en Anti-microbial agents, resistance and treatment protocols Anti-microbial agents, resistance and treatment protocols Original article Original article <div style="text-align: justify;"><b><span style="font-size:10.0pt"><span style="line-height:107%"><span new="" roman="" style="font-family:" times="">Introduction: </span></span></span></b><span style="font-size:10.0pt"><span style="line-height:107%"><span new="" roman="" style="font-family:" times="">The rising prevalence of extended-spectrum &beta;-lactamase (ESBL)-producing Gram-negative bacteria and carbapenem-resistant <i>Enterobacteriaceae</i> (CRE) pose significant threats to public health. <b>Methods: </b>This study investigated the prevalence and antibiotic resistance patterns of ESBL-producing Gram-negative bacteria isolated from various clinical samples in Peshawar, Pakistan. Out of 400 samples, 150 (37.5%) were ESBL-positive, including 50 (33.3%) from urine, 50 (33.3%) from indwelling devices, 25 (16.7%) from pus, and 25 (16.7%) from blood. <i>Escherichia coli</i> was the most prevalent species (57.3%), followed by <i>Klebsiella pneumoniae</i> (34.0%). Antibiotic susceptibility testing revealed that all ESBL-producing isolates (n=150) were resistant to ampicillin, cefoperazone, and ceftazidime, excluding intermediate results as per CLSI 2023 guidelines. PCR assays characterized &beta;-lactamase genes, with <i>bla-</i>CTX-M<i> </i>detected in 95.3% of ESBL-producing isolates, followed by<i> bla-</i>TEM (80.6%) and <i>bla-</i>SHV<i> (70.6%). </i>The Modified Hodge Test showed that 14.7% (22/150) of ESBL isolates were carbapenem-resistant, of which a concerning 86.4% (19/22) carried the<i> bla</i>NDM gene. <b>Results: </b>Statistical analysis revealed a significant association between sample type and ESBL positivity (&chi;&sup2;=16.67, <i>P</i>=0.0008), with the highest prevalence in urine samples. However, no significant difference in carbapenem resistance was found between <i>E.</i> <i>coli</i> and <i>K.</i> <i>pneumoniae</i> (&chi;&sup2;=0.0, <i>P</i>=1.000). The presence of the <i>bla</i>_NDM gene was very strongly associated with carbapenem resistance (&chi;&sup2;=101.42, <i>P</i><0.0001; Cramer&rsquo;s <i>V</i>=0.82). <b>Conclusion: </b>The high prevalence of ESBL-producing isolates, predominantly carrying the <i>bla</i>CTX-M gene, and the emergence of <i>bla</i>NDM-mediated carbapenem resistance in Peshawar highlight an urgent need for enhanced surveillance and targeted antimicrobial stewardship to guide effective treatment strategies and public health interventions.</span></span></span></div> &nbsp; Antimicrobial resistance, blaCTX-M, blaNDM, Carbapenem resistance, Escherichia coli, ESBL, Klebsiella pneumoniae, Modified Hodge Test, Pakistan, PCR 291 302 http://jommid.pasteur.ac.ir/browse.php?a_code=A-10-636-1&slc_lang=en&sid=1 Jawad Ahmad 0009-0007-4381-7263 No Institute of Biotechnology and Genetic Engineering, University of Agriculture, Peshawar, Pakistan Hammad Ahmed Abbasi hammadahmedabbasi403@gmail.com 0009-0004-1159-6337 Yes Institute of Biotechnology and Genetic Engineering, University of Agriculture, Peshawar, Pakistan Muhammad Atif 0009-0005-9323-6278 No Center for Applied Molecular Biology, University of the Punjab, Lahore, Pakistan Fawad Khan 0009-0005-8271-5177 No Center of Biotechnology and Microbiology, University of Peshawar, Peshawar, Pakistan