Antibiotic resistance is a major global health concern that caused by the overuse and misuse of antibiotics. Mobile genetic elements have a roles in the transmission of antibiotic resistance genes. The distribution and diversity of antibiotic resistance genes and mobile genetic elements in the microbiome of Sof Umer Cave have yet to be explored. To map the distribution and diversity of antibiotic resistance genes and mobile genetic elements in the microbiome of Sof Umer Cave using high-throughput shotgun sequencing. High-molecular-weight DNA was extracted from homogenized sample using the GeneAll DNA Soil Mini Kit. Purified environmental DNA was sequenced using a NovaSeq PE150. Analysis of the pathogen host interaction database revealed the predominance of pathogenic organisms such as Xanthomonas oryzae, Acinetobacter baumannii, Erwinia amylovora, and Mycobacterium tuberculosis. Similarly, analysis of the virulence factor database confirmed the presence of Type IV pili (VF1240), lipopolysaccharides, capsules, heme biosynthesis (VF0758), and alginate. More than 800 antibiotic resistance genes were identified, with 50% related to glycopeptide resistance, followed by antibiotic resistance genes associated with multidrug efflux pumps (30%), aminoglycoside resistance genes (10%), and unknown genes. A variety of mobile genetic elements were also identified, highlighting their importance in the genetic diversity and adaptation of the microbiome of Sof Umer Cave. These findings underscore the importance of the Sof Umer Cave habitat as a reservoir for antibiotic resistance, emphasizing the need for ongoing monitoring to enhance the understanding and control of antibiotic resistance genes. • Mobile genetic elements play a significant role in the distribution and wide range of antibiotic resistance genes of Sof Umer Cave microbiomes. • The most prevalent pathogens in Sof Umer Cave are Xanthomonas oryzae, Acinetobacter baumannii, and Erwinia amylovora. • A total of over 800 antibiotic resistance genes were identified, including glycopeptide resistance (50%), efflux pumps (30%), aminoglycosides (10%), and unknown resistance mechanisms (10%).

Soda lakes are extreme saline–alkaline environments that harbor metabolically versatile microbial communities with significant biotechnological potential. This study employed shotgun metagenomics (NovaSeq PE150) to investigate the functional diversity and metabolic potential of microbial communities in Ethiopia’s Chitu and Shala Lakes. An analysis of gene content revealed 554,609 and 525,097 unique genes in Chitu and Shala, respectively, in addition to a substantial fraction (1,253,334 genes) shared between the two, underscoring significant functional overlap. Taxonomic analysis revealed a diverse phylogenetic composition, with bacteria (89% in Chitu Lake, 92% in Shala Lake) and archaea (4% in Chitu Lake, 0.8% in Shala Lake) as the dominant domains, alongside eukaryotes and viruses. Predominant bacterial phyla included Pseudomonadota, Actinomycetota, and Gemmatimonadota, while Euryarchaeota and Nitrososphaerota were prominent among archaea. Key genera identified in both lakes were Nitriliruptor, Halomonas, Wenzhouxiangella, Thioalkalivibrio, Aliidiomarina, Aquisalimonas, and Alkalicoccus. Functional annotation using the KEGG, eggNOG, and CAZy databases revealed that the identified unigenes were associated with various functions. Notably, genes related to amino acid, carbohydrate, and energy metabolism (KEGG levels 1–2) were predominant, indicating that conserved core metabolic functions are essential for microbial survival in extreme conditions. Higher-level pathways included quorum sensing, two-component signal transduction, and ABC transporters (KEGG level 3), facilitating environmental adaptation, stress response, and nutrient acquisition. The eggNOG annotation revealed that 13% of identified genes remain uncharacterized, representing a vast untapped reservoir of novel enzymes and biochemical pathways with potential applications in biofuels, bioremediation, and synthetic biology. This study identified 375 unique metabolic pathways, including those involved in pyruvate metabolism, xenobiotic degradation, lipid metabolism, and oxidative stress resistance, underscoring the microbial communities’ ability to thrive under fluctuating salinity and alkalinity. The presence of carbohydrate-active enzymes (CAZymes), such as glycoside hydrolases, polysaccharide lyases, and oxidoreductases, highlights their role in biomass degradation and carbon cycling. Enzymes such as alkaline proteases (Apr), lipases (Lip), and cellulases further support the lakes’ potential as sources of extremophilic biocatalysts. These findings position soda lakes as reservoirs of microbial innovation for extremophile biotechnology. Future research on unannotated genes and enzyme optimization promises sustainable solutions in bioenergy, agriculture, and environmental management.