Methicillin-Resistant Staphylococcus aureus Nasal Colonization among Healthcare Workers at Kampala International University Teaching Hospital, Southwestern Uganda

Whereas Staphylococcus aureus is a pathogen, it colonizes healthy people as normal flora without causing any symptoms or illness. Probably because of greater exposure, healthcare workers (HCWs) are more colonized, serving as reservoir for endogenous infections as well as dissemination. In developing countries including Uganda, there is scarcity of the literature on S. aureus carriage among HCWs, making infection control difficult. This study aimed at determining the nasal carriage rate and comparing the antimicrobial susceptibility profiles of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible Staphylococcus aureus (MSSA) isolates from HCWs of Kampala International University Teaching Hospital. Nasal swab specimens from HCWs were screened for MRSA using both phenotypic and genotypic methods. Antimicrobial susceptibility testing of the MRSA and MSSA isolates was performed using the Kirby–Bauer disc diffusion method. Out of the 97 participants, 28 (28.8%) participants were nasal carriers of S. aureus of which 13 (46.4%) were phenotypically MRSA (resistant to cefoxitin) and 8 (28.6%) were genotypically MRSA (had mecA gene). Only 6 isolates of the 13 isolates (46%) which showed resistance to cefoxitin had mecA gene detectable while 2 (13.3%) of the 15 cefoxitin susceptible isolates were found to carry mecA gene. The study thus shows that methicillin resistance in S. aureus may not only be determined by mecA gene.


Introduction
S. aureus is a very common bacterium that is both a pathogen and normal flora. It can be isolated from many body parts, mostly the nasal cavity and has ability to survive on inanimate objects such as beds, trays, and toilet seats [1,2]. Approximately 30% of the world human population is persistent carriers of S. aureus [3,4]. e carriage rate is even higher in healthcare workers and clinical students [5]. Factors that determine colonization without showing clinical symptoms are largely unknown [3,4]; however, variability in host adhesins, immune response, reduced expression of antimicrobial peptides in nasal secretions, polymorphisms in the genes encoding the glucocorticoid receptor, C-reactive proteins, interleukin-4, and complement inhibitor proteins have been associated with persistent nasal carriage [6][7][8][9]. Also, studies by Brown et al. demonstrated that after decolonization, persistent carriers often become recolonized with their prior S. aureus strain, whereas noncarriers resist experimental colonization [10]. is shows that certain host traits determine colonization.
Colonization of healthcare workers with S. aureus is a prerequisite for subsequent endogenous infection and dissemination of the strains to the hospital environment [11]. In 1944, most Staphylococci were susceptible to penicillin G; however, due to the misuse of penicillin, many isolates became resistant to the drug by production of β-lactamases (penicillinases), coded by blaZ gene regulated in an operon manner by a regulatory gene called BlaR1 [12]. ese enzymes degrade the β-lactam ring of the antibiotic, making it harmless to the microorganisms [13]. e discovery of β-lactamase-resistant penicillins (e.g., nafcillin, oxacilin, cefoxitin, and methicillin) provided a temporary respite [13][14][15]. However, later methicillin-resistant strains evolved mecA gene coding for refractory penicillin binding proteins (PBP2a) which are cell wall-synthesising enzymes that have reduced affinity for penicillins [2]. Currently, MRSA is the most commonly identified antibiotic-resistant pathogen in many parts of the world, both in hospital and community environments [16].
Although the literature is still scarce, MRSA has been reported in different African countries at different prevalences, for instance, 12.7% in Ethiopia [16], 35.8% in Botswana [17], and 46% in Uganda [18]. Colonized healthcare workers have been implicated as major reservoirs of MRSA by different studies [16,19]. e present study aimed at detecting S. aureus and MRSA in HCWs as well as determining antimicrobial susceptibility profile of the isolates.

