Outbreak Investigation of Phytobacter diazotrophicus Using Fourier Transform Infrared Spectroscopy compared to cgMLST

Kulek, D. N.; Goncalves, G.; Silveira, M. C.; D?Espindula, H. R. S.; da Cunha Neto, J. F.; Lin, A. D.; Gaio, T. O.; Kraft, L.; Carvalho-Assef, A. P. D.; Mira, M. T.; Pillonetto, M.

doi:10.1101/2025.09.17.676977

bioRxiv · 10.1101/2025.09.17.676977

Outbreak Investigation of Phytobacter diazotrophicus Using Fourier Transform Infrared Spectroscopy compared to cgMLST

Kulek, D. N.·Goncalves, G.·Silveira, M. C.·D?Espindula, H. R. S.·da Cunha Neto, J. F.·Lin, A. D.·Gaio, T. O.·Kraft, L.·Carvalho-Assef, A. P. D.·Mira, M. T.·Pillonetto, M.

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Abstract

IntroductionPhytobacter diazotrophicus is an emerging opportunistic pathogen associated with hospital outbreaks and a reservoir of antimicrobial resistance genes; however, its clonal epidemiology remains poorly understood. Accurate identification and rapid clonal typing are crucial for infection control. Core genome MLST (cgMLST) is the genomic gold standard; however, it is both costly and time-consuming. Fourier-transform infrared (FT-IR) spectroscopy using the IR Biotyper(R) emerges as a rapid and accessible typing alternative. ObjectiveThis study evaluated the performance of the IR Biotyper(R) in discriminating P. diazotrophicus outbreak isolates, compared to cgMLST. MethodologyEight epidemiologically relevant isolates, including two from a total parenteral nutrition (TPN) outbreak and four from a hemodialysis clinic, along with two outliers, were analyzed. Its identification was confirmed by API20E, MALDI-TOF MS, nifL gene qPCR, and WGS. Both cgMLST, based on 7,755 conserved genes, and the Fourier Transform Infrared (FT-IR) Spectroscopy by IR Biotyper(R) were used for clonal typing, with the spectral cutoff empirically determined based on known clonal isolates. ResultsBoth methodologies demonstrated remarkable agreement in detecting clonal clusters. The two TPN outbreak isolates showed 23 allele differences (ADs) by cgMLST and consistently clustered according to the IR Biotyper. Similarly, the four isolates from the outbreak at hemodialysis clinic formed homogeneous clusters by both methods (5-27 ADs by cgMLST). The outlier isolates were consistently discriminated. ConclusionFT-IR spectroscopy (IR Biotyper(R)) proved promising and complementary to cgMLST for typing P. diazotrophicus in outbreak scenarios, offering a rapid and cost-effective alternative for detecting clonal events. Its applicability in routine epidemiological surveillance is significant. Statement of ImportancePhytobacter diazotrophicus is an emerging opportunistic pathogen of increasing concern in clinical environments, associated with hospital outbreaks and carrying antimicrobial resistance genes. Rapid detection and precise clonal characterization are essential for infection control and epidemiological surveillance. However, these are challenging for less-understood species. Genomic methods, such as cgMLST, are the gold standard, but their cost and time limitations hinder routine application in outbreak response. This study evaluated the IR Biotyper system, a rapid and accessible phenotypic tool that uses Fourier Transform Infrared Spectroscopy, compared to cgMLST for clonal typing of P. diazotrophicus. Our findings demonstrate a remarkable agreement between the two methods in identifying clonal clusters. This validation suggests the potential of the IR Biotyper as an effective tool for real-time screening and active surveillance of P. diazotrophicus in clinical settings. This enables a more agile response to outbreaks and aids in managing antimicrobial resistance, reserving high-resolution genomic analyses for more in-depth investigations.

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Kulek, D. N., Goncalves, G., Silveira, M. C., D?Espindula, H. R. S., da Cunha Neto, J. F., Lin, A. D., Gaio, T. O., Kraft, L., Carvalho-Assef, A. P. D., Mira, M. T., Pillonetto, M.. 2025-09-19. Outbreak Investigation of Phytobacter diazotrophicus Using Fourier Transform Infrared Spectroscopy compared to cgMLST. https://doi.org/10.1101/2025.09.17.676977

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