Hydrogen sulfide (H₂S) is a gas produced by bacteria in the gastrointestinal tract (GI) that has many unique properties and is a known gasotransmitter. Recently, a new breath test (BT) has been developed that can measure H₂S in addition to other bacterial gases. Here, we use shotgun sequencing to examine the duodenal microbiome in patients undergoing BT and determine whether H₂S gas levels correlate with specific H₂S producers.
Methods: Human subjects aged 18-85 years undergoing upper endoscopy without colon prep were recruited and duodenal aspirates were collected using a double-lumen sterile aspiration catheter. DNAs were extracted using MagAttract PowerMicrobiome DNA/RNA Kit. Libraries were prepared using Illumina DNA Prep kit and IDT for Illumina–DNA/RNA UD Indexes (Illumina). Shotgun sequencing was performed on a NovaSeq platform (Illumina), using the NovaSeq 6000 S2 Reagent Kit v1.5 (300 cycles). The Dragen Metagenomics Pipeline, using kraken2, was used for data analysis. Pathways were built based on metaseq. Breath H₂S levels (ppm) were measured by gas chromatography (Gemelli Biotech).
Results: Duodenal aspirates from 109 subjects (female=64%, age=56.6±15.8yrs, BMI=26.1±6.2kg/m²) were sequenced. The duodenal relative abundances (RA) of classic H₂S producers were found to be associated with BT findings. RA of Desulfovibrio desulfuricans was positively correlated with H₂S levels on BT (R=0.210, P=0.028), as did the RA of five other species from phylum Thermodesulfobacteriota and one from phylum Chrysiogenota, including Desulfobulbus oligotrophicus (R=0.202, P=0.035), Desulfobulbus oralis (R=0.194, P=0.043), Desulfoluna limicola (R=0.189, P=0.049), Desulfosarcina widdelii (R=0.243, P=0.011), Desulfurispirillum indicum (R=0.229, P=0.017), and Desulfuromonas sp. DDH964 (R=0.242, P=0.01). Subjects with 3 or more of these species had higher H₂S levels on BT compared to subjects with none of these (Fig 1). Another well-known H₂S producer, Proteus mirabilis, also correlated positively with H₂S levels on BT (R=0.313, P=0.001). Importantly, the sulfoacetaldehyde degradation IV pathway correlated with H₂S levels on BT (R=0.313, P=0.027). Moreover, one of the end products of this pathway is sulfite, and sulfite is converted to H₂S via the dissimilatory sulfate reduction pathway, which is present in D. desulfuricans. Lastly, sulfite is also an end product of the thiosulfate disproportionation IV (rhodanese) pathway, and the association between this pathway and H₂S levels on BT also approached significance (R=0.249, P=0.081).
Conclusions: The duodenum contains a number of H₂S producing bacteria. Both the RA of these species and the number of different H₂S producers present are associated with higher H₂S levels on BT. Understanding these H₂S producers and their locations in the GI tract may be important in future microbiome based treatments for GI illness.

<b>Figure 1.</b> Comparison of breath H<sub>2</sub>S levels in subjects (N=109) who have zero to three or more of the following H<sub>2</sub>S producers present in their small bowel (by shotgun sequencing): <i>Desulfovibrio</i> <i>desulfuricans</i>, <i>Desulfobulbus</i> <i>oligotrophicus</i>, <i>Desulfobulbus</i> <i>oralis</i>, <i>Desulfoluna</i> <i>limicola</i>, <i>Desulfosarcina</i> <i>widdelii</i>, <i>Desulfurispirillum</i> <i>indicum</i>, and<i> Desulfuromonas </i>sp. DDH964.

Figure 1. Comparison of breath H₂S levels in subjects (N=109) who have zero to three or more of the following H₂S producers present in their small bowel (by shotgun sequencing): Desulfovibrio desulfuricans, Desulfobulbus oligotrophicus, Desulfobulbus oralis, Desulfoluna limicola, Desulfosarcina widdelii, Desulfurispirillum indicum, and Desulfuromonas sp. DDH964.

Daniel Brimberry

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