Study Design.
is was a cross-sectional study which involved collection of nasal swab specimens from healthcare workers between September 2016 and July 2017. e participants included nurses, paramedical officers, laboratory technicians, and medical doctors. Nasal swab specimens were collected following previously described procedure [16]. Isolation of S. aureus from the samples was done following described bacteriological methods [20]. Antimicrobial susceptibility testing of the isolates was done using the Kirby-Bauer disc diffusion method on Mueller-Hinton agar. Screening for MRSA was done using a cefoxitin disc and PCR amplification of mecA gene [21,22].

Study Area.
e samples were collected from Kampala International University Teaching Hospital located in the Ishaka town along Mbarara-Kasese road in Bushenyi District, Southwestern Uganda. Kampala International University Teaching Hospital is sectioned into different departments including Medical, General Surgery, Obstetrics and Gynecology, Pediatrics, Orthopedics, Psychiatry, Dentistry, and Ear Nose and roat (ENT). e hospital is staffed with about 249 healthcare workers comprising nurses, clinical officers, laboratory technologists, pharmacists, medical officers, and consultants.

Sample Size Determination.
e minimum sample size was determined by Slovin's formula stated as n � N/(1 + N(e) 2 ) [23], where n � sample size, N � population size, and e � margin error. In this study, N � 249, e � 0.08, and the minimum number of participants n � 249/(1 + 249 (0.05) 2 ) � 96. is formula was preferred because it is the best formula when the study involves determination of proportion at confidence level 95% and optimal when the proportion is suspected to be close to 0.5.

Isolation and
Identification of S. aureus. Nasal swab specimens were collected from consenting healthcare workers using a sterile cotton tip swab (Zhejiang Gongdong Medical Technology, China).
is was done by rotating sterile cotton swabs in both nares of the participants. e specimens were then transported to Microbiology Laboratory of Kampala International University Teaching Hospital for immediate culture. e specimens were inoculated on 5% blood agar and then incubated at 37°C for 18-24 hours. e colonies showing β-hemolysis were subjected to Gram staining, catalase test, mannitol fermentation, and tube coagulase [20,21]. e isolates that showed positive results for all the above tests were confirmed using Slidex Staph Plus (Biomerieux, France) as S. aureus.

Determination of Antimicrobial Susceptibility Pattern.
Antibiotic susceptibility testing was carried out using the Kirby-Bauer disc diffusion method on Mueller-Hinton agar (MHA) (Himedia M173-500G, India).

DNA Extraction.
e DNA was extracted using the boiling method as previously described [21,22]. Briefly, it involved centrifuging 1 ml of bacterial culture in Luria Bertani (LB) medium at 6800 × g for 3 minutes at room temperature. e pellet was then resuspended in 100 μl of molecular biology grade water and centrifuged at 15000 × g for 10 min. e supernatant was discarded, and the sediment resuspended in 40 μl of molecular biology grade water and boiled at 100°C in a water bath for 10 minutes. is was followed by cooling on ice and centrifuging at 15000 × g for 10 seconds. e supernatant was then used for PCR.

mecA Gene PCR Amplification.
is was done following the protocol previously described by Elhassan et al. [26]. Briefly, a total volume of 25 μl consisting of 12.5 μl of master mix (containing 2x taq polymerase, dNTPs, and buffer) (Bioline, UK), 0.5 μl of the forward primer, 0.5 μl of the reverse primer, 7.75 μl of PCR water, 1.25 μl MgCl 2 , and 2.5 μl of the template DNA was used for PCR. e 533 bp segment of mecA gene was amplified using the primer pair F: 5′-AAAATCGATGG-TAAAGGTTGGC-3′ and R: 5′-AGTTCTGCAGTACCG-GATTTTGC-3′ (Eurofins Genomics, Germany), as previously reported [27]. e PCR conditions were in accordance with the protocol by Kateete et al. [20] with some modifications. Briefly, it involved initial denaturation at 94°C for 5 minutes followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 50°C for 1 minute, extension at 72°C for 1 minute followed by 7 min of final extension at 72°C. e PCR products were resolved by electrophoresis at 125 V for 30 minutes through 2% agarose gel prepared with TAE buffer containing 0.5 mg/ml ethidium bromide [28,29]. DNA bands on the gel were viewed under the UV digital imaging system. e size of PCR mecA amplicons was estimated at 533 bp in comparison with their motilities with those of 50 bp ladder standard.

