Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) represents a major public health threat. It is responsible for a broad spectrum of infections in both hospitalized patients and individuals in the community, encompassing conditions from mild, self-limiting skin infections to severe and potentially fatal diseases such as sepsis and pneumonia [Reference Tong1]. Treatment of MRSA infection is often complicated by resistance to multiple antimicrobial agents [Reference David and Daum2] or by the production of virulence factors, like Panton-Valentine leucocidin (PVL), which is frequently found in community-associated MRSA (CA-MRSA) strains [Reference David and Daum3]. PVL is a bi-component exotoxin targeting human neutrophils, implicated in severe skin and soft tissue infections (SSTIs) and, in rare cases, necrotizing pneumonia [Reference Saeed4]. In Pakistan, MRSA is a common cause of both hospital-acquired and community-acquired SSTIs [Reference Ahmad and Asrar5]. A high prevalence of PVL has previously been reported among MRSA isolates from SSTIs in Pakistan, with diverse MRSA clones carrying PVL identified, including epidemic CA-MRSA lineages CC8-MRSA-IV (“USA300”) and ST772-MRSA-V (“Bengal Bay Clone”) [Reference Madzgalla6].
A detailed description of the molecular structure of MRSA is essential to understand its global transmission and evolutionary dynamics [Reference Baede7]. Several molecular typing methods for MRSA typing have been developed, including multilocus sequence typing (MLST), SCCmec typing, and staphylococcal protein A (spa) typing. Among these, spa typing is particularly valued for its high discriminatory capacity, internationally standardized nomenclature, and relative cost effectiveness [Reference Stefani8].
As data on the clonal structure of MRSA circulating in Pakistan are scarce, the aim of this study was to investigate the prevalence of distinct spa types, SCCmec types, and the presence of PVL among clinical MRSA isolates obtained from SSTIs at the Lady Reading Hospital (LRH), Peshawar, Pakistan.
Methods
Collection of MRSA isolates
Non-repetitive MRSA isolates were obtained from pus and/or wound swabs originating from SSTIs in patients attending both inpatient and outpatient departments at Lady Reading Hospital (LRH), Peshawar, Pakistan, between August 2021 and May 2022, at the Department of Pathology. Following culture on blood agar, presumptive S. aureus colonies were identified using the catalase test, mannitol fermentation, the tube coagulase test, and a latex agglutination test (Staphaurex, Thermo Scientific). Methicillin resistance was initially screened by the cefoxitin disc diffusion test, and the minimum inhibitory concentration for cefoxitin was determined by the E-test. The presence of the methicillin resistance determinant mecA was confirmed using multiplex PCR [Reference Kondo9].
Antimicrobial susceptibility testing
Susceptibility to ten commonly used anti-staphylococcal agents was determined by the disc diffusion method in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines [10]. Isolates with intermediate susceptibility were classified as resistant. Quantification of multiresistance was based on the categorization of antimicrobial classes as defined for multidrug resistance in S. aureus [Reference Magiorakos11].
SCCmec typing
SCCmec typing of MRSA isolates (types I–VI) was performed using Multiplex PCRs as described previously by Kondo et al. [Reference Kondo9] (Supplementary Figures S1 and S2). The PCR reactions were carried out in the final volume of 50 μl, containing 2 μl template DNA, 10 μl primer mixture (10 μM), 25 μl Dream Taq Green PCR Master Mix (2x), and 13 μl nuclease-free water. Amplification involved an initial denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 2 min, annealing at 57°C for 1 min, and extension at 72°C for 2 min, with a final elongation at 72°C for 2 min. PCR products were separated on 1% agarose gel.
