Study area, design and period
An institution based cross sectional study was conducted in the College of Medicine and Health Sciences, AMU, southern Ethiopia from 01 August through 30th November 2020, located in Arba Minch town which enrolls 1,598 students in total in the health sciences and medical programs (regular). In 2019/2020 academic year, among the new entrants, 1,029(64.4%) were males. The campus consists of a number of departments such as medicine, nursing, pharmacy, anesthesia, midwifery, radiology, medical laboratory science, environmental health, health informatics and public health.
Study population and eligibility criteria
All undergraduate students belonging to clinical medicine and health sciences, who had a clinical exposure for eight hours per day for more than 3 months in any hospital setting during the study period, are considered. The inclusion criteria set for the study comprises; 1. all medical and health science graduating class students who have exposure for 8 hours per day for the previous 3 months and who are available during the study period; 2. students without a history of hospitalization in the preceding six months (ie., they are not exposed because of admission, rather exposed during care giving). The exclusion criteria include; 1. participants who had nasal infections during the study period, 2. those who received intranasal antibiotic ointment or other antibiotics within the previous two weeks of the commencement of the study or who underwent a nasal decolonization procedure. The study protocol was approved by the Institutional Research Ethics Review Board of the College of Medicine and Health Science of Arba Minch University (IRB/464/12). This study was conducted in accordance with the declaration of Helsinki. Besides, separate permission was procured from Arba Minch University, College of medicine and health sciences. Formal written consent was obtained from each study participant. Confidentiality was strictly maintained from sample collection up to the final report writing.
Sample size determination and Sampling technique
The sample size was calculated by using a single population proportion formula, where, p value of 50% was chosen due to the dearth of prior studies conducted among similar study population. Calculations were done using an open Epi version 3 by assuming 95% confidence level with 5% degree of precision, as followed in the case of sample size for frequency .
Population size (for finite population correction factor or fpc)(N):
Hypothesized % frequency of outcome factor in the population (p):
Confidence limits as % , (absolute +/- %)(d):
Design effect (for cluster surveys-deff):
Accordingly, the sample size determined was 224
Then by assuming a non-response rate of 15%, the total subjects included in the study was 258
(n= 224; n+15% of 224 = 258); and it is the final sample size.
Clinical medical and other graduating health science students at the verge of completion of their courses during the study year and who were also attending clinical practices in different departments of AMGH or any other hospitals were stratified, based on academic years (for medicine students) and departments (for other graduating class students). A proportional allocation sampling technique was followed for each stratum; as clinical-I, clinical-II, medical intern, graduating class of anesthesia, medical laboratory, midwifery, nursing, radiology and health officer; also a systematic sampling technique was used to recruit the rest. Probability proportional to size /PPS (proportional allocation technique)  was obtained by using the formula,
Where: ni = Required sample size of students from each ith stratum
Ni = Number of medical students in the ith stratum
n= Over all sample size
N= Total number of students in the ith stratum
Absolute proportions were found in anesthesia, 4.7% (n=12), clinical – I medical students, 13.2% (n=34), clinical - II medical students, 13.9% (n=36), medical interns, 25.2% (n=65), health officers, 10.5% (n=27), medical laboratory science, 8.5% (n=22), midwifery, 8.9% (n=23), nursing, 10.8% (n=28) and radiology, 4.3% (n=11).
Data Collection tool and procedure
A pre-tested structured questionnaire with three sections was used to collect the data; it is formulated after an extensive literature survey. The first section consisted of socio-demographic questions, the second part comprised clinical data and the third section corresponded questions linked to behavioral peculiarities. Informed consents were sought for all participants prior to study. Socio-demographic and other related information were accumulated by the data collector through a face-to-face interview.
Sample collection, Transportation, Processing and analysis
Nasal samples were collected by inserting a sterile cotton swab (moistened with sterile normal saline prior to sampling, to avoid discomfort) into both nostrils . A single specimen was obtained from each participant from both anterior nares consecutively, using the same swab and it was then placed in a sterile normal saline, transported to the Microbiology and Parasitology Laboratory, Department of Medical Laboratory Sciences, using a cold chain within an hour of collection.
Isolation and Identification of S. aureus
All the samples were processed immediately to avoid any possible contamination. Each sample was directly inoculated into mannitol salt agar (Oxoid, Hampshire, UK). The inoculated plates were then incubated for 24 h at 37 °C, and the yellowish colonies obtained were subsequently subjected to species identification and confirmation. Morphological, physiological and biochemical characteristics (positive catalase and coagulase tests), of bacteria were ascertained by adopting standard laboratory methods including Gram staining and examination of morphology on different media such as DNase and blood agar . Corresponding American Type Culture Collection (ATCC) strains were utilized as references.
