported resistance of MRSA to trimethoprim-sulfamethoxazole to vary worldwide, in general being low in the industrialized world and higher in developing countries.
Overall the annual antibiotic S. aureus resistance has declined in the past 5 years. One potential explanation for the trend of increasing S. aureus antibiotic susceptibility is a shift in antibiotic pressures. The decline in the use of narrow-spectrum beta-lactams such as Oxacillin and Penicillin G since 2000 and the inpatient use of rst-generation cephalosporins since 2006 may select against hospital acquired MRSA in favor of PSSA (Kanjilal et al., 2018).
There are several limitations to our study. First, the testing for inducible beta-lactamase production was not performed, raising the possibility that specimens reported as penicillin susceptible from this time period were in fact penicillin resistant. In addition, we did not collect information on a number of vital aspects such as history of antimicrobial use and duration of hospital admission. Furthermore, in the absence of genotyping of specimens, phenotypic MRSA identi cation methods may not re ect an accurate picture.

Conclusion
The prevalence of MRSA was low when compared with the prevalence rates obtained in previous studies conducted in Kenya. MRSA strains were non-multidrug-resistant and there were no strains that were resistant to Vancomycin, the drug of choice for MRSA. There has been a decline in resistance of S. aureus to Penicillin and Amoxicillin clavulanic acid in the past ve years. Good infection control practices and prudent antibiotic use can help to further reduce the burden of MRSA in our setting. Authors' contributions CO participated in the study design, literature search, data collection, data analysis, manuscript writing and is the corresponding author, CM, FCFO, LAO and EO participated in the study design, writing the manuscript, interpretation of data, supervision of the microbiological diagnosis and served as scienti c advisors. All authors read and approved the nal manuscript.

Funding
This study did not bene t from any funding Tests of difference are two-sided and comprised t tests for mean age, chi-squared tests for sex and site of infection.