spa typing and PVL detection
Multiplex PCR was employed to detect the PVL and mecA and spa genes following the protocol of Stegger et al. [Reference Stegger12], with a modification (Supplementary Figure S3). Briefly, the PCR reaction was carried out in the final volume of 25 μl, containing 2 μl template DNA, 10 μl primer mix (10 μM), 11.5 μl of Qiagen Multiplex PCR Master kit, and 2.5 μl Milli-Q water. Amplification was performed in the T100 thermocycler (Bio-Rad, USA). The PCR products were separated on 1% agarose gel (E-Gel 48, Invitrogen, Grand Island, CA, USA).
spa typing was performed as described previously [Reference Harmsen13]. spa types of the MRSA isolates were determined using Ridom StaphType software; subsequently, all spa types were clustered into spa-Clonal Complexes (spa_CCs) using Based Upon Repeat Pattern (BURP) algorithm. MLST clonal complexes (CCs) were inferred from the results of spa typing.
Statistics
Statistical analyses were conducted to assess differences in both continuous and categorical variables. For continuous variables such as age, the distribution of values between groups was compared. Specifically, the Mann–Whitney U test was used to compare the distributions of age between two independent groups (e.g., patient age in PVL-positive vs. PVL-negative isolates). When comparing age across more than two groups (e.g., across MRSA clonal lineages), the non-parametric Kruskal–Wallis test was applied. Patients were stratified into four age groups: children (0–14 years), youth (15–24 years), adults (25–59 years), and the elderly (60+ years). Categorical variables, including the proportion of PVL-positive isolates, the distribution of patients across age groups, resistance phenotypes, and sex distribution, were compared using the chi-squared test. When the expected frequency in any cell was less than five, Fisher’s exact test was used instead. All tests were two-tailed, and a p-value <0.05 was considered statistically significant.
Ethic statement
The study was conducted as a laboratory-based investigation, and ethical approval or informed consent was not required. Basic demographic data (sex and age) were anonymized, and the result of the study has no impact on the patient treatment.
Results
MRSA isolates
Between August 2021 and May 2022, 154 non-repetitive MRSA isolates from inpatients and outpatients presenting with SSTI were collected. A total of 91 (59.1%) isolates were from males; and the median age of the patients was 24 years (ranging from 2 months to 86 years). Among the study population, there were 41 isolates from children (0–14 years), 37 from youth (15–24 years), 57 from adults (25–59 years), and 19 from elderly patients (60+ years). (Tables 1 and 2).
Table 1. Statistical analysis of patient age, gender, and antimicrobial resistance according to PVL status and clonal background
Demographic and antimicrobial resistance characteristics of MRSA isolates stratified by PVL status and by the three most common clonal lineages. Data are presented as numbers (%) for categorical variables and as averages (medians) for continuous variables. p values were calculated using chi-squared or Fisher’s exact test for categorical variables and Mann–Whitney U test or Kruskal–Wallis test for continuous variables, as appropriate. Multiresistance* is defined as non-susceptibility to at least one agent in three or more antimicrobial categories; the numbers represent the median number of antimicrobial categories to which the isolates were resistant. Statistically significant values are highlighted in bold.
IQR, interquartile range; PVL, Panton–Valentine leucocidin; MRSA, methicillin-resistant S. aureus.
Table 2. Distribution of antimicrobial resistance among MRSA isolates across different age groups
The association between age group and resistance to each antibiotic was assessed using a chi-squared test (or Fisher’s exact test when expected counts <5). Statistically significant values are highlighted in bold.
IQR, interquartile range.
