Prevalence of Methicillin-Resistant Staphylococcus aureus and Associated Risk Factors among Patients with Wound Infection at Referral Hospital, Northeast Ethiopia

Background The spectrums of infections due to methicillin-resistant Staphylococcus aureus are manifold and are associated with worse outcomes. A study on the prevalence of these pathogens and their sensitivity patterns will give updated information which is very helpful for health personnel responsible in the management of patients and timely monitoring of the emergence of resistant bacteria. Hence, the study aimed at assessing the prevalence of methicillin-resistant Staphylococcus aureus and associated factors among patients with wound infection at Dessie Referral Hospital. Method A cross-sectional study was conducted among 266 patients at Dessie Referral Hospital from February to May 2016. Wound swab samples were collected aseptically using Levine's technique and transported to Dessie Regional Laboratory by using brain-heart infusion transport media. Isolation of Staphylococcus aureus was done based on cultural and biochemical profiles. Drug susceptibility test was performed using the disc diffusion technique as per the standard and interpreted based on the Clinical and Laboratory Standards Institute guidelines. The data were entered and analyzed by using SPSS version 20. Result Staphylococcus isolates from 266 processed wound swabs were 92 (34.58%). Of these, 26 (28.3%) were identified as methicillin-resistant S. aureus and 66 (71.7%) were methicillin-sensitive S. aureus. The overall prevalence of methicillin-resistant S. aureus among the study population was 9.8%. The isolated methicillin-resistant S. aureus showed full resistance to penicillin (100%) followed by erythromycin and ciprofloxacin (16, 61.5%) and cotrimoxazole and gentamicin (14, 53.8%). From the total S. aureus isolates, 20 (21.7%) of them showed multidrug resistance. Of these methicillin-resistant S. aureus, 18 (69.8%) showed high multidrug resistance. Patients who are farmers in occupation (AOR = 6.1, 95% CI (1.086–33.724)), admitted in the hospital (AOR = 3.56, 95% CI (1.429–8.857)), and have low BMI (<18.5) (AOR = 13.89, 95% CI (4.919–39.192)) were among the risk factors significantly associated with wound infection due to methicillin-resistant S. aureus. Conclusion All methicillin-resistant S. aureus isolates were 100% resistant to penicillin and showed high multidrug resistance. Therefore, antibiotic susceptibility test should be performed prior to treatment.


Background
Wound is a break in the skin and exposes the underlying tissue to the outside environment. Loss of skin integrity by wounding provides a moist, warm, and nutritious environment for microbial colonization, proliferation, and infection [1,2]. Common bacterial skin infections include Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pyogenes, Proteus species, Streptococcus species, and Enterococcus species [3,4]. Among the most frequent members of wound infection, Staphylococcus aureus [5] is a leading cause of nosocomial infections (NI) and surgical wound infections [6]. It develops resistance to many antibiotics in recent years.
Methicillin-resistant S. aureus acquires its resistance via the methicillin resistance gene mecA, which encodes a lowaffinity penicillin-binding protein (PBP2a) that is absent in susceptible S. aureus strains [7,8]. is resistant penicillinbinding protein receptor does not bind well to most β-lactams and therefore allows MRSA to grow in their presence [8]. Methicillin-resistant S. aureus strains were recently classified as two groups by epidemiologic as well as molecular characteristics, namely, community-associated (CA) MRSA and healthcare-associated (HA) MRSA. Community-associated MRSA isolates are usually less resistant than HA-MRSA isolates [9].
Methicillin-resistant S. aureus is a major problem worldwide causing hospital-acquired infections [10]. It is estimated that MRSA infections within the healthcare setting alone affected more than 150,000 patients annually in the European Union, with an additional cost of 380 million euros [11]. e widespread and prolonged use of antibiotics leads to the emergence of resistant bacterial pathogens during wound infections contributing to high morbidity and mortality [12]. e spectrums of infections due to MRSA are manifold [13,14] and are associated with worse outcome in addition to prolonged hospital stay, higher cost of treatment, and increased mortality [15,16].
In some parts of Africa, 80% of S. aureus infection was resistant to methicillin, rendering treatment with standard antibiotics ineffective [17]. Even though different studies across the region in Ethiopia showed that the burden of MRSA constitutes a major public health problem [18][19][20], prevention and control strategies are not well established to minimize MRSA. In addition, antibiotics are widely and inappropriately used results in the increased prevalence of drug resistance strain bacteria such as MRSA, so that a study on the prevalence of these pathogens and their sensitivity patterns in healthcare facility will give updated information which is very helpful for health personnel responsible in the management of patients and timely monitoring of the emergence of resistant bacteria. In general, the current study results can also be used as input data to establish a guideline to minimize the burden of MRSA.

