Prevalence and Antibiotic Susceptibility of Clinical Staphylococcus Aureus Isolates in Various Specimens Collected from a Tertiary Care Hospital, Hayatabad, Peshawar, Pakistan.

Staphylococcus aureus is a notorious Gram-positive, non-spore-forming, opportunistic bacterium that causes a variety of infections including bacteremia, endocarditis, pneumonia, skin and soft tissue infections, and several others. Also, the overuse and misuse of drugs attributed to the crises of multidrug resistance especially in MRSA. Objective: Therefore, the aim of this study was to determine the prevalence rate of MRSA, antimicrobial susceptibility pro�les of S . aureus , MRSA, and MSSA isolates to a variety of commonly used antibiotics, and its multidrug resistant patterns. Methods : Samples were collected from the microbiology department of HMC Peshawar, Pakistan. Antibiotic susceptibility patterns were determined under CLSI and EUCAST guideline, 2021 by following the Kirby-Bauer disc diffusion method. Results: Out of 106 S . aureus clinical isolates, 83 (78.3%) isolates were identi�ed as MRSA and 23 (21.7%) were MSSA. In MRSA high resistance was exhibited to Penicillin G and cefoxitin (100%), followed by erythromycin 84.34% and cipro�oxacin 79.52%. Meanwhile low resistance was observed to doxycycline 19.28% followed by chloramphenicol 14.46%, teicoplanin and linezolid 2.41% for each respectively. High sensitivity in MRSA isolates was exhibited to linezolid 97.59% followed by teicoplanin 95.18%, chloramphenicol 85.54%, doxycycline 80.72% and fusidic acid 74.70%. A total of n=94 (88.67%) isolates were characterized as MDR. Conclusions: In conclusion, the most effective antibiotics used to treat S. aureus infections were linezolid, teicoplanin, chloramphenicol, doxycycline, fusidic acid, and gentamycin. In addition, the current study also noticed a signi�cant prevalence of resistance to several antibiotics, emphasizing the importance of antibiotic usage monitoring.

Then followed by incubation for 24 hours at 37 C. After the incubation, S. aureus isolates were identi ed through Gram-staining and isolated colonies were further subjected to biochemical tests including catalase, tube coagulase test and DNase test. S. aureus isolates were further processed for Antibiotic Susceptibility Testing (AST) through the Kirby-Bauer disc diffusion method [20]. Following 11 antibiotics were inoculated on Muller Hinton Agar (MHA) (OXOID CM0377, England) for antimicrobial testing: penicillin G (P) 10μg, chloramphenicol (C) 30μg, cefoxitin (FOX) 30μg, cipro oxacin (CIP) 5μg, clindamycin (DA) 2μg, doxycycline (DO) 30μg, erythromycin (E) 15μg, fusidic acid (FD) 10μg, gentamicin (CN) 10μg, linezolid (LZD) 30μg and teicoplanin (TEC) 30μg. Results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, 2021 while for teicoplanin CLSI guidelines, 2016 were followed. The breakpoints of fusidic acid were interpreted according to European Committee on Antibiotic Susceptibility Testing (EUCAST) guidelines, 2021. The AST growth suspension was prepared in 5ml normal saline solution with the turbidity adjusted to match the 0.5 McFarland standards to obtain the estimated 6 amount of organism number of 1x10 colony forming units (CFU) per milliliter. After 15 minutes of inoculation, antibiotics discs were placed on MHA, seeded with each 0 isolate, and were cultured at 37 C for 24 hours. After incubation, the antibiotics zones of inhibitions were measured using a ruler and the results were interpreted according to the CLSI and EUCAST guidelines, 2021. For the determination of MRSA, FOX discs of 30μg were used to screen all the S. aureus isolates. S. aureus isolates were 0 grown on MHA agar at 37 C for 18-24 hours with a growth suspension calibrated to 0.5 McFarland standards and inhibition zone equal to or less than 21mm on MHA was considered as MRSA while inhibition zone equal to or greater than 22mm on MHA was considered as MSSA by following CLSI guidelines, 2021. The D-test method was used to determine the inducible clindamycin resistance in MRSA isolates. Brie y, a 0.5 McFarland standards equivalent bacterial culture was seeded on MHA plates, followed by 15mm apart insertion of erythromycin (15μg) and clindamycin (2μg) discs. After that, the plate was incubated for an overnight period and positive inducible clindamycin resistance was determined by a "D" shaped clindamycin zone of inhibition towards an erythromycin disc. For the detection of MDR, Magiorakos et al., [11]. de nition of non-susceptibility to at least one antimicrobial agent out of three or more antimicrobial classes was used. The chi-square technique was used to establish statistical signi cance in the age, gender, and specimen type, using GraphPad Prism 9.1.2.226. A p-value

