Temporal Dynamics of Microbial Community Composition and Antimicrobial Resistance in a Mass Gathering Setting Using Culturomics and Metagenomic Approaches.

Introduction: Antimicrobial resistance (AMR) is one of the major global concerns in the current scenario. Mass-gathering events in fast-developing and densely populated areas may contribute to antibiotic resistance. Despite meticulous planning and infrastructure development, the effect of mass gatherings on microbial ecosystems and antibiotic resistance must be investigated. This study used culture and metagenome-based methods to investigate and compare the bacterial diversity, AMR profile & mechanism of resistance for bacteria in water samples collected from the mass gathering event (2019 Prayagraj Kumbh Mela in Uttar Pradesh, India) with the control samples, collected during no mass gathering.

Methods: This study analyzed the water samples collected from a mass gathering event held in February 2019. Water samples collected in this study were grouped into "Test" (mass gathering event) and "Control" (no mass gathering event) groups. This study involved methods including culturomics, antibiotyping, phenotypic & genotypic identification methods, and metagenomics.

Results: There is a significant variation observed in the evenness and richness of bacterial diversity and MDR profile, expressed in terms of the relative abundance of the bacterial species between test and control samples. Out of the total multi-drug resistant (MDR) strains identified in the Prayagraj sample, the majority were derived from the test sample. A pathway-based analysis of MDR strains revealed the highest levels of acquired resistance were related to the inhibition of cell wall synthesis primarily in Pseudomonas spp., followed by resistance to protein synthesis and nucleic acid synthesis pathways. Additionally, the overall resistance profile of the test sample demonstrated a significantly elevated resistome for beta-lactams, particularly in the Pseudomonas spp. Additionally, several ESBL (Extended-spectrum beta-lactamase)-associated gene variants were identified. The test sample showed a two-fold increase in the prevalence and diversity of common beta-lactam gene variants in addition to the presence of unique variants. Using the metagenomics approach, we investigated the mechanism of antibiotic resistance, and it revealed a dominant trend in antibiotic efflux and inactivation pathways within both the test and control samples. Overall, the bacterial diversity, abundance (including AMR strains of human origin), and ARGs were relatively higher in the Test sample compared to the control sample which was collected 3 months after the mass gathering event.

Conclusion: Our study found significant variations in microbial communities, MDR strains, and ARGs due to environmental and human influences. Pseudomonas spp. was the most abundant MDR strain, primarily resistant to cell wall synthesis inhibitors. The test sample showed an increased resistome for beta-lactams, while the control sample had reduced bacterial species, ARGs, and MDR strains linked to human microflora. This shift could be due to the re-establishment of native bacterial communities in the Ganges River which may be attributed to its bacteriophage activity, biomolecules, and inherent antimicrobial properties. The study highlights the need for surveillance, monitoring AMR emergence to develop new strategies to combat it.