Antimicrobial resistance
All isolates exhibited resistance to cefoxitin (MRSA), and the presence of mecA gene was confirmed by a multiplex PCR (Supplementary Figure S1). The MIC of cefoxitin ranged from 4 to >256 mg/L (Supplementary Table). Overall, a high level of resistance was observed. For six out of the ten antimicrobials tested, more than 50% of isolates were resistant. Isolates were frequently resistant to ciprofloxacin (n = 132; 85.7%), erythromycin (n = 117; 76.0%), sulfamethoxazole (n = 106; 68.8%), gentamicin (n = 106; 68.8%), fusidic acid (n = 89; 57.8%), and clindamycin (n = 65; 42.2%). Tetracycline resistance was present in 86 (55.8%) isolates; however, only 33 (21.4%) were resistant to doxycycline (Figure 1). Resistance to quinupristin/dalfopristin was rare (8 isolates; 5.1%), and no isolate was resistant to linezolid. The median number of antimicrobial classes to which the isolates were resistant was six. One isolate was resistant to all tested antimicrobials except linezolid. Conversely, three isolates were fully susceptible to all antimicrobials except for beta-lactams. Except for the high tetracycline resistance observed in children, there were no statistically significant differences in the distribution of resistance between age groups (Table 2).
Figure 1. The proportion of resistant isolates among the major MRSA lineages, Peshawar, Pakistan 2021-2022. Frequencies of antimicrobial resistance in the three dominant clones in comparison with all isolates. Abbreviation: CIP, ciprofloxacin; CN, gentamicin; DA, clindamycin; DO, doxycycline; E, erythromycin; FD, fusidic acid; SXT, sulphamethoxazole; QD, quinupristin/dalfopristin; TET, tetracycline. No isolate was resistant to linezolid.
Clonal structure
Among the MRSA isolates, four SCCmec types were identified: II (n = 4; 2.6%), III (n = 5; 3.3%), IV (n = 89; 57.8%), and V (n = 56; 36.4%). A total of 27 spa types, grouped into six spa_CCs (Figure 2 and Supplementary Table) and ten clonal complexes (CC1, CC1/ST772, CC5, CC8, CC8/ST239, CC22, CC30, CC88, CC121, CC361) were detected. Only three clonal complexes comprised more than 10 isolates: CC8 (n = 48); CC1/ST772 (n = 46); and CC1 (n = 32). These were assigned to 16 MRSA clones based on CC and SCCmec type. The three most frequent clones were CC8-MRSA-IV, CC1/ST772-MRSA-V, and CC1-MRSA-IV (Table 3). Statistical analysis indicated that CC1/ST772-MRSA-V isolates were resistant to fewer antimicrobial classes overall (p = 0.008). In particular, resistance to tetracycline (p < 0.001) and fusidic acid (p < 0.001) was less frequent in CC1/ST772-MRSA-V than in other lineages. Conversely, resistance to gentamicin (p < 0.001) and sulfamethoxazole (p = 0.005) was more common in CC1/ST772-MRSA-V, as well as in CC8-MRSA-IV, than in CC1-MRSA-IV. No significant differences were observed between the three clones with respect to patient gender, age, or age group. (Table 1).
Figure 2. Cluster analysis of MRSA isolates based on similarity of their spa types. spa-Clonal Complexes (CC) were identified using (BURP) algorithm (Ridom StaphType software) that clusters spa types into groups, based on the similarity of their repeat patterns. The maximum cost for distances was four, and spa types shorter than five repeats were excluded. The founder of the spa-CC is indicated in blue. The size of the circle is proportional to the number of isolates within the corresponding spa-CC.
Table 3. Clonal analysis of MRSA, Peshawar, Pakistan, 2021–2022
The table shows the distribution of MRSA clones in the study, along with their corresponding spa clonal complexes (spa_CC), spa types, and the proportion of PVL-positive isolates. spa types of isolates carrying PVL are in bold.
a Other and undetermined clonal complexes detected in this study include: CC8-MRSA-V (t304); CC121-MRSA-V(t314); CC8/ST239-MRSA-V (t030); CC88-MRSA-IV(t2526); CC361-MRSA-IV (t1309); CC5-MRSA-IV (t6100); undetermined (t363; t525).
CC8-MRSA
Overall, the most common MRSA clonal lineage detected was CC8-MRSA-IV (n = 42; 27.3%). The isolates belong to spa types t064 (n = 33), t008 (n = 7), and t304 (n = 2). PVL-negative t064 isolates resemble an American USA500 (CC8-MRSA-IV-t064) HA-MRSA clone, also by exhibiting a high level of antimicrobial resistance.
