Prevalence of Methicillin-ResistantStaphylococcus aureus and Multidrug-Resistant Strains from Patients Attending the Referral Hospitals of Amhara Regional State, Ethiopia

Background Staphylococcus aureus (S. aureus) causes different types of human infections and can develop resistance to many antibiotics. There is a scarcity of data on the mecA gene and multidrug-resistant (MDR) strain distribution of this organism in developing countries, such as Ethiopia. This study investigated the presence of mecA gene and MDR profile of S. aureus among patients attending referral hospitals of Amhara regional state. Methods Of the total of 110 isolates collected from Amhara regional referral hospitals, 70 MDR isolates were further processed for isolation of S. aureus mecA gene. Genomic DNA was isolated using a Sigma-Aldrich genomic DNA isolation kit for Gram-positive bacteria. Amplification of S. aureus mecA gene was performed with the amplicon size of 533 bp. Antimicrobial susceptibility test including methicillin resistance was determined by the Kirby–Bauer disc diffusion method. Results The majority of the isolates were recovered from patients aged less than 5 years (51; 36.7%) and the least number of isolates was recorded in age group greater than 60 years (6; 4.3%). Most of the isolates were from blood (61; 43.9%), followed by wounds (45; 32.4%). A high resistance rate was observed in penicillin (81; 73.6%), followed by cotrimoxazole (78; 70.9%), ceftriaxone (76; 69%), erythromycin (66; 60%), and tetracycline (65; 59.1%). Phenotypically, considering cefoxitin as a surrogate marker, 38 (34.5%) of the isolates were methicillin-resistant. The overall MDR isolates were 80 (72.7%). The PCR amplification result of the mecA gene was 14 (20%). Conclusions and Recommendations. High rates of MDR and methicillin-resistantS. aureus were reported. PCR amplification indicated that 20% of MRSA isolates were the mecA gene carriers. Large-scale studies for the detection of MDR strains of S. aureus including MRSA using molecular techniques should be encouraged in the Amhara region.


Introduction
Staphylococcus aureus (S. aureus) is one of the most common causes of bacterial infection in humans that causes both community-and hospital-acquired infection of the skin, urinary tract, surgical site infections, osteomyelitis, septicaemia, and endocarditis [1]. Staphylococcus aureus has an extraordinary ability to develop resistance to many antibiotics. Tis was frst revealed by the acquisition of β-lactamase on "penicillinase plasmids" and the subsequent response to β-lactamase stable derivatives by acquisition of staphylococcal cassette chromosome (SCCmec) elements by methicillin-resistant S. aureus (MRSA) [2].
Penicillin has been used as a drug of choice for S. aureus as it was discovered by Fleming in the 1940s, but with the widespread use of penicillin in the 1950s, penicillin-resistant S. aureus appeared in the hospitals [3,4]. Penicillin-resistantS. aureus can produce penicillinase, which can hydrolyze the penicillin β-lactam ring, leading to resistance to penicillin. Later, scientists developed a new penicillinaseresistant semisynthetic penicillin named methicillin, which is resistant to the hydrolysis of β-lactamase [3,5]. Terefore, in the widespread appearance of penicillin-resistant S. aureus, methicillin was used as a drug of choice for penicillin-resistant S. aureus. However, soon later MRSA strain was reported; this resistance was produced by a gene encoding the penicillinbinding protein 2a or 2′ (PBP2a or PBP2′) (mecA) which was integrated into the chromosomal element (SCCmec) of methicillin-sensitive S. aureus [6]. Available data show that the structural gene, mecA, is present in the resistant strains of S. aureus, but not in the susceptible ones [7]. Tis achievement has enabled the development of an alternative method for identifying methicillin-resistant S. aureus by detecting the mecA gene. Moreover, other than macA gene, mecB, mecC, and macD have been documented as responsible for methicillin resistance in the Staphylococcaceae family. Te mecB and mecD genes were reported at frst on the chromosome and/or on a plasmid of Macrococcus caseolyticus. Recently, the mecB gene was also documented on a plasmid of one MRSA isolated from a human patient [8].
In the present study, the polymerase chain reaction (PCR) was used to detect the methicillin resistance determinant by amplifying a 533-bp region of the mecA gene. Te gold standard to determine MRSA genotypes is to detect conserved genes constantly found in the mecA gene, which is within the range of a particular chromosome in staphylococcal cassette chromosome (SCCmec) [9]. Terefore, amplifcation of mecA can be performed by using PCR, which is the gold standard for the detection of mecA gene [10]. No information on the distribution of the mecA gene on MRSA in Amhara region is available. Terefore, this study aimed at investigating the presence of mecA gene and multidrug-resistant (MDR) strain distribution of S. aureus among patients attending referral hospitals of Amhara regional state.

