PM₂.₅-Associated Variation in Airborne Antimicrobial Resistance Gene Abundance During Winter Smog Episodes in Urban Pakistan (2022–2023)

Abstract Airborne particulate matter (PM₂.₅) is a major component of winter smog and has been increasingly recognized as a potential carrier of microbial contaminants, including antimicrobial resistance genes (ARGs). However, limited data are available on the distribution of airborne ARGs associated with PM₂.₅ in urban environments of Pakistan. Objective This study aimed to quantify the relative abundance of clinically relevant ARGs in PM₂.₅-associated bacterial communities across three cities of Pakistan and to evaluate their association with air pollution and meteorological conditions. Methodology PM₂.₅ samples were collected from Lahore, Islamabad, and Okara during the winter months (November–December 2022 and 2023). External DNA (eDNA) associated with particulate matter was extracted and analysed using quantitative real-time PCR (qPCR). The relative abundance of selected ARGs ( sul1, blaTEM, blaCTX-M-32, blaNDM-1, vanA, vanRA, tnpA, intI1 , and blaTEM-F ) was determined using the 2^(-ΔCt) method and normalized against the 16S rRNA gene. Environmental parameters, including PM₂.₅ concentration, Air Quality Index (AQI), temperature, and humidity, were also assessed. Results Lahore exhibited the highest pollution levels, with PM₂.₅ concentrations reaching 276.6 ± 27.4 µg/m³ and AQI values up to 289.3 ± 42.7. PM₂.₅ showed strong positive correlations with AQI (r = 0.92) and relative humidity (r = 0.91) and a negative correlation with temperature (r = − 0.38). Distinct spatial variation in ARG relative abundance was observed across the study sites. The blaNDM-1 gene demonstrated the highest relative abundance, particularly in Lahore (0.077) and Islamabad (0.056), while vanA showed a comparatively higher abundance in Okara. Other ARGs, including blaTEM-F, sul1 , and intI1 , exhibited variable distribution patterns across cities, indicating heterogeneous environmental resistome profiles. Conclusion The findings reveal significant spatial variability in airborne ARG relative abundance associated with PM₂.₅ and highlight the influence of environmental conditions on resistome distribution. These results underscore the importance of monitoring airborne antimicrobial resistance in polluted urban environments and provide baseline data for future environmental and public health assessments.