All seven t008 isolates were PVL-positive, resembling characteristics of the epidemic USA300 CA-MRSA clone. CC8-MRSA included four PVL-positive t008 isolates belonging to SCCmec type II and two PVL-negative t304 isolates with SCCmec type V.
CC1/ST772-MRSA
This CC was represented by PVL-positive isolates (n = 40) resembling ST772-MRSA-V, the Bengal Bay Clone, and a group of six PVL-positive CC1/ST772 isolates sharing the same spa types (t657 and t345) and spa_CC657 as CA-MRSA Bengal Bay Clone isolates, however, carrying SCCmec type IV.
CC1-MRSA
Distinct from the previous lineage, CC1-MRSA was represented by spa types belonging to spa_CC127: t114 (n = 1); t127 (n = 27); t177 (n = 1); t273 (n = 1); t398 (n = 1); and t1784 (n = 1). SCCmec type IV was detected in 25 isolates, none of which carried PVL, resembling the multidrug-resistant but PVL-negative CA-MRSA clone originating in the South-Eastern Europe (CC1-MRSA-IV-t127). Another six PVL-negative t127 isolates carried SCCmec type V, and a single PVL-positive t273 isolate was identified.
Other MRSA lineages
The remaining lineages were represented by seven or fewer isolates (≤4%). These included CC22-MRSA-IV (n = 4), similar to the EMRSA-15 (HA-MRSA) or Gaza Clone (CA-MRSA); CC8/ST239-MRSA-III (n = 4), the Brazil/Hungarian clone (HA-MRSA); PVL+ CC30-MRSA-V (n = 5), Western Australia (WA) MRSA-124 (CA-MRSA); and PVL+ CC30-MRSA-IV (n = 3), the Southwest Pacific clone (CA-MRSA) (Table 1).
PVL-positive isolates
Almost half of the isolates (n = 70; 45.5%) harboured PVL (Supplementary Table). The most common PVL-positive lineage was the Bengal Bay Clone (CC1/ST772-MRSA-V; n = 40), along with the related CC1/ST772-MRSA-IV (n = 6) and the USA300 clone (CC8-MRSA-IV; n = 7). Minor lineages among PVL-positive isolates included CC30-MRSA-IV/V (n = 8; t021, t1749, and t4109), CC8-MRSA-II (n = 4; t008), and isolates of t363 (n = 2), t273 (CC1-MRSA-V), t314, and t2526 (Table 1). PVL-positive isolates were statistically less resistant than PVL-negative isolates, showing an average resistance to five versus six antimicrobial classes, respectively. Specifically, PVL-positive isolates exhibited significantly lower frequencies of resistance to clindamycin, fusidic acid, and tetracycline. No significant differences were observed between the groups with respect to patient gender, age, or age group (Table 1).
Discussion
A high prevalence of methicillin resistance among S. aureus isolates from clinical samples was reported in studies from South Asia. In Pakistan, the MRSA prevalence ranges from 36.1% to 61.8% [Reference Idrees14–Reference Ullah16] and in India from 22.6% to 80.8% [Reference Chen and Huang17]. Previous reports on MRSA epidemiology in South Asia have documented the presence and the co-occurrence of multiple epidemic clones, including ST239-MRSA-III as the major healthcare-associated MRSA (HA-MRSA) clone and ST772-MRSA-V and ST22-MRSA-IV as major clones among CA-MRSA [Reference Chen and Huang17]. However, the number of studies on the molecular epidemiology of MRSA in Pakistan remains limited, often involving a small number of isolates. Here, we present data on the molecular epidemiology of a large, recent collection of 154 SSTI MRSA isolates collected in Peshawar, Pakistan, during 2021–2022.