Methods
A cross-sectional study was conducted from February 2016 to April 2016 to assess the antibiotic resistance pattern of methicillin-resistant Staphylococcus aureus isolated from wound infection and associated risk factors in Dessie Referral Hospital (DRH). Dessie Referral Hospital is found in Dessie town with a distance of 400 km from the capital city of the county Addis Ababa and 471 km far from Bahir Dar which is the capital city of Amhara regional state. In Dessie town, there are one referral hospital, three private general hospitals, three health centers, five higher private clinics, and one regional health research laboratory where culture and susceptibility tests are performed. All patients suspected of wound infections and who have not taken antibiotics for the last two weeks prior to the study period were included in this study. e sample size was determined using a single population proportion formula at 19.6% [18] and 95% confidence interval (CI), and the total sample size was 266.
Sociodemographic related data and associated risk factors were collected by using a structured questionnaire.

Sample Collection and
Processing. Wound samples were collected using Levine's technique [21]. e wound surface was cleaned with sterile gauze moistened with 70% alcohol. Dressed wounds were cleansed with sterile normal saline after removing the dressing. Aseptically, the end of a sterile cotton-tipped applicator was rotated over 1 cm 2 area for 5 seconds with sufficient pressure to express fluid and bacteria to surface from within the wound tissue as technique stated by Levine and Gardner [22]. Samples from closed wound were collected after the skin was cleansed with 70% alcohol. Double wound swabs were taken from each wound at a point in time to increase the chance of recovering bacterial pathogens. All collected specimens were labeled and transported by using brain-heart infusion transport media to Dessie Regional Health Research Laboratory for culturing and antimicrobial susceptibility testing within 1 hour. Each wound specimen was inoculated on blood (Oxoid, Ltd., Basingstoke, Hampshire, England) and subcultured on mannitol salt agar. All plates were incubated in aerobic atmosphere at 35-37°C for 24 h.
Staphylococcus aureus was identified based on Grampositive cocci in clusters, β-hemolytic colonies on blood agar, catalase and coagulase production, and yellow colony surrounded by yellow zone on mannitol salt agar [21].

Antimicrobial Susceptibility Test.
Antimicrobial susceptibility test was carried out on each bacterial isolate using the disc diffusion method on Muller Hinton agar (MHA). ree to five pure colonies of each bacterium were picked and transferred to a tube containing 5 ml sterile nutrient broth. e preparation was mixed thoroughly to make the suspension homogeneous. e suspension was incubated at 37°C until the turbidity of the suspension adjusted to a 0.5 McFarland turbidity standard (bacterial concentration of 1.5 × 10 8 colony-forming unit/ml) [23]. A sterile swab was dipped in the suspension, and the entire surface of the MHA plates was uniformly flooded with the suspensions and allowed to dry for about 15-30 minutes. e antimicrobial impregnated disks were placed on the media using sterile forceps in such a way that each disk was placed at least 24 mm away from each other to avoid the overlapping zone of inhibition. After the disk was placed on the inoculated media, the plates were allowed to stand for 30 minutes so that the antibiotic will diffuse into the media. e plates were inverted and incubated at 35 ± 2°C for 24 h and observed for the zone of inhibition. e selected antibiotic disks used were (Oxoid UK) penicillin (10IU), ciprofloxacillin (5 μg), cotrimoxazole (1.25/23.75 μg), doxycycline (30g), erythromycin (15 μg), clindamycin (2 μg), chloramphenicol (30 μg), and gentamicin (10 μg). Susceptibility pattern was interpreted by comparison of the zone of inhibition according to the Clinical and Laboratory Standards Institute (CLSI, 2014) guideline and reported as sensitive, intermediate, and resistant [24]. Standard strains of S. aureus (ATCC25923) were used as controls on the biochemical tests and agar plates including MHA with antimicrobial discs to assure the testing performance of antimicrobial discs.
Data were entered and analyzed using SPSS version 20 for Windows. Stepwise logistic regression model was considered to determine factors associated with wound infection. Adjusted odds ratio and 95% CI were calculated to measure the strength of the association. p values <0.05 were considered as statistically significant.