Quality Control.
To avoid false positives, gloves and surgical mask were put on to avoid contamination of the samples. All samples collected were cultured immediately after collection. e reference strain S. aureus ATCC 25923 was used as quality control strain during identification and antibiotic susceptibility testing. S. aureus ATCC 25923 and S. aureus ATCC 43300 were used as negative and positive controls, respectively, during PCR assay.

Data Management and Analysis.
Data were entered in Epidata version 4.2 and were analysed using IBM SPSS version 20. e nasal carriage rate of MRSA was calculated as the proportion of individuals positive for MRSA out of the sample population. e chi-square test was used to compare the different groups of healthcare workers. All results with p < 0.05 were taken as significant.

Ethical Considerations.
e ethical approval was obtained from Institutional Review Board of Mbarara University of Science and Technology (no. 12/09-15). e permission was sought from Directorate of MEDICAL Services of Kampala International University Teaching Hospital. Informed consent was obtained from the participant before starting the study. e identity of the participants was highly concealed.

Participants' Baseline Characteristics.
In total, 97 participants who included doctors, paramedical officers, nurses, and laboratory personnel were involved in the study. Of these, 61 (63%) participants were males and 36 (37%) were females. e participants were stratified according to age and working ward/department. Results are shown in Table 1.

Prevalence of Staphylococcus aureus.
28.7% (28/97) of the participants were found to be nasal carriers of S. aureus.
When the nasal carriage rate was compared across sex, no significant difference (p � 0.458) was observed between males (26.2%) and females (33.3%). e nasal carriage rate significantly increased with age (p < 0.01), being highest (75%) in individuals above 35 years (Table 1). Comparisons of nasal carriage rates across different professions did not show any significant differences (p � 0.225). However, laboratory staff (50%) and doctors (30%) were more colonized. Similarly, the ward/department where the samples were collected did not significantly (p � 0.433) impact the S. aureus positivity ( Table 2).  Table 3.

Genotypic MRSA Screening.
e S. aureus isolates were analysed for mecA gene using PCR. Among the 28 isolates, 8 (28.6%) isolates had mecA gene (Figure 1). Only 6 isolates of the 13 isolates (46%) which showed resistance to cefoxitin had mecA gene detectable. On the other hand, 2 (13.3%) isolates of the 15 cefoxitin susceptible isolates were found to carry mecA gene. e participants' profession and the ward/department of the work significantly affected the carriage of mecA positive strains (p < 0.05) while sex and age of the participants did not have any statistically significant effect (p > 0.05). e summary of this analysis is presented in Table 4.

Antimicrobial Susceptibility Pattern of MRSA and MSSA Isolates
. When the isolates were tested for the susceptibility to different antibiotics, MRSA isolates (mecA positive) showed a higher resistance rate ( Figure 2) than MSSA isolates. Since cefoxitin is used as a predictor of MRSA, the resistance rate among cefoxitin resistant and susceptible isolates was compared. Cefoxitin-resistant isolates showed higher resistance rate to the tested antibiotics than cefoxitin susceptible ones (Figure 3).
S. aureus whether MRSA or MSSA isolates showed a high resistance rate to ceftazidime, amoxicillin/clavulanic acid, penicillin G, and cotrimoxazole. On the other hand, the isolates were very susceptible to vancomycin, amikacin, and levofloxacin.

