Methods

Study setting

This descriptive cross-sectional study was conducted on clinical specimens collected from patients attending selected hospitals in Hyderabad, Sindh, Pakistan, including Taluka Hospitals in Latifabad (Shah Bhittai), Kotri, and Qasimabad, as well as Liaquat University Hospital. All microbiological processing, antimicrobial susceptibility testing, data compilation, and analysis were performed at the Department of Physiology, University of Sindh, Jamshoro.

The study was carried out over a 1-year period from August 2023 to August 2024. All laboratory records were anonymized, and no personal identifiers were included at any stage.

Study population and sampling

The study included patients of all age groups, both outpatients and patients hospitalized for more than 48 hours, from major clinical units including the Medical, Surgical, Pediatric, Obstetrics and Gynecology departments; intensive care unit; and the cardiac care unit. Convenience sampling was employed by including all eligible clinical specimens received from the participating hospitals during the study period, based on their availability and predefined inclusion and exclusion criteria, without random selection.

Inclusion and exclusion criteria

Patients with clinical features suggestive of urinary tract, upper or lower respiratory tract, and gastrointestinal tract infections were included, as well as those with sepsis, wound infections, abscesses, pus discharge, prolonged fever, or other clinically significant signs of infection. Patients who had received antimicrobial therapy within 2 weeks before specimen collection were excluded to reduce false-negative culture results due to suppressed bacterial growth.

Specimen collection and transport

Clinical specimens included blood, urine, body fluids, sputum, tracheal aspirates, high vaginal swabs, pus, and wound swabs. Blood samples were collected aseptically and processed using the BacT/ALERT 3D automated blood culture system. Urine and other specimens were collected using standard aseptic techniques and transported in sterile containers. All procedures for specimen collection, handling, and transport followed Clinical and Laboratory Standards Institute (CLSI) guidelines (M40-A2 and GP41) [10,11].

Laboratory methods

Culture isolation and identification

Positive blood cultures and other specimens were inoculated onto appropriate selective and differential media, including MacConkey agar, CLED agar, Mannitol Salt agar, Blood agar, and Salmonella-Shigella agar (Oxoid, Basingstoke, UK), according to specimen type, and incubated aerobically at 37°C for 24 hours. Bacterial identification was performed using Gram staining and conventional biochemical tests. Gram-positive organisms were identified by catalase and coagulase tests, while Gram-negative organisms were identified using standard biochemical reactions, including lactose fermentation, oxidase, citrate utilization, hydrogen sulfide production, Triple Sugar Iron test, motility, indole, urease, and Voges-Proskauer tests, following routine clinical microbiology protocols.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed using the modified Kirby-Bauer disc diffusion method on Mueller-Hinton agar. Minimum inhibitory concentrations for selected antibiotics were determined by Mueller-Hinton broth dilution. Inoculum preparation, incubation, and interpretation were conducted according to CLSI guidelines (M100, 2023) and cross-checked with EUCAST 2023 breakpoints. Results were reported as susceptible, intermediate, or resistant based on standardized zone diameter and minimum inhibitory concentration criteria [12,13].

Resistance category definitions

Following the categorization of Magiorakos et al. [8], isolates showing resistance to at least one agent in three antimicrobial classes, in both Gram-positive and Gram-negative groups, were defined as multidrug-resistant (MDR). In S. aureus, MRSA isolates were classified as MDR due to resistance to oxacillin, penicillin, and cefoxitin, which indicates non-susceptibility to all β-lactam agents except anti-MRSA cephalosporins (ceftaroline/ceftobiprole), as per the MDR classification [8]. Extensively drug-resistant (XDR) was defined as resistance to one or more agents in all but two or fewer categories, whereas pandrug-resistant (PDR) was defined as resistance to all agents in all classes recommended by CLSI and EUCAST [8,12,13].

According to the Centers for Disease Control and Prevention (CDC), carbapenem resistance (CR) was defined as intermediate or resistant to one or more carbapenem agents in P. aeruginosa and A. baumannii, and resistant to E. coli, Klebsiella spp., and Enterobacter spp. (ertapenem only in Enterobacterales). ESCR included isolates resistant to one or more extended-spectrum cephalosporins (ceftazidime, cefepime, ceftriaxone, or cefotaxime). Fluoroquinolone resistance (FQR) was defined as resistance to one or more fluoroquinolones (ciprofloxacin, levofloxacin, or moxifloxacin) [14].

Difficult-to-treat resistance (DTR) applied to isolates that were intermediate or resistant to all tested agents within the following groups: carbapenems (imipenem, ertapenem, or meropenem; ertapenem only in Enterobacterales), β-lactams (ceftazidime, cefotaxime, cefepime, ceftriaxone, or piperacillin/tazobactam—excluding cefotaxime and ceftriaxone in P. aeruginosa), ampicillin/sulbactam (A. baumannii only), and fluoroquinolones (ciprofloxacin or levofloxacin) [9].

The term wild type was applied to isolates that did not exhibit any acquired resistance mechanisms phenotypically and were pansusceptible to tested agents, excluding those to which the species is intrinsically resistant [15]. In contrast, isolates resistant to all antibiotics tested in this study were designated as possible PDR. Since polymyxins and tigecycline were not included in the testing panel, the isolates could not be classified as true PDR but were designated as possible PDR according to standard definitions [8].

Statistical analysis

The data were sorted and extracted using Microsoft Excel 2024, and were then analyzed using SPSS version 25.0 software (IBM) to determine the percentages, differences, and categorization of resistant isolates.