Genetic Diversity of Staphylococcus aureus Strains from a Tertiary Care Hospital in Rawalpindi, Pakistan

Staphylococcus aureus is an important healthcare-associated bacterium that causes a multitude of infections in humans such as superficial skin and soft tissue infections, necrotizing pneumonia, foodborne illnesses and postsurgical infections. Treatment of S. aureus infections has become more complicated due to the emergence of Methicillin-Resistant Staphylococcus aureus (MRSA), some of which are multidrug resistant. The present study aimed to characterize S. aureus isolates from a tertiary care hospital in the Rawalpindi district of Pakistan. Staphylococci were isolated from 300 clinical samples collected from January 2018 to January 2019 and S. aureus isolates were tested for antimicrobial susceptibility and analyzed using Pulsed-Field Gel Electrophoresis (PFGE), Multi-Locus Sequence Typing (MLST), staphylococcal cassette chromosome mec (SCCmec) and spa typing. Approximately 25.3% (76/300) of the clinical samples were positive for S. aureus; of those, 88.2% (67/76) were mecA+ (MRSA). In addition to the β-lactam antibiotics, high levels of resistance were also found to the fluoroquinolones (ciprofloxacin, gatifloxacin and levofloxacin (73.7% each)). Of the 23 different spa types identified, the majority of isolates belonged to spa type t632 and t657 (9/66; 13.6% each spa type). ST772-t657 (Bengal Bay clone) was the most commonly identified clone in this study although other clones circulating around different regions of the world were also found indicating the diversity in MRSA isolates from this area of Pakistan. This study emphasizes the need to monitor MRSA in the clinical setting for improved infection control and treatment options.


Introduction
Staphylococcus aureus is a well-known commensal found on different parts of the human body such as skin, skin glands, nose and gut mucus membranes [1]. However, S. aureus is a major cause of skin diseases and invasive infections, such as endocarditis, pneumonia and osteomyelitis, in both healthcare and community settings. It has been estimated that 20% of humans are persistent nasal carriers for S. aureus, while 30% are intermittent and 50% are non-carriers for S. aureus [2]. Persistent and intermittent colonization increases the opportunity for infection, which is primarily caused by the same strains present as a commensal on an individuals' body [3][4][5]. Different infections, skin lesions and injuries, catheters, implants and chronic diseases (diabetes, AIDS, innate and acquired deficiencies of immune system) favor S. aureus infections [6,7].
Emergence of resistant phenotypes is usually linked with injudicious use of antimicrobial agents. A study by Schentag et al. (1998) reported changes in normal biota of a patient within 24-48 h under antibiotic pressure [8]. Some studies have supported causal relationships between antibiotic administration and emergence of Methicillin-Resistant S. aureus (MRSA) [9,10]. MRSA emergence was first observed in 1961, soon after the clinical application of penicillinase-resistant penicillin [1]. Patients infected with these resistant bacteria take longer to recover as compared to those infected with other staphylococcal species, especially those that are susceptible to antibiotics [9]. As such, MRSA isolates have been recognized as a source of infections with resistance to antibiotics in the β-lactam antibiotic class as a main characteristic contributing to its disease-causing ability along with other virulence factors in the bacterium [9].
The presence of mobile genetic elements also plays a major role in conferring resistance to antibiotics in MRSA such as resistance to vancomycin [7]. Mobile genetic elements include plasmids, transposons, bacteriophage and pathogenicity islands [5]. MRSA isolates also contain a mobile genetic element, Staphylococcal cassette chromosome (SCCmec), that may be horizontally disseminated among S. aureus isolates resulting in spread of antimicrobial resistance genes among the isolates [11]. SCCmec is composed of two parts, namely the mec gene complex and cassette chromosome recombinase (ccr) gene complex both of which contribute to production of different variants of MRSA.
Studies conducted in Pakistan in the last decade have reported high prevalence of MRSA [12][13][14][15][16]. Recent studies in our laboratory conducted on MRSA isolates from Peshawar and Malakand cities using microarray technology showed epidemiological links to the Middle Eastern/Arabian Gulf region [17,18]. However, available data for this region is still limited and there is a need for continued surveillance of S. aureus and characterization of isolates from local hospitals for control and better treatment options. The aim of the present study was to characterize clinical S. aureus isolates from a tertiary care hospital in Rawalpindi city of Pakistan. S. aureus from clinical samples were isolated and analyzed using antimicrobial susceptibility testing, presence of antimicrobial resistance genes, Pulsed-field Gel Electrophoresis (PFGE), Multi-Locus Sequence Typing (MLST), SCCmec and spa typing.

