Discussion

This study provides a comprehensive overview of antimicrobial resistance trends among ESKAPEE pathogens isolated from diverse clinical specimens in Hyderabad, Pakistan. Although Escherichia coli is not formally classified within the ESKAPEE group, it was the predominant pathogen in this study, followed by K. pneumoniae, A. baumannii, P. aeruginosa, Enterococcus spp., S. aureus, and Enterobacter spp. These organisms are well recognized for their ability to acquire and disseminate resistance determinants, facilitating evasion of antimicrobial therapy [[16], [17], [18]]. The ESKAPEE pathogens were designated by the IDSA as major contributors to health care-associated infections [17] and were subsequently included among the World Health Organization priority pathogens requiring urgent antibiotic development [18]. Consistent with reports from Europe documenting high levels of resistance in E. coli, particularly to β-lactams, fluoroquinolones, aminoglycosides, and sulfonamides [18], our findings similarly demonstrate the predominance of E. coli in the local clinical setting.

E. coli exhibited high resistance to ampicillin (81.2%) and third-generation cephalosporins (68.7%), consistent with reports from South and Southeast Asia [19]. Carbapenems retained partial activity, with 11% to 18% resistance to imipenem and meropenem, while 25.7% of isolates were resistant to ertapenem, indicating early erosion of last-resort options. Based on Magiorakos et al. [8] and IDSA [9] criteria, 56.9% of E. coli isolates were MDR, 13.9% XDR, and 6.9% possible PDR, with CR, ESCR, FQR, and DTR rates of 25.7%, 54.2%, 55.6%, and 11.1%, respectively. These findings align with regional studies, including Hashem et al. [20] from Iraq, Camacho-Ortiz et al. [14] from Mexico, and Gondal et al. [21] from Pakistan, underscoring the persistent burden of multidrug and extended-spectrum resistance in E. coli and highlighting the emerging threat of carbapenem and fluoroquinolone resistance to empirical therapy and infection control.

K. pneumoniae demonstrated high resistance rates, particularly to β-lactam/β-lactamase inhibitor combinations (76.3%) and cephalosporins (81.6%), consistent with reports from Eastern India and the Middle East [22,23]. MDR, XDR, possible PDR, and DTR rates were 7.9%, 28.9%, 44.7%, and 18.4%, respectively, indicating a predominance of XDR and possible PDR strains. The XDR prevalence was comparable to that reported in Greece (26.8%) [24], while the MDR rate was lower than that reported from southeastern Romania (24.4%) [25]. Resistance phenotypes including CR (50%), ESCR (81.6%), FQR (71.1%), and DTR (18.4%) partially align with findings from China [25], while the FQR rate is similar to that reported in a recent study from Hyderabad (64.29%) [26]. Although CR rates were broadly consistent, higher ESCR and FQR rates and lower DTR rates in this study highlight regional differences in resistance dynamics. These findings underscore the global dissemination of ESBL- and carbapenemase-producing K pneumoniae, with concurrent ESCR and FQR phenotypes supporting plasmid-mediated spread of resistance determinants such as _bla_CTX-M, _bla_KPC, and qnr genes [27].

Enterobacter spp. were isolated in low numbers but displayed MDR (20%), XDR (60%), and possible PDR (20%) phenotypes, along with CR (40%), ESCR (60%), and FQR (40%). Comparable trends have been reported from a hospital in Mexico, with MDR (26.6%), ESCR (45.2%), and FQR (21.5%), although CR and DTR rates were notably lower (5.1% and 2.3%) [14], likely reflecting sample size or regional variation. No DTR isolates were identified in the present study. Despite the limited number of isolates, these findings indicate the potential emergence of highly resistant Enterobacter strains in clinical settings.

A. baumannii exhibited extensive resistance, with CR and ESCR each at 67.9%, FQR at 57.1%, and the highest DTR rate (32.1%) among ESKAPEE isolates. MDR, XDR, and possible PDR rates were 14.3%, 17.9%, and 32.1%, respectively, consistent with reports from Greece (XDR/PDR 34.3%) [24] and China (CR, ESCR, FQR, and DTR >80%) [28].

