p-Cresol and C. difficile: A Love-Hate Story Revealed by Raman Spectroscopy

Abstract

Clostridioides difficile is known to produce p-cresol, a phenolic compound with selective antimicrobial properties, which may contribute to its competitive advantage within the gut microbiome. In this study, we investigated the interaction between Clostrioides difficile and Escherichia coli in coculture to assess the role of p-cresol in modulating interspecies dynamics. Raman spectroscopy was employed as a label-free, nondestructive analytical technique to profile the molecular signatures of both species in mono- and coculture. Excitation wavelengths at 244 and 532 nm were used to enhance complementary vibrational features, including those associated with aromatic compounds like p-cresol. Our results demonstrate distinct spectral changes in coculture conditions suggesting the involvement of p-cresol and its impact on the biochemical composition of E. coli. This dual-wavelength Raman approach offers a powerful means of characterizing microbial interactions and identifying metabolic markers that may drive microbial competition and survival.

1. Co-Culture Plating Arrangement for E. coli (Horizontal) and C. difficile (Vertical)

1

Mean UVRR spectra of E. coli monocultures cultivated either aerobically (green) or anaerobically (blue) and C. difficile (red) on BHI agar supplemented with (a) p-cresol, (b) p-HPA, (c) l-tyrosine, and (d) control. The inset gives a detailed view of the 1500–1300 cm^–1 region, specifically highlighting the peaks at 1478 cm^–1 and 1364 cm^–1.

2

Mean Raman spectra of E. coli monocultures cultivated either aerobically (green) or anaerobically (blue) and C. difficile (red) on BHI agar supplemented with (a) p-cresol, (b) p-HPA, (c) l-tyrosine, and (d) control. Raman spectra were recorded using an excitation wavelength of 532 nm. The inset gives a detailed view of the 1500–1300 cm^–1 region, specifically highlighting the peaks at 1478 and 1364 cm^–1.

3

(A) 244 nm and (B) 532 nm LD plots of monocultures of E. coli (EC) cultivated either aerobically (blue) or anaerobically (green) and C. difficile (CD; red) on supplemented BHI agar.

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Mean Raman spectra of aerobic E. coli cultivated in liquid BHI agar supplemented with varying concentrations of p-HPA (left) and p-cresol (right), recording after 3h (red) and 6h (blue) of incubation.

5

Mean UVRR spectra (A) and mean Raman spectra (B) of E. coli (EC) and C. difficile (CD) cocultures. LD plots of the cocultures measured using excitation wavelengths of (C) 244 nm and (D) 532 nm. The insets give a detailed view of the 1600–1300 and 1500–1300 cm^–1 regions, specifically highlighting the peaks at 1589, 1478 cm^–1 and 1364 cm^–1.

6

(A) UVRR and (B) 532 nm LD plots of monocultures grown on supplemented BHI agar (tetrahedron) and cocultures (sphere) of C. difficile (CD; red) and E. coli (EC; blue).