biostudies-literatureUnknown14(1)Markkanen MThe introduction of the first broad-spectrum antibiotics, sulfonamide drugs, fundamentally revolutionized medicine in the 1930s. Shortly after, and ever since, sulfonamide resistance genes (<i>sul</i> genes) have been widely detected. Still, the most recent variant of these genes, <i>sul4</i>, 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 <i>sul4</i> gene in wastewater. Furthermore, we extended our description of <i>sul4</i> carriers to previously published data sets. Our results indicate that <i>sul4</i> is prominently found in sludge and hosted by various bacteria, such asMicrobiology spectrume0085725https://www.ebi.ac.uk/biostudies/studies/S-EPMC12772301biostudies-literatureSulfonamide resistance gene &lt;i&gt;sul4&lt;/i&gt; is hosted by common wastewater sludge bacteria and found in various newly described contexts and hosts.PMC12772301Markkanen MKarkman AVirta MPezzutto DfalseSulfonamide resistance gene &lt;i&gt;sul4&lt;/i&gt; is hosted by common wastewater sludge bacteria and found in various newly described contexts and hosts.The introduction of the first broad-spectrum antibiotics, sulfonamide drugs, fundamentally revolutionized medicine in the 1930s. Shortly after, and ever since, sulfonamide resistance genes (<i>sul</i> genes) have been widely detected. Still, the most recent variant of these genes, <i>sul4</i>, 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 <i>sul4</i> gene in wastewater. Furthermore, we extended our description of <i>sul4</i> carriers to previously published data sets. Our results indicate that <i>sul4</i> is prominently found in sludge and hosted by various bacteria, such as2026-01-01T00:00:00Z2026 Jan2026-06-06T10:51:01.378Z2026-05-29T03:12:00.509ZS-EPMC127723014125871810.1128/spectrum.00857-25