P. aeruginosa showed CR, ESCR, and FQR >50%, with DTR at 22.2%, and MDR, XDR, and possible PDR rates of 18.5%, 22.2%, and 33.3%, respectively, comparable to reports from Mexico, Lebanon, and Iran [14,29,30]. DTR and CR rates in Lebanon were 30% to 50% lower than those observed here, reflecting geographic variation and differences in nosocomial resistance gene prevalence. These findings align with global data identifying carbapenem-resistant Acinetobacter and Pseudomonas as major nosocomial pathogens, often associated with intensive care unit and ventilator-related infections, driven by antibiotic selection pressure and broad-spectrum agent use [31,32]. The emergence of possible PDR isolates among these species underscores their significant clinical threat, particularly in critical care settings.

S. aureus showed 65.2% resistance to oxacillin, indicating a high MRSA prevalence, along with 91.3% resistance to erythromycin and penicillin. MDR, XDR, and possible PDR rates were 73.9%, 4.3%, and 4.3%, respectively. Similar findings have been reported by Pandey et al. [33] from Nepal, with MRSA at 58%, MDR at 68.2%, and erythromycin resistance at 75%. Linezolid and vancomycin remained largely effective in the present study (91.3% and 73.9%), consistent with those reported by Pandey et al. [33], confirming their continued reliability against MRSA infections. MRSA-associated XDR prevalence reported in Indonesia and India ranged from 0.77% to 15.1%, supporting the XDR rate observed here [34,35] and reflecting regional variability likely driven by antibiotic overuse and inadequate antimicrobial stewardship.

Enterococcus spp. exhibited MDR in 66.7% and XDR in 12.5% of isolates, with vancomycin and linezolid resistance at 20.8% and 12.5%, respectively. These trends are comparable to findings from India, where MDR and XDR rates showed an inverse pattern (28.9% and 35.6%) [35], likely reflecting differences in VRE gene distribution. Vancomycin resistance was also reported at 20% by Pandey et al. [33], supporting regional patterns of VRE prevalence in E faecium across Asian clinical centers. The presence of VRE highlights the need for continued surveillance, given its potential as a reservoir for transmissible resistance genes.

ESKAPEE pathogens were most frequently recovered from urine samples (69.6%), particularly among females (34.3%) and patients over 65 years of age (46%). Similar patterns were observed in India, where urine was the predominant source and recovery was higher in females (55.1%) and older patients (>61 years, 16.4%) [33]. These findings indicate increased susceptibility in the elderly, likely due to comorbidities and greater antibiotic exposure, and highlight the urinary tract as a major reservoir for resistant Enterobacterales, facilitating potential transmission from the community to hospital settings.

The study underscores multiple drivers of escalating antimicrobial resistance in Pakistan, including unregulated antibiotic access, empirical prescribing, limited diagnostic capacity, and inadequate infection control. Previous studies have reported widespread dispensing of antibiotics without a prescription in community pharmacies across Pakistan. In one survey, 67% of individuals obtained antibiotics without a physician's prescription, highlighting weak regulatory enforcement and inappropriate antibiotic use [36]. Qualitative evidence from Pakistani migrants in New Zealand also indicates that antibiotics purchased in Pakistan are frequently carried abroad for subsequent self-medication, further illustrating their easy availability without prescription [37]. These practices collectively contribute to inappropriate antimicrobial use and increase the selection pressure for resistant organisms.

The rising prevalence of carbapenem resistance and possible PDR [8] among ESKAPEE pathogens emphasizes the need for urgent national and institutional interventions. These findings reflect regional and global resistance trends and call for integrated strategies combining molecular and epidemiological surveillance, antimicrobial stewardship, and public health education. Strengthening national AMR frameworks and enforcing rational antibiotic use are critical to preserving the efficacy of last-resort antimicrobials.