Sociodemographic Characteristics of Study Participants.
In this study, a total of 266 study participants were included. Of these, 180 (67.7%) were male and 86 (32.3%) were female. e mean ages of the study participants were 33.2 ± 17.8 years (range from 5 to 81 years). One-fourth of the study participants had no formal education, while the majorities were lived in urban (205, 77.1%) ( e overall prevalence of MRSA in inpatients and outpatients was 19.5% (16/82) and 5.4% (10/ 184), respectively ( Figure 1).

Antibiotic Resistance Pattern.
Out of the 92 S. aureus isolated from wound swab including MRSA, 78 (84.8%) showed a high level of resistance to penicillin and 4 (4.3%) showed a low level of resistance to clindamycin while MRSA showed full (100%) resistance rate to penicillin followed by erythromycin and ciprofloxacin (16, 61.5%) and cotrimoxazole and gentamicin (14, 53.8%) ( Table 2).

Multidrug Resistance Pattern of MRSA.
In this study, a high prevalence of multidrug resistance (MDR) to MRSA was observed as compared to methicillin-sensitive Staphylococcus aureus (MSSA) which accounted for 18 (69.2%) and 2 (3%), respectively; none of the strains were resistant to all antibiotics tested. However, 10 (15.2%) MSSA were sensitive to all antibiotics tested (Table 3).

Bivariate and Multivariate Analysis of Factors Associated
with MRSA among Wound Infection Individuals. In a bivariate logistic regression analysis, wound infection due to MRSA showed significant association with occupation, history of recent admission, history of recent surgery, being diagnosed in the inpatient department, and low body mass index (BMI) (<18.5). However, other factors such as age, sex, education, residence, history of previous antibiotic use, and chronic illness did not show statistically significant association.
In a multivariate logistic regression analysis, the abovementioned associated factors remained associated with wound infection due to MRSA except recent history of