Detection of MRSA
Identification of MRSA was performed in accordance with the criteria set by Clinical Laboratory Standard Institute (CLSI), using cefoxitin disk diffusion assay. Bacterial suspension (5 ml) of 0.5 McFarland (1 × 108 CFU/ml) was prepared and swabbed into Mueller Hinton agar (MHA) (Hi-media, India), and cefoxitin disk were placed. After incubation for 24 hours at 37°C, the zone of inhibition was measured. Strains showing zone of inhibition ≤21 mm were extrapolated as MRSA .
Antimicrobial susceptibility testing
Antibiotic susceptibility profiling of all isolates of MRSA were achieved by Kirby–Bauer disk diffusion technique according to the criteria set by CLSI . Inoculums equivalent to the opacity of 0.5 McFarland standards were prepared and swabbed over MHA surface; exposed to a concentration gradient of antibiotic, and then incubated face up at 37 °C for 24 hours. Diameter of zones of inhibition were measured to the nearest millimeter and categorized as sensitive, intermediate, and resistant according to the table described in CLSI. Following antibiotics were used to examine the susceptibility patterns of MRSA, viz., penicillin G (10 units), ciprofloxacin (5 μg), clindamycin (2 μg), gentamicin (10 μg), erythromycin (15μg), chloramphenicol (30 μg), ampicillin (10 μg), ceftriaxone (30 μg), tetracycline (30 μg) and trimethoprim-sulfamethoxazole (1.25/25.75 μg) and cefoxitin (30 μg) . In addition, inducible clindamycin resistance was also analyzed by disk diffusion using D-zone test. Erythromycin disk (15 μg) was placed at a distance of 15–26 mm (edge to edge) from a clindamycin disk (2 μg) on MHA. After overnight incubation, plates were examined for the formation of flattened zone of inhibition adjacent to the erythromycin disk. Formation of D-shape with erythromycin indicated a positive clindamycin inducible resistance; this method is applied only to organisms which were resistant to erythromycin and susceptible or intermediate to clindamycin .
Detection of biofilm
Micro-titer plate assay was used for the detection of biofilm forming MRSA isolates in this study . Well standardized bacterial suspensions were prepared in sterile normal saline from pure colonies and adjusted to a 0.5 McFarland turbidity standard. Standardized bacterial suspensions were diluted with tryptic soy broth (TSB), supplemented with 1% glucose to a final volume of 200 µl per well. This experiment was performed for each isolate in triplicate and incubated at 37°C for 24 hours in flat-bottomed polystyrene micro-titer plates, the contents of the wells were then removed and washed with 300μl (0.3 ml) of phosphate buffered saline (PBS). Finally, the wells were fixed with 150μL of 99% methanol for 30 minutes and stained with crystal violet (0.1% w/v) for 15 minutes. Excess stain was rinsed off with distilled water. After drying, the wells were treated with 150μL of 95% ethanol for 30 minutes at room temperature to solubilize the dried crystal violet stain adhered to the biofilm. Optical densities (OD) were then determined by an automated micro ELISA reader at a wavelength of 570 nm. These OD values were considered as an index of bacterial adhesion and biofilm formation. Six wells (A1 to A6) of micro-titer plates were used as negative controls which contained only TSB + 1% glucose without bacteria. Average of three wells used for each bacterial isolate is considered as the actual OD since each of them was added to three wells. The cut-off value of optical density (ODc) was calculated and defined as three standard deviations above the mean OD of the negative control.
ODc = Average OD of Negative controls + 3 x Standard deviation of negative controls
Isolates were classified as follows: as described elsewhere .
1. Bacterial OD ≤ ODc = non-biofilm former;
2. OD>ODc, but ≤ 2 ODc= weak biofilm former;
3. OD>2 ODc, but ≤4 ODc= moderate biofilm former and
4. OD >4ODc = strong biofilm former
A pre-test was done on 5% of the total sample size before one week of the actual study in Wolita Sodo University to validate the variables and data collection tools were modified accordingly. Standard quality measures were implemented throughout the entire process of data collection and the laboratory works and also the completeness, accuracy, clarity, and consistency of data were checked. Standard Operating Procedures (in-house SOP manual) for each operation were strictly followed. All culture media were prepared following the instructions of manufacturers and sterility was tested by incubating 5% of each batch at 35–37°C overnight, for the evaluation of any possible contamination. Moreover, positive control (standard) strains of S. aureus, (ATCC 25923) were used as the quality controls (reference) for biochemical tests and agar plates including MHA with vancomycin disk; this ensured the testing performance, ie., the potency of the disk, as per CLSI 2019 guidelines.
Data were checked, cleaned and coded for its completeness and entered into the Epi data version 22.214.171.124 software, and analyzed by the Statistical Package for Social Sciences (SPSS) version 25. Descriptive statistics including frequency, mean and percentages were used. Binary logistic regression model was used to analyze the association among dependent and independent variables. Those variables with p value < 0.25 in bivariable analysis were considered as candidates for further multivariable analysis; p value ≤0.05 was considered as statistically significant. Adjusted odds ratio (AOR) and 95% confidence interval (CI) were used to determine the strength of association among variables.