In the present study, we applied spa typing and SCCmec typing to characterize the clonal structure of MRSA. We identified 27 distinct spa types, including 16 that, to our knowledge, are reported for the first time in Pakistan (t304, t1309, t6100, t223, t309, t363, t114, t1749, t177, t1784, t2526, t273, t3596, t398, t4109, and t525). Using SCCmec typing, the isolates were assigned to 16 MRSA clonal lineages.
The highest number of isolates belonged to CC8-MRSA-IV (n = 42). This lineage comprised a group of PVL-negative isolates, predominantly represented by spa type t064 (n = 33), which exhibited relatively higher levels of antimicrobial resistance compared with other MRSA isolates in this study, suggesting a possible healthcare-associated origin. Notably, the same spa type was reported as dominant among 26 isolates from Rawalpindi/Islamabad hospitals during 2006–2008 [Reference Arfat18]. However, more recent studies indicate that PVL-negative CC8-MRSA-IV was rare in Islamabad and Peshawar between 2015 and 2018 [Reference Khan19, Reference Monecke20]. The same characteristics – t064; SCCmec type IV; and resistance to gentamicin, tetracycline, and sulfamethoxazole-trimethoprim – are common in the USA500 clone, which is considered healthcare-associated [Reference Frisch21] but exhibits higher virulence potential than typical HA-MRSA clones [Reference Li22]. Another seven PVL-positive CC8-MRSA-IV isolates, all characterized by spa type t008, showed lower levels of resistance, with one isolate susceptible to all antimicrobials except β-lactams. These isolates resemble the epidemic USA300 CA-MRSA clone, the predominant CA-MRSA lineage in the United States [Reference David and Daum3], which has also been reported at high frequency in Central Europe [Reference Tkadlec23] and northern Taiwan [Reference Huang and Chen24].
The second-most common clone in this study, PVL-positive CC1/ST772-MRSA-V (n = 40), exhibited resistance to fewer antimicrobials compared with the other dominant clones identified. Nevertheless, these isolates were still frequently resistant to multiple agents, including ciprofloxacin, gentamicin, erythromycin, and sulfamethoxazole/trimethoprim – a resistance profile otherwise uncommon among CA-MRSA [Reference David and Daum3]. This clone represents a common South Asian CA-MRSA lineage, known as the Bengal Bay Clone [Reference Steinig25]. With a relatively low fitness cost, it has successfully combined the high virulence typical of CA-MRSA with resistance to multiple antibiotics, a trait more characteristic of HA-MRSA. Multiple introductions of this clone into other countries have been documented, often linked to travel or migration from South Asia [Reference Steinig25]. Unusually, six CC1/ST772-MRSA isolates carried SCCmec type IV. One such isolate, harbouring an SCCmec IV composite element, was identified among MRSA collected in 2017 in Rawalpindi, Pakistan [Reference Jamil26]. Interestingly, a large phylogenetic study of the ST772 lineage reported an isolate from Bangladesh, within the basal clade of the Bengal Bay Clone, which retained remnants of SCCmec type IV [Reference Steinig25].
PVL-negative CC1-MRSA-IV (n = 25) was the third-most common MRSA in this study, represented mainly by t127. This clone is present, albeit rare, in Pakistan [Reference Khan19, Reference Monecke20]. It is a multidrug-resistant CA-MRSA, originating in South-Eastern Europe, where it is endemic in countries such as Germany, Italy, Ireland, and Romania and has more recently spread to the Middle East [Reference Earls27].
Among the minor lineages detected in this study, ST239-MRSA-III (n = 4) represents the globally disseminated HA-MRSA clone previously reported in Pakistan [Reference Arfat18] and remains the predominant MRSA clone in Indian hospitals [Reference Abrudan28]. However, a recent study in hospitals in Afghanistan found it to be rare [Reference Naimi29]. Four PVL-negative CC22-MRSA-IV were detected, resembling EMRSA-15, a common European HA-MRSA clone [Reference Grundmann30]. PVL-negative CC22-MRSA-IV (t223) has also been reported as CA-MRSA in Russia [Reference Gostev31] and was recently found to be a common MRSA in a large multicentric study in India [Reference Abrudan28]. Two additional CA-MRSA clones detected in this study included PVL-positive CC30-MRSA carrying SCCmec IV (n = 3; Southwest Pacific CA-MRSA clone) and SCCmec V (n = 5; WA MRSA-124), which were recently reported as major MRSA lineages in Kabul, Afghanistan [Reference Naimi29].
