DOI: 10.6084/m9.figshare.26395601
title: Bacterial discrimination by Fourier transform infrared spectroscopy, MALDI-mass spectrometry and whole-genome sequencing

description: <b>Aim:</b> Proof-of-concept study, highlighting the clinical diagnostic ability of FT-IR compared with MALDI-TOF MS, combined with WGS. <b>Materials & methods:</b> 104 pathogenic isolates of <i>Neisseria meningitidis</i>, <i>Streptococcus pneumoniae</i>, <i>Streptococcus pyogenes</i> and <i>Staphylococcus aureus</i> were analyzed. <b>Results:</b> Overall prediction accuracy was 99.6% in FT-IR and 95.8% in MALDI-TOF-MS. Analysis of <i>N. meningitidis</i> serogroups was superior in FT-IR compared with MALDI-TOF-MS. Phylogenetic relationship of <i>S. pyogenes</i> was similar by FT-IR and WGS, but not <i>S. aureus</i> or <i>S. pneumoniae</i>. Clinical severity was associated with the zinc ABC transporter and DNA repair genes in <i>S. pneumoniae</i> and cell wall proteins (biofilm formation, antibiotic and complement permeability) in <i>S. aureus</i> via WGS. <b>Conclusion:</b> FT-IR warrants further clinical evaluation as a promising diagnostic tool. We tested a technique (FT-IR) to identify four different, common bacteria from 104 children with serious infections and compared it to lab methods for diagnosis. FT-IR was more accurate. We tested if it could identify subtypes of bacteria, which is important in outbreaks. It was able to subtype two species, but not the two other species. However, it is a much faster and cheaper technique than the gold standard. It may be useful in certain outbreaks. We also investigated the trends between genes and the length of hospital stay. This can support further laboratory research. As a fast, low-cost test, FT-IR warrants further testing before it is applied to clinical labs. FT-IR spectroscopy is a low cost, rapid vibrational technique that does not degrade the sample and provides a ‘molecular fingerprint’. This has been studied in other areas of life sciences, for example, environmental contamination, but not in pathogenic clinical isolates. A total of 104 clinical pathogens from serious bacterial infections in children from 4 species were studied. This is a proof-of-concept design to evaluate FT-IR diagnostic accuracy, clinical severity and ability to subtype pathogenic bacteria. FT-IR spectroscopy diagnostic accuracy was 99.6% compared with the currently used clinical technique of MALDI-TOF MS (95.8%) on purified isolates. Further research is required to assess this directly on patient samples. FT-IR analysis clustered <i>N. meningitidis</i> and group A <i>Streptococcus</i> in a similar manner to gold standard whole-genome sequencing. However, it produced significantly different phylogenetic relationships to WGS for <i>S. pneumoniae</i> or <i>S. aureus</i> subtypes. This is consistent with other laboratory strain studies and suggests FT-IR has limited application in outbreak settings, but may be beneficial in certain bacterial species as it is much more rapid than WGS. <i>S. aureus</i> WGS variants were significantly associated with longer length of hospital stay, after controlling for multiple comparisons, including known virulence factors (FnbA/B) and cell wall proteins (Ebh, SraP). Comparing <i>S. pneumoniae</i> WGS data with clinical metadata, genes involved in DNA repair mechanisms were found to be significantly associated with the need for non-invasive ventilation. Loss of a zinc ABC transporter lipoprotein was associated with a greater PICU length of stay and has been described a hyperencapsulated phenotype. In contrast with the <i>S. aureus</i> variants, these were typically more conservative. PLS-R modeling of FT-IR spectra with clinical demographics (e.g., age, length of stay) did not show significant correlation. However pediatric risk of mortality (PRISM) severity scores correlated with FT-IR spectra for those admitted to pediatric intensive care. FT-IR is a promising technique for clinical microbiology application and warrants further study in a wider range of pathogenic species, with antimicrobial resistance and direct patient specimens before it is routinely applied to clinical workflows.