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resistant bacterial diseases are responsible for more than 35,900 deaths per year and is still a major public health issue in US, with 2.8 million antibiotic-resistant pathogenic infections [10]. Multi-drug resistant (MDR) bacteria are resistant to at least three or more antibiotic classes and are the most prominent trait of MRSA [11]. In Nigeria, β-lactam antibiotics are used for the treatment of MRSA infections, but they are highly (88%) ineffective against them. Even in India and Pakistan, 95% of adults carry β-lactam antibiotic-resistant pathogens [12]. In 2010, a study was conducted at a tertiary care hospital in Mangalore, South India, where a total of 237 isolates were studied, in which 29.1% were methicillin-resistant, while erythromycin, gentamicin, and chloramphenicol resistance were found in 40-50% of isolates. Inducible clindamycin resistance was observed in 18.8% of MRSA strains, with less than 30% resistance to cipro oxacin and amikacin [13]. Another study from Rawalpindi, Pakistan aimed to determine the prevalence rate of MRSA. Out of 350 staphylococcal isolates, 60.40% were identi ed as MRSA isolates. All the β-lactam antibiotic drugs were 100% resistant to MRSA followed by nalidixic acid 89.18%, cotrimoxazole 86.48%, erythromycin 85.81%, levo oxacin 80.4%, gentamicin 76.35%, tetracycline 59.45%, cipro oxacin 44.59%, chloramphenicol 18.24%, and rifampicin 10.13% [14]. Several reports had highlighted a signi cant proportion of nosocomial and community-acquired MRSA infections in Pakistan [15][16][17][18]. The rst case of MRSA in Pakistan was discovered in 1989, and the prevalence has been steadily increasing since then. According to researches, the percentage of MRSA isolates grew from 5% in 1989 to 69% in 2020 [17][18][19]. Therefore, this study aimed to determine the prevalence rate of MRSA, antimicrobial susceptibility pro le of S. aureus, MRSA and MSSA isolates to various antibiotics, and its MDR pro le. These ndings with respect to resistant phenotypes will help in the development of an appropriate hospital antibiotic stewardship policy to reduce the risk of S. aureus-associated infections. It would further highlight the importance of local surveillance in providing useful antibiotic-resistant data that can guide empiric therapy.
A total of 106 samples were collected from the microbiology laboratory of HMC, Peshawar, Khyber Pakhtunkhwa (KPK), Pakistan. Various clinical samples including pus, uids, blood, sputum, throat swab, and tracheal aspirate were collected from December 2020 to May 2021. The clinical isolates were randomly collected from patients who came to the hospital or were already admitted. The randomly collected clinical isolates were processed for bacterial culturing on Mannitol Salt Agar (OXOID CM0085, England), which is a selective and

Gram staining
All of the 106 bacterial isolates were identi ed as Grampositive cocci under a light microscope.

Identi cation of isolates
The Gram-positive bacterial isolates on Mannitol Salt Agar plates changed the medium color from pink to yellow, con rming that the bacteria belong S. aureus species.

Biochemical tests
All the clinical isolates were tested positive for catalase test, tube coagulase test and DNase test. Antibiotic susceptibility pro les of S. aureus isolates All of the 106 strains were 100% resistant to Penicillin G. High resistance was obser ved among cefoxitin, cipro oxacin, and erythromycin i.e., 78.3% (for each) whereas low resistance was found in linezolid 1.9% followed by teicoplanin 2.8% and chloramphenicol 13

Resistant phenotype of S. aureus
Ninety-four (88.67%) of the isolates were MDR. MDR strains ranged from resistance to three classes of antibiotics (n=10, 9.43%) to 9 classes of antibiotics (n=1, 0.94%). The high resistance rate for MDR was observed among s4-5 classes of antibiotics (n=25, 23.58%). The detailed resistance of the MDR pattern is given in Table 4. S. aureus is one of the most leading causes of hospital and community-acquired infections around the world due to its enhanced virulence and continuous development of