Discussion
Wound infection due to MRSA was a major concern in resource-limited countries, in particular, Ethiopia, where there are poor infection prevention and control measures [25]. In this study, the prevalence of S. aureus wound infection was 34.5%. is finding is in line with the study conducted in Debre Markos (39.7%) [18] and Cameroon (28.9%) [26]. On the other hand, this finding is higher than studies conducted in Jimma (23.6%) [19], Nigeria (26.6%) [27], Tanzania (26.7%) [28], and Brazil (20%) [29]. However, the prevalence reported in the current study is lower than a study reported in Addis Ababa (57.8%) [30] and Uganda (41%) [31]. e variation in prevalence might be due to variation in the study subjects, study conducted time, and the method employed for the detection of S. aureus. In our study, the overall prevalence of MRSA was 9.8%. is finding is similar to the results reported from studies in Addis Ababa (13.2%) [20], Eretria (9%) [32], and Cameroon (13.16%) [26] and lower than the results reported from previous studies in Ethiopia such as Debre Markos (19.6%) [18] and Jimma (17.4%) [19] and other African countries: Uganda (41%) [31] and Libya (31%) [33]. On the other hand, this study finding is higher than study reports from Nigeria (5.8%) [27], Brazil (5.6%) [29], and Tanzania (4.3%) [28]. e observed high prevalence of MRSA in our study may be due to the high rate of certain antibiotics use either due to availability or cost-effectiveness issues.
Regarding the possible associated risk factors, MRSA wound infections were significantly associated with occupation (farmers), patients with low BMI, and those patients who are currently admitted (inpatient) as compared to their counterparts. is might be because farmers may not have knowledge of utilizing healthcare services; in addition, their occupation may expose them to wound infection and make them use antibiotics without prescription. High prevalence of MRSA in admitted patients may be attributed by resistant strain bacterial cross-contamination in health institutions. Patients who have low BMI had higher odds of developing wound infection due to MRSA. Healthy people may carry MRSA asymptomatically for long periods of time, but patients with compromised immune system are at a significantly greater risk of symptomatic infections [13,14].
Concerning the antimicrobial resistance profile of the isolates, in the present study, S. aureus isolates showed resistance to penicillin (84.8%), gentamicin (15.2%) ciprofloxacillin (18.4%), clindamycin (4.3%), erythromycin (26.1%), cotrimoxazole (16.3%), doxycycline (9.7%), and chloramphenicol (8.7%). e resistance profile of S. aureus to penicillin in our study is similar to the results obtained within DRH (82.2%) [18]. In other studies, resistance to penicillin done in Tanzania [28] and Jimma, Ethiopia [34], is slightly higher and reported as 97% and 100%, respectively. e resistance to clindamycin in the current study is similar to other studies in Ethiopia in which the resistance is less than 50% [20,30]. We noticed that the resistance rate of other antibiotics listed above is varied in different studies conducted in Ethiopia [18,19].  Journal of Pathogens e current study showed MRSA isolates were resistant to penicillin (100%), gentamicin (53.8%), ciprofloxacillin (61.5%), clindamycin (7.7%), erythromycin (61.5%), cotrimoxazole (53.8%), doxycycline (30.8%), and chloramphenicol (26.9%). Similarly, studies conducted in different areas showed MRSA isolates were 100% resistant to penicillin [18,35]. In this study, the resistance of MRSA isolates to gentamycin is higher compared to the study done in Yekatit 12 Hospital in Addis Ababa (38.2%) [20]. Similarly, the resistance to ciprofloxacin is slightly higher compared to the results reported in Tanzania (54%) [28]. In contrast, the resistance to clindamycin, erythromycin, cotrimoxazole, and chloramphenicol is lower compared to the studies done in Ethiopia [18,20]. e main variation in drug resistance patterns among different studies might be due to the indiscriminate use and availability of these antibiotics in a certain area. e variation of resistance rate among different areas indicates the resistance pattern of antibiotics varies according to regional and geographical location and also changes through time.
Furthermore, high prevalence of multidrug-resistant MRSA (69.2%) was reported in the study area. is finding is concordant with the study conducted in northern India whereby 73% of MRSA strains were multidrug-resistant [36]. Likewise, a study conducted in Debre Markos showed all MRSA strains isolated were resistant to ≥3 antibiotics [18]. High prevalence of multidrug resistance may predispose patients to infection with intractable isolates, emphasizing the need for improved infection control practices and guidelines for the use of antibiotics in this setting. e current study results could not show MRSA is community-associated or healthcare-associated.

Conclusion
Out of 266 patients suspected with wound infection, 92 (34.58%) have culture-confirmed S. aureus. Of these, 26 (28.3%) were MRSA. Wound infection due to MRSA showed significant association with occupation, being diagnosed in the inpatient department, and body mass index. Greater than 50% of MRSA isolates were resistant to gentamicin, ciprofloxacillin, cotrimoxazole, and erythromycin.  Data Availability e data used to support the results of this research are available from the corresponding author upon request.

Ethical Approval
e study was ethically approved by the ethical review committee of the School of Biomedical and Laboratory Sciences, University of Gondar.

Consent
Permission was obtained from DRH. Informed written consent was obtained from participants before data collection. All the information obtained from the study subjects was coded to maintain confidentiality. When the participants were found to be positive for S. aureus, they were informed by the hospital clinician and received proper treatment.

Conflicts of Interest
e authors have no conflicts of interest.

Authors' Contributions
YT and TG conceived this research topic and objectives. YT, TG, AA, MA, and BG participated in the designing of the study and data analysis and performed statistical analysis. MM and ST prepared and critically revised the manuscript for its scientific content. All authors read and approved the final manuscript.