The prevalence of PVL in this study was relatively high (45.5%), while an even higher prevalence of 70.8% PVL-positive MRSA was reported in Kabul, Afghanistan [Reference Naimi29]. In both studies, the isolates originated exclusively or predominantly (65.3%) from SSTIs. A high prevalence of PVL among MRSA from community-associated infections, mostly skin and soft tissue, was similarly reported in a European multicentric study [Reference Rolo32], highlighting the general association of PVL with SSTIs and the community setting. Interestingly, in Pakistan, as many as 71.0% of bloodstream MRSA isolates were PVL-positive in a recent study [Reference Monecke20], an observation that is otherwise uncommon among MRSA from infections other than community SSTIs [Reference Grundmann30, Reference Hetem33].
The high level of antimicrobial resistance observed among MRSA in this study, including atypically multidrug-resistant CA-MRSA clones such as CC1/ST772-MRSA-V and CC1-MRSA-IV, may be linked to the recent increase in linezolid consumption in Pakistan [Reference Mustafa34]. The excessive and inappropriate use of antibiotics in hospitals and by the community in Pakistan promotes the dissemination of multiresistant MRSA clones [Reference Mirha35]. Antimicrobial consumption, measured as Defined Daily Doses per 1,000 inhabitants per day, nearly doubled between 2019 and 2021 in the wake of the COVID-19 pandemic, with the highest levels recorded in the Peshawar region [Reference Mustafa34], where the present study was conducted. Frequently used antimicrobials – mostly administered orally – include beta-lactams, macrolides, sulphamethoxazole-trimethoprim, fluoroquinolones, and tetracyclines [Reference Mustafa34], corresponding to the agents to which resistance was commonly observed in our isolates.
The limitations of this study include the inability to analyse potential epidemiological links between MRSA cases or to determine the community versus healthcare origin of the isolates due to the lack of detailed clinical data. Additionally, as the isolates were collected from a single hospital, the findings may not fully represent the nationwide clonal distribution of MRSA.
In conclusion, our study provides a snapshot of the fragmented picture of MRSA epidemiology in Pakistan and South Asia. Taken together with other regional studies, Pakistan and neighbouring countries appear to be hotspots for the emergence of highly virulent, multidrug-resistant MRSA clones circulating in both community and healthcare settings. This is likely facilitated by insufficient epidemiological measures, high antimicrobial consumption, and dense human populations. Although multiple introductions of these clones to other countries have been documented, local spread has so far been limited. With the projected rise in global antimicrobial use [Reference Klein36], such clones could potentially disseminate widely, posing serious health risks due to limited treatment options. To mitigate this threat, systematic and detailed surveillance of MRSA epidemiology and more prudent antibiotic use in the region are urgently needed.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0950268825100575.
Data availability statement
All relevant data are provided in the tables and figures within the main manuscript and the Supplementary Material.
Author contribution
AU and DF conceived and designed the study; SFJ and MM performed the experimental work; AU, DF, JT, and SFJ performed analysis and interpretation of results; AU and MM wrote the initial draft; DF, SF, and JT performed the critical analysis of the initial draft.
All the authors participated in the manuscript review. All the authors approved the final version of the manuscript.
Competing interests
The authors declare none.
Funding statement
This work was supported by the Higher Education Commission of Pakistan under the international research support initiative programme and by the Ministry of Health of the Czech Republic in cooperation with the Czech Health Research Council under project no. NU23–09-00080.
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