Discussion
is study determined S. aureus nasal colonization rate among HCWs, proportion of the phenotypically and  [16,18,[30][31][32]. ese differences probably are due to differences in the relative abundance of S. aureus in the respective study sites. Age was associated with S. aureus nasal colonization rate. e significance of age in the colonization rate has also been reported by other studies by Hogan et al. and Shibabaw et al. [16,33]. is may be due to cumulative exposures to the organism which happens with time in hospital setting. e prevalence of S. aureus in this study varied according to professions ranging from 50% among laboratory workers to 22.9% in paramedical officers, although no statistical difference could be observed. is observation agrees with what was reported by Omuse et al. [30]who observed higher prevalence of S. aureus nasal carriage among phlebotomists.
is suggests that healthcare workers may acquire organisms from patients as they collect laboratory samples. Laboratory staff are also exposed to isolates in the labs, especially if they do not adhere to safety precautions during handling samples/isolates. In this study, the colonization rate among nurses was 25% (3rd highly colonized) as compared to 21.2% in a study in Ethiopia, by Shibabaw et al. [16]. Of the S. aureus isolates, 46.4% showed resistance to cefoxitin (hence phenotypic MRSA) and 8 (28.6%) possessed mecA gene (hence genotypic MRSA).
is may be suggestive that cefoxitin is more sensitive than mecA detection even though 3 of the cefoxitin-sensitive isolates carried mecA gene. Comparing these findings with other studies, it can be shown that there are slight differences in the prevalence; for example, Zorgani [16,35]. All these differences in the findings may be due to different geographical distributions of S. aureus and relative prevalence of MRSA in different places.
Both the participants' profession and the ward/ department where they work statistically affected the genotypic MRSA carriage rate (p < 0.05), with only nurses and laboratory staff carrying mecA-positive strains. is study concurs with other studies as observed by Shibabaw et al. and Nabil et al. who established nurses to be more MRSA carriers than other professionals [16,32]. e higher prevalence of genotypic MRSA isolated from lab staff and nurses than other professions could probably be due to frequent exposure to patients.
In the current study, five cefoxitin-resistant isolates were negative for mecA.
is was also been observed in other studies by Olayinka et al. and Broekema et al. When they performed nitrocefin assay on these isolates, they showed hyperproduction of type A β-lactamases [36,37]. Genome sequencing of another S. aureus strain with this trait (called A and E, accident and emergency; Obs and Gyne, Obstetrics and Gynecology; OPD, outpatients department; * statistically significant. A and E, accident and emergency; Obs and Gyne, Obstetrics and Gynecology; OPD: outpatients department; * statistically significant. LGA251) found a mecA homologue which was 69% identical to mecA. To date, it has been called mecC and also confers resistance to methicillin drugs [38]. "Auxiliary genes" identified by Tn551 mutagenesis have also been shown to confer resistance to methicillin drugs in addition to mecA gene [39]. is shows that methicillin resistance is complex and changing as new strains are evolving different mechanisms distinct from classical mecA gene. Despite many studies reporting cefoxitin as a surrogate maker for mecA, in the current study, 2 (7%) of S. aureus isolates were susceptible to cefoxitin but carried mecA gene.
is trend has been reported by other studies [40,41]. is could be explained in terms of structural differences in the mecA regulatory genes causing low expression [42].

Conclusions
e present study indicates a high nasal carriage rate of S. aureus (28.8%), of which 46% were phenotypically MRSA and 28.6% genotypically MRSA.
ere is need for more A and E, accident and emergency; Obs and Gyne: Obstetrics and Gynecology; OPD: outpatients department; * statistically significant.   follow-up studies to identify carriers and establish risk factors for colonization in order to put in place decolonization measures. irty-eight percent of the S. aureus isolates were resistant to cefoxitin but did not carry mecA gene while 13% of isolates susceptible to cefoxitin carried mecA gene. erefore, future studies detect not only mecA gene when studying genetic basis for methicillin resistance but also other markers such mecC gene as well as whole genome sequencing to detect homologues genes that might also cause resistance.
Data Availability e tables and figures data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
ABJ designed the study and collected and analysed samples. JB and CDK supervised the study. ABJ analysed data and wrote the manuscript. All the authors revised and approved the manuscript prior to submission.