Sulfonamide resistance gene sul4 is hosted by common wastewater sludge bacteria and found in various newly described contexts and hosts.

UNLABELLED: The introduction of the first broad-spectrum antibiotics, sulfonamide drugs, fundamentally revolutionized medicine in the 1930s. Shortly after, and ever since, sulfonamide resistance genes (sul genes) have been widely detected. Still, the most recent variant of these genes, sul4, was first described only in 2017, and its host range and transmission mechanisms are still largely unknown. Here, we applied PacBio long-read metagenomic sequencing and bacterial methylation signals to investigate the genetic contexts and bacterial carriage of the sul4 gene in wastewater. Furthermore, we extended our description of sul4 carriers to previously published data sets. Our results indicate that sul4 is prominently found in sludge and hosted by various bacteria, such as the species from the phyla Myxococcota and Chloroflexota and genera Trichlorobacter and Desulfobacillus, which are commonly found in activated sludge. Additionally, according to our results, sul4 has already spread into multiple strains of opportunistic human pathogens, such as Aeromonas and Moraxella, in addition to the previously described Salmonella. The sequence region flanking sul4 included a truncated folK gene and an ISCR28 element and exhibited a high degree of conservation across the investigated sequences. Furthermore, the module was associated with various integron integrase genes. Also, other mobility-related elements that could further increase the likelihood of sul4 mobilization were detected. Altogether, our results describing the sul4 hosts of bacteria from distant lineages indicate the efficient mobility of sul4 by genetic elements that traverse both clinical and environmental bacteria. Finally, we suggest that wastewater may provide favorable conditions for such horizontal gene transfer events.

IMPORTANCE: Antibiotic resistance is an ancient phenomenon and a common trait of many environmental bacteria. However, human activities in the post-antibiotic era, coupled with the bacteria's ability to exchange genetic material across different lineages, have drastically increased the spread of resistance traits among bacteria from various niches. The primary concern is the resistance genes encoded by infections causing pathogens, already causing over 1 million deaths annually and indirectly contributing to nearly 4 million more. Therefore, understanding the bacteria that harbor ARGs and the genetic mechanisms driving their mobilization is crucial for understanding the dynamics and emerging trends of resistance. Here, we focus on revealing these crucial aspects of the newly discovered sulfonamide resistance gene, sul4. Given the limitations of the metagenomic approach in linking the functional genes to their host genomes, the significance of our research lies in our workflow, which allows this linkage through the identification of shared methylation profiles.