Profiling of volatile organic compounds in clinical isolate cultures and stools from children with <i>Clostridioides difficile</i> infection by HS-SPME-GC-MS

Bacterial volatile organic compounds (VOCs) have been investigated as a non-invasive approach to diagnosis of infectious diseases. Here, we aimed to explore potential diagnostic markers by profiling VOCs in cultures of unique clinical<i>Clostridioides difficile</i>isolates and stool samples from pediatric patients with<i>C. difficile</i>infection (CDI) using headspace solid-phase microextraction coupled with gas chromatography combined with mass spectrometry (HS-SPME-GC-MS). A total of 106 individual compounds were detected in 39<i>C. difficile</i>isolate cultures, of which 1-hexanol, ethanol and 4-methylvaleric acid were detected in all bacterial cultures, and 2-methyl-butanoic acid, 1-pentanol, 2-methyl-1-propanol,<i>p</i>-xylene and 6-methyl-2-heptanone were found in 38 (97.4%), 37 (94.9%), 34 (87.2%), 34 (87.2%) and 32 (82.1%) isolate cultures, respectively. The most abundant compound was 4-methylvaleric acid (relative abundance 14.71%, interquartile range 11.73%, 16.38%). A direct comparison of six paired isolates and stools revealed the transfer of<i>C. difficile</i>isolate VOCs to feces: ethanol was detected in all six pairs, 4-methylvaleric acid was in five pairs and 1-hexanol in four pairs. Fecal VOC patterns between the CDI children and healthy children were significantly different. Receiver operating characteristic analysis showed that 1-propanol, 6-methyl-2-heptanone, phenylethyl alcohol and ethanol presented the highest discrimination value for differentiating feces of CDI children from healthy children. Our data indicate that fecal 1-propanol, 6-methyl-2-heptanone, phenylethyl alcohol and ethanol may be used as potential screening biomarkers for diagnosing CDI.