Bacterial Isolates.
A total of 139 isolates of S. aureus were isolated between the periods of 2017 and 2018 from Amhara region referral hospitals (University of Gondar Comprehensive Specialized Hospital, Felege Hiwot Comprehensive Specialized Hospital, Dessie Referral Hospital, and Debre Markos Referral Hospital). Detailed data of sample size determination, sampling technique, and specimen collection were found from the previous two studies [11,12]. All isolates were clinical isolates from diferent specimens such as blood, urine, wounds, discharges, and body fuids. Each clinical sample was cultured on mannitol salt agar and incubated at 37°C for 24 h. Further identifcation of S. aureus isolates was performed by colony morphology, Gram stain, and standard biochemical characteristics such as catalase, coagulase, and novobiocin susceptibility tests. Te ATCC 25923 of S. aureus was used as reference strain.

Extraction of DNA from S. aureus.
Te clinical isolates were subcultured using nutrient agar medium and incubated for 24 h at 37°C. A single colony was taken from the previously subcultured medium and inoculated in to 10 ml Luria-Bertani (LB) broth medium, incubated at 37°C with a shaker incubator for 24 h. After 24 h incubation, genomic DNA was isolated using Sigma-Aldrich genomic DNA extraction kit for Gram-positive bacteria, and the isolation protocols were followed according to the manufacturer's instructions of Sigma-Aldrich. Finally, the extracted DNA was dissolved with Tris-EDTA bufer (10 mM Tris-Cl and 1 mM EDTA bufer), and the quality of isolated genomic DNA was confrmed by using NanoDrop and 1.5% agarose gel electrophoresis, and then it was stored at −21°C till use.
Te specifc oligonucleotide primers for mecA genes were diluted by using nuclease-free water according to the manufacture company information to get primary concentration equal to 100 pmol. Termal cycler and the reaction mixtures were prepared accordingly. Te PCR was performed with a total volume of 25 μl containing a mixture of 2 μl of template DNA, 2.5 μl of 10x PCR bufer, 2.5 μl (10 pmol/μl) of each mecA gene forward and reverse primers, 0.5 μl of dNTPS (10 mM), 1.5 μl of MgCl 2 , and 0.5 μl of Taq polymerase, and the remaining volume was flled by nuclease-free water to get a fnal volume of 25 μl. PCR mixture without DNA template was used as a negative control. After preparation of mixtures, the PCR program was as follows: initial denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 60 s, annealing at 62°C for 30 s, extension at 72°C for 35 s, and fnal extension at 72°C for analysis by agarose gel electrophoresis.

Agarose Gel Electrophoresis.
Agarose gel electrophoresis was prepared with 1.5% agarose in 1x trice acetate EDTA (TAE) bufer, and 0.5 μg/mL of ethidium bromide was added and mixed. A 12 μl volume of PCR-amplifed products was mixed with 3 μl loading dye and then loaded into wells of agarose gel. Electrophoresis was carried out for 90 min (70 Volts/cm 2 ) in 1x TAE bufer. DNA ladder (100 bp) was used to assess the PCR product size, then PCR products were visualized by UV light at 336 nm, and photographs were taken using a digital camera.
Among 110 isolates tested for 10 diferent commonly used antibiotics, 7 isolates were sensitive to all drugs tested and 23 isolates were resistant to one or two antibiotics. However, S. aureus isolates resistant to 3 or more antibiotic classes were 80 (72.7%) ( Table 4).
For molecular detection of methicillin-resistant genes, out of 80 (72.7%) MDR isolates, we randomly selected 70 isolates of S. aureus and included from all study sites. Accordingly, we considered 40 isolates (13 were cefoxitinresistant, 3 of them were intermediate, and 24 were sensitive) from the University of Gondar Comprehensive Specialized Hospital, 14 isolates (5 were cefoxitin-resistant, 2 of them were intermediate, and 7 were sensitive) from Felege Hiwot Comprehensive Specialized Hospital, 11 isolates (4 were cefoxitin-resistant, 3 of them were intermediate, and 4 were sensitive) from Dessie Referral Hospital, and 5 isolates (4 were cefoxitin-resistant and 1 was sensitive) from Debre Markose Referral Hospital (Table 5). In all cases, the isolates taken for mecA gene detection were phenotypically MDR.
Te PCR amplifcation result of the mecA gene was performed in all 70 clinical isolates of S. aureus. However, among the total of 70 isolates, mecA gene was detected only in 14 (20%) S. aureus isolates. Te mecA gene positive in 14 isolates was phenotypically from MRSA and MSSA, but both of them were MDR (Figure 1). Although its distribution is diferent, mecA gene producing methicillin-resistantS. aureus was reported in all study sites.