Sample Collection and Bacterial Identification
Three hundred clinical samples (urine, pus, tracheal tubes, vaginal swabs, body fluids, blood and cannula) were collected from January 2018 to January 2019 from Fauji Foundation hospital, Rawalpindi, Punjab, Pakistan. Samples were collected from patients for routine procedures to more urgent conditions including infected wounds, abscesses, burns and serious or life-threatening medical conditions. The samples were streaked onto Mannitol Salt Agar (MSA) using sterile cotton swabs and incubated at 37 • C for 24 h. For liquid samples (such as urine, blood, etc.), 50 µL of the sample was placed onto the surface of the MSA plate and then spread on the media surface using a sterile swab. Initial bacterial identification was performed by microscopy and biochemical tests (catalase test, tube coagulase test, mannitol fermentation and DNase). Confirmation of the isolates was performed by PCR using previously reported species-specific primers for S. aureus [19]. Pure cultures were preserved in glycerol stock at −80 • C. The work was approved by the Ethics Review Committee of Department of Microbiology of The University of Haripur.

Discussion
Like other geographical regions around the globe, MRSA is a serious health concern in Pakistan [28]. MRSA emergence was reported for the first time in the 1960s in the United Kingdom and since then its prevalence has been increasing throughout the world [8]. In the present study, high prevalence of MRSA (88%) among the collected S. aureus was observed while only 12% isolates were MSSA. Although high prevalence of mecA in clinical S. aureus isolates has also been reported in Pakistan previously, this work is one of those reporting the highest frequency of MRSA [15,17,28]. For example, a study conducted by Siddiqui et al. on clinical isolates in Karachi reported 52% prevalence of MRSA among S. aureus isolates, while 68% of the isolates were MDR S. aureus [29]. A similar study conducted in Peshawar city of Pakistan on clinical isolates by Ullah et al. reported prevalence of 36% [28]. Furthermore, a recent study conducted at a tertiary care hospital of Islamabad city of Pakistan reported 65% prevalence of MRSA isolates; 83% of those MRSA isolates were MDR S. aureus [13]. However, it is important to note that MRSA prevalence data may differ from town to town and even between hospitals of the same city [16]. Therefore, additional studies will provide needed data to inform decisions in specific clinical settings and to increase positive outcomes with infections caused by MRSA. The results of this study are alarming, since the majority of the isolates were resistant to drugs commonly used to treat S. aureus infections. Inappropriate use of antibiotics in Pakistan is widespread as physicians prescribe the antibiotics without performing sensitivity tests [30]. Self-medication along with inappropriate dispensing of antibiotics among patients are some of the other weaknesses in the clinical health system in Pakistan that further enhance the prevalence of antibiotic resistance [31,32]. In contrast to the previous reported results of different studies [12,33,34], no vancomycin resistant S. aureus were identified in the present study. Vancomycin intermediate resistant and vancomycin resistant S. aureus (VISA and VRSA, respectively) are a serious public health concern. In Pakistan, VISA was reported

