Host-dependent resistance of Group A Streptococcus to sulfamethoxazole mediated by a horizontally-acquired reduced folate transporter - MS and NMR data

Metabolic profiling of MH-Bm and MH-Ox media. The differences in the susceptibility of TB08-2-14 to SXT on MHF-Ox and MHF-Bm media suggest differences in the composition of MH media from these different suppliers. To investigate this, we first examined the composition of MH base medium from each supplier using nuclear magnetic resonance (NMR) spectroscopy, revealing differences in the composition of MH base medium, with the MH-Ox medium containing higher levels of adenine, uracil and TRIS, while the MH-Bm medium contained higher levels of uridine, glucose and an unknown guanine moiety containing compound. However, we were unable to identify any major differences in folate pathway compounds. To further investigate the composition of MH-Ox and MH-Bm, untargeted metabolomics analysis was performed using LC-MS. As with NMR, LC-MS data showed differences in the medium following positive and negative ionisation, but did not detect differences in folate pathway intermediates. Untargeted LC-MS profiling followed by multivariate statistical analysis (principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) and database peak annotation revealed significant differences in a number of analytes, including higher levels of guanosine, uridine, methionine and peptides Pro-Ile-Ile and Pro-Val-Ile in the MH-Bm medium, with the MH-Ox medium containing higher levels of adenine, uracil, pyrrolidonecarboxylic acid and citric acid. Further comparison of spectra against reference standards for folate pathway intermediates revealed no significant differences other than a slight increase in 5,10-methylene-THF in MH-Bm. Thus, while there were major differences in the gross composition of MH media from each supplier, we were unable to identify substantial differences in the concentration of folic acid or reduced folate derivatives that might explain the relative performance of each media for detecting ThfT-mediated SMX resistance.

1H NMR sample preparation: 1H NMR was performed on a 600 MHz Bruker Avance III HD spectrometer, equipped with a 5 mm BBI probe and fitted with a Bruker SampleCase set to 6 °C. Time domain data were Fourier transformed and processed manually using Bruker TopspinTM 3.6.2 or Bruker TopspinTM 4.1.3 to obtain phase and baseline corrected spectra.

Standard preparation. Stock solutions were prepared at 1 mg/mL in DMSO, and further diluted to 10 ug/mL in methanol containing 0.1 % butylated hydroxytolunene (BHT) for LC-MS analysis. Sample preparation. Media samples were filtered and either directly injected for the detection of targeted tetrahydrofolic acid metabolites, or diluted 1 in 10 (v/v) with LC-MS grade water for untargeted profiling. Preparation was identical for both methods.