Discussions
S. aureus is a main pathogenic bacterium which causes severe human health problems globally [16], and its antimicrobial resistance characteristics have made it more rebellious in the health institutions [17].
Te isolation rate of S. aureus in the current study was 139/1365 (10.2%) which is lower than a study conducted in Ethiopia (79/94 (84.0%)) [18] and Nigeria (55/360 (15.3%)), and the occurrence of S. aureus was the highest in wound swabs [19], but in the present study, the highest isolates were recovered from blood sample followed by wound specimen.
Majority of the isolates were recovered from patients aged less than 5 years (51 (36.7%)), followed by 16-30 years (42 (30.2%)), while the least number of isolates was from patients greater than 60 years. Tis is in line with an observation from previous Ethiopian report where the rate of isolation of S. aureus was higher in lower age (15-24 years)   (46/210 (21.9%)) [20] and from Eritrean study where it was signifcantly associated with lower age, 13 to 18 years (78.6%) and <13 years old (85.0%), and lower rate of isolation was recorded in older age (≥61 years old) [21].
Phenotypically, considering cefoxitin as surrogate marker for methicillin test, 38 (34.5%) of the isolates of S. aureus were methicillin-resistant in the current study which is in agreement with the pooled prevalence of MRSA reported in Ethiopia (32.5%) [23]. However, the current fnding of MRSA is lower than a report from Ethiopia, where 54 (68.4%) of the isolates were MRSA [18]; from Eritrea, 59 (72.0%) of the isolates were MRSA [21]; from Nigerian studies, 44.0% [17]; and 40.4% of the isolates were MRSA [24]; and from Iran, 133/279 (47.6%) of the isolates were MRSA [15]. On the other hand, the present report is higher than another previous report from Ethiopia where 34/194 (17.5%) of the S. aureus isolates were found to be MRSA [20]; in Iraq, the prevalence of MRSA was 114/429 (26.54%) [25]. Te possible explanation for the observed discrepancies across the literature might be associated with the variation of the methods used to detect methicillin resistance. Some studies used cefoxitin and others used oxacillin as a surrogate marker for the detection of methicillin resistance.
Te MDR isolates observed in the current study was 80/ 110 (72.7%) which is in line with a previous report in Ethiopia (65 (82.3%)) [18]. However, the MDR S. aureus observed in the present study is higher than a previous study reported from Ethiopia where 98 (50.5%) of the S. aureus isolates were MDR [20], from Eritrea where 17/43 (39.5%) isolates were MDR [21]; and from Saudi Arabia where 47% of MRSA were MDR [26].
Te PCR amplifcation result of mecA gene, a gene that confers resistance to methicillin and most β-lactam antibiotics, was obtained in 70 clinical isolates of S. aureus. However, among the total of 70 isolates, mecA gene was detected only in 14 (20.0%) S. aureus isolates with an amplicon of 533 bp considered as indicative with the presence of mecA gene. Although its distribution is diferent, mecA gene producing MRSA was reported in all study sites. Tis is similar with a study from Nigeria that phenotypic resistance to cefoxitin was 46.5%, while the mecA gene was 19.2% [24]. Another study from Nigeria indicated that S. aureus isolates with phenotypic resistance to methicillin (oxacillin) were tested for mecA gene and none of the isolates contained the mecA gene [27]. Nwaogaraku et al. from Nigeria showed that all isolates of MRSA from blood samples of pigs were mecA negative on PCR [28]. However, the present study is diferent from many studies performed elsewhere [26,29,30]. Te possible explanation why phenotypically MRSA-positive isolates did not show mecA gene might be due to loss of the mecA gene during prolonged storage [31] or other mechanisms other than the presence of mecA gene (mecC and mecB) responsible for methicillin-resistantStaphylococcus aureus [32,33].

Conclusion and Recommendation
Phenotypic resistance to cefoxitin was 34.5%. Tis prevalence overestimated the prevalence of MRSA, as the mecA gene that encodes resistance to methicillin was detected by PCR in 20.0% of the S. aureus isolates. A large-scale study for mecA gene detection is important to re-assure the

Data Availability
Te data used and analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval
Ethical approval was obtained from the Institutional Review Board of the University of Gondar with reference number O/VIP/RCS/05/478/2015.

Consent
Informed written consent was obtained from each study participant. Children less than 18 years who are not able to give consent were also asked an assent and/or written consent taken from their parents or guardians.

Disclosure
A preprint has previously been published by Moges et al. in Research Square on 16 February 2022 [34].

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
FM was involved in conception of the research idea, study design, data collection, analysis and interpretation, and the drafting of the manuscript; TTand AA performed laboratory work; GM performed data analysis; SE contributed to study design and data analysis and interpretation; MD was involved in data analysis and write up; TF and MG performed data collection and laboratory work; WA contributed to conception of the research idea, study design, data analysis and interpretation, and write up. All authors read and approved the fnal manuscript.