Discussion
Like other geographical regions around the globe, MRSA is a serious health concern in Pakistan [28]. MRSA emergence was reported for the first time in the 1960s in the United Kingdom and since then its prevalence has been increasing throughout the world [8]. In the present study, high prevalence of MRSA (88%) among the collected S. aureus was observed while only 12% isolates were MSSA. Although high prevalence of mecA in clinical S. aureus isolates has also been reported in Pakistan previously, this work is one of those reporting the highest frequency of MRSA [15,17,28]. For example, a study conducted by Siddiqui et al. on clinical isolates in Karachi reported 52% prevalence of MRSA among S. aureus isolates, while 68% of the isolates were MDR S. aureus [29]. A similar study conducted in Peshawar city of Pakistan on clinical isolates by Ullah et al. reported prevalence of 36% [28]. Furthermore, a recent study conducted at a tertiary care hospital of Islamabad city of Pakistan reported 65% prevalence of MRSA isolates; 83% of those MRSA isolates were MDR S. aureus [13]. However, it is important to note that MRSA prevalence data may differ from town to town and even between hospitals of the same city [16]. Therefore, additional studies will provide needed data to inform decisions in specific clinical settings and to increase positive outcomes with infections caused by MRSA. The results of this study are alarming, since the majority of the isolates were resistant to drugs commonly used to treat S. aureus infections. Inappropriate use of antibiotics in Pakistan is widespread as physicians prescribe the antibiotics without performing sensitivity tests [30]. Selfmedication along with inappropriate dispensing of antibiotics among patients are some of the other weaknesses in the clinical health system in Pakistan that further enhance the prevalence of antibiotic resistance [31,32]. In contrast to the previous reported results of different studies [12,33,34], no vancomycin resistant S. aureus were identified in the present study. Vancomycin intermediate resistant and vancomycin resistant S. aureus (VISA and VRSA, respectively) are a serious public health concern. In Pakistan, VISA was reported for the first time in 2004 while VRSA was reported for the first time in 2009 [14,35]. Concern for emergence of VISA increased after the study conducted by Hakim et al. (2007) which reported an incidence rate of 13% of VISA isolates [33]. According to some of the studies conducted on usage of antibiotics in Pakistan, the most commonly used antibiotics are penicillin, cephalosporins, fluoroquinolones, aminoglycosides, ceftriaxone, cefradine, ciprofloxacin and tetracycline [32,[36][37][38], which would account for the resistance patterns among MRSA observed in this study.
The hospital sampled in this study receives patient samples from different neighboring cities, which was reflected in the genetic diversity of the population structure of the isolated strains. In the present study, MRSA isolates exhibited 23 different spa types. Although a paucity of data is available on spa typing in Pakistan, approximately 10 spa types from this study have been previously reported in other studies. For example, Syed et al. (2018) reported spa type t657 in Pakistan from table egg samples [39] while spa types t632, t030, t021, t008, t5414, t314, t345, t127 and t064 detected in the present study have also been reported in Pakistan, primarily from clinical samples [13,39,40]. To the best of our knowledge, no previous record is available for Pakistan on most spa types identified for the first time in this study (t657, t304, t1309, t223, t852, t345, t790, t525, t331, t10234, t14125, t711, t1991 and t6100).
ST772-CC1-t657 or the Bengal Bay clone was one of the most identified clones in this study. This clone was first reported in Bangladesh and has spread to other locations due to travel to India or contact with travelers to that region [41,42]. It is known to be endemic in regions around the Bay of Bengal and resulted in the discovery of its first clinical case which was associated with surrounding regions [18,43]. This clone was first classified as HA-MRSA and caused health care issues in multiple countries as defined in other studies [12,28,41,44,45]. It is now also associated with community infections and has linked Pakistan with the Arabian Peninsula epidemiologically as described previously [18]. The diversity of MRSA clones in this study may also be due to travel or the expansion of clones within the hospital or community. Two other prevalent clones in this study, ST239-CC8-t632 and ST239-CC8-t030, have also been recently identified as dominant epidemic MRSA clones from clinical specimens from hospitals in China [46]. According to that study, ST239-t030 has been a dominant epidemic clone in China for the last 20 years, displacing the previous dominant clone, ST239-t037. ST239 was also once considered to be hospital-associated MRSA (HA-MRSA), but it is no longer confined to hospitals only and has been reported as a commonly circulating strain in Middle East and Gulf countries [17,18].
Like many of the MRSA clones detected in this study, ST30-CC30-t021 has been described in different regions of the world and from various sources. For example, it was one of four major MRSA clades detected in Argentina between 2004 and 2015 while also circulating among a veterinarian and a dog from the same veterinary clinic in Japan [47,48]. Recently, the USA300 pulsotype (ST8-CC8-t008) has been detected in studies of clinical MRSA isolates from Pakistan [17,18,49]. Some of these same clones from Pakistan show a close epidemiological association of Pakistan with Arab Gulf countries and the Middle East, as approximately 3.6 million Pakistanis are employed there [17]. The proportion of CC5 and CC22 in isolates from the present study was 9.0% each while CC30 was the fourth most abundant clonal complex found. This clonal complex was reported for the first time in the UK but has now disseminated to Australia, Belgium, Canada, Greece, Finland, Denmark, Ireland, Spain, Sweden and the US [50]. The CC30/ST30 clonal type was also previously reported in Pakistan [51]; six isolates in the present study were found to belong to this clonal type. However, other MRSA clones reported previously in studies conducted in Pakistan, such as CC509 and CC6, were not found in this study [17,18].

Conclusions
Antibiotic resistance remains a serious health concern globally as well as in local hospitals. The results of the current study present an alarming situation as frequency of MRSA was very high among the S. aureus isolated. The genetic fingerprinting results indicate that the MRSA population of the present study is diverse and comprised of several global and regional clones. Monitoring evolution and circulation of antimicrobial resistant bacterial clones in the clinical setting allows for: (1) making informed decisions in prescribing the most effective antimicrobial treatment therapy for patients and (2) forming strategies to develop effective control measures to limit dissemination of these clones.  Institutional Review Board Statement: The study was approved by Advance Study and Research Board of the University of Haripur. Ethical review and approval were waived for this study, as the study did not require IRB approval since it did not involve experimentation on humans and the identity of the subjects whose samples were collected was kept confidential.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Each subject had the right to withdraw from participation at any time during the study. However, the identity of subjects whose clinical samples were used in the study was kept confidential.

Data Availability Statement:
Data is contained within the article.