Prayagraj, a city steeped in history and spirituality, famously hosts the Maha Kumbh Mela, drawing millions seeking purification in the confluence of its rivers. But beyond the visible throngs and ancient rituals, an invisible threat hangs in the city's air–Antimicrobial-resistant bacteria. Recent research from the Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj, explored the presence of antibiotic-resistant bacteria floating within the air people breathe across Prayagraj.
These airborne biological particles, collectively known as bioaerosols, are like tiny dust particles or mots, but they can carry living microorganisms, including bacteria. The concern arises when these bacteria are superbugs, meaning they've evolved to survive treatments with antibiotics that we rely on to fight infections. Understanding where these resistant bacteria linger in the air and their prevalence is crucial for safeguarding public health, especially in densely populated areas.
Scientists conducted an aerial survey during January and February 2021 to capture the airborne inhabitants across eleven distinct locations in Prayagraj. Their sampling sites were diverse, reflecting the city's varied environments: busy city crossroads, a municipal wastewater treatment plant (WWTP), a landfill area, religious sites like the Bade Hanuman Temple, the Ganga River Sangam (the confluence), parks, and near a hospital.
An air petri dish designed to gently suck a specific volume of air (500 litres) through a lid pierced with hundreds of tiny holes was used to collect samples. The air impacts a Petri dish containing a nutrient-rich jelly called Mueller Hinton Agar, which acts as a welcoming environment for bacteria to grow. These samples were carefully transported to the lab and incubated, allowing any captured bacteria to multiply and form visible colonies. By counting these colonies, researchers could estimate the concentration of bacteria in the air at each site, measured in Colony Forming Units per cubic meter (CFU/m³).
The results presented a varied picture of Prayagraj's aerial microbiome. The air wasn't uniformly infected; some places had significantly higher concentrations of airborne bacteria than others. The wastewater treatment plant emerged as a hotspot, with the highest bacterial count (around 1,500 CFU/m³). Wastewater is teeming with microbes, and processes like aeration can churn them into the air. Hospitals and landfill areas also showed elevated levels, likely due to waste handling and the potential presence of pathogens. Busy city crossroads, perhaps surprisingly, also harboured higher concentrations, possibly stirred up by traffic and large population densities. In contrast, the air near the Ganga River Sangam had the lowest bacterial load (around 140 CFU/m³), suggesting relatively cleaner air in that open, riverside environment. The researchers cultivated 129 distinct bacterial isolates from these diverse air samples.
The next step was to test these captured bacteria for antibiotic resistance. The researchers used a standard method called the Kirby-Bauer test. They spread each bacterial isolate onto a fresh agar plate and placed small paper discs, each soaked with a different common antibiotic, onto the surface. The antibiotics tested included workhorses like ampicillin, amoxicillin, tetracycline, and trimethoprim. After incubation, if a bacterium was sensitive to an antibiotic, it wouldn't grow near the disc, creating a clear zone of inhibition. But if it were resistant, it would grow unfazed up to the disc.
The test revealed that out of 129 isolates, 41 showed resistance to at least one antibiotic. The most common resistance was against trimethoprim, seen in over 23% of resistant isolates, followed by ampicillin and amoxicillin, around 10% and 9%, respectively. Worryingly, 12 isolates, or about 9.3% of the total, were multi-drug resistant (MDR), meaning they could fend off attacks from two or more different types of antibiotics. These are the superbugs that pose a significant treatment challenge. Encouragingly, no bacteria showed resistance to certain powerful antibiotics like gentamicin and ciprofloxacin in this study.
The researchers selected the 12 MDR isolates for genetic fingerprinting to identify the resistant bacteria. They extracted DNA from these bacteria and used a technique called PCR (Polymerase Chain Reaction), essentially a DNA photocopier, to amplify a specific gene called the 16S rRNA gene. This gene acts like a barcode for bacteria, allowing scientists to identify them by comparing their sequence to vast databases. The results revealed a dominance of the Bacillus genus, with eight of the twelve MDR isolates belonging to this group. These included species like Bacillus cereus and Bacillus anthracis. Bacillus species are known for forming tough spores, like seeds, that can survive harsh conditions, which might explain their prevalence in the air across various sites.
The remaining MDR isolates included Klebsiella aerogenes (one isolate) and three different Aeromonas species, found primarily in the air samples from the wastewater treatment plant. These Gram-negative bacteria (Klebsiella and Aeromonas) have thinner cell walls compared to Gram-positive Bacillus, making them generally less hardy in the environment but still significant, especially near sources like wastewater. Many of these identified species are known opportunistic pathogens capable of causing infections, particularly gastrointestinal and respiratory illnesses, in humans.
Beyond identifying the bacteria, the researchers also looked for the specific genetic instructions or the antibiotic resistance genes (ARGs) that give these bacteria their defensive capabilities. Using PCR again, they screened the MDR isolates with primers designed to find known ARG sequences. They found several genes responsible for resistance, particularly against β-lactam antibiotics, a major class including penicillin derivatives like ampicillin and amoxicillin. Genes like blaSHV, blaTEM, and blaKPC were detected. These genes are the blueprints for building the antibiotic-destroying enzymes or efflux pumps that protect the bacteria. They also detected intI1, an integron gene often associated with the capture and spread of multiple resistance genes. The presence of these specific ARGs, especially intI1, in airborne bacteria, particularly near the WWTP and crossroads, confirms that the air can act as a vehicle for spreading the genetic tools of resistance.
This study builds upon previous work globally that has highlighted wastewater treatment plants, landfills, and agricultural activities as sources of airborne ARB and ARGs. While researchers knew these bacteria could become airborne, this study provides crucial, location-specific data for Prayagraj, a major city with unique environmental pressures. It confirms that common urban environments, not just specialised facilities, contribute to the aerial load of resistant bacteria. Compared to some studies, like one in Poland, which found much higher rates of MDR bacteria (over 60%) in waste sorting plants, the 9.3% MDR rate found here might seem lower, but it still represents a significant presence of difficult-to-treat bacteria in public airspaces.
The identification of specific dominant genera (Bacillus widespread, Aeromonas and Klebsiella near WWTP) and the detection of key resistance genes like blaSHV and intI1 provide valuable molecular details about the nature of the resistance circulating in Prayagraj's air. The study acknowledges its limitations, including a relatively small sample size and incomplete identification for every single bacterial colony observed, meaning this is likely a snapshot rather than the full, complex picture.
The presence of pathogenic bacteria resistant to commonly used antibiotics means that simply breathing in certain areas could expose people to infections that are harder to treat. Workers at facilities like WWTPs and landfills and residents living near these hotspots or frequenting busy crossroads might face increased exposure. It underscores the urgent need for continuous air quality monitoring, including biological components like ARB and ARGs. It provides evidence for policymakers to strengthen regulations and improve management practices for wastewater treatment and solid waste disposal to minimise the release of these microbes into the air. Just as we monitor chemical pollutants, understanding the biological quality of our air, especially concerning antibiotic resistance, is becoming increasingly vital for protecting community health in Prayagraj and cities worldwide.
This research article was written with the help of generative AI and edited by an editor at Research Matters.