Background Pythium insidiosum is a member of the oomycetes class of aquatic fungus-like microorganisms. It can infect humans and animals through skin wounds and the eyes, causing pythiosis, an infectious disease with high morbidity and mortality rates. Antifungal agents are ineffective as pythiosis treatments because ergosterol, the target site of most antifungal agents, is not found in the P. insidiosum cytoplasmic membrane. The best choice for treatment is surgical removal of the infected organ. While natural plant products or secretory substances from bacterial flora have exhibited in vitro anti- P. insidiosum activity, their mechanism of action remains unknown. Therefore, this study hypothesized that the mechanism of action could be related to changes in P. insidiosum biochemical composition (such as lipid, carbohydrate, protein or nucleic acid) following exposure to the inhibitory substances. The biochemical composition of P. insidiosum was investigated by Synchrotron radiation-based Fourier-transform infrared (FTIR) microspectroscopy. Results Fraction No.6 from the crude extract of P. stutzeri ST1302, fraction No.1 from the crude extract of K. pneumoniae ST2501 and xanthyletin were used as anti- P. insidiosum substances, with MFCs at 3.125, 1.57–1.91, 0.003 mg/ml, respectively. The synchrotron FTIR results show that the deconvoluted peak distributions in the amide I, amide II, and mixed regions were significantly different between the treatment and control groups. Conclusions Xanthyletin and the secondary metabolites from P. stutzeri ST1302 and K. pneumoniae ST2501 exerted anti- P. insidiosum activity that clearly changed the proteins in P. insidiosum . Further study, including proteomics analysis and in vivo susceptibility testing, should be undertaken to develop a better understanding of the mechanism of anti- P. insidiosum activity.

Pythiosis is a harmful disease caused by Pythium insidiosum, an aquatic oomycete. Therapeutic protocols based on antifungal drugs are often ineffective because the cytoplasmic membrane of P. insidiosum does not contain ergosterol. Therefore, the treatment of pythiosis is still challenging, particularly making use of natural products and secondary metabolites from bacteria. In this study, xanthyletin and substances obtained from Pseudomonas stutzeri ST1302 and Klebsiella pneumoniae ST2501 exhibited anti-P. insidiosum activity and, moreover, xanthyletin was non-toxic against human cell lines. The hyphae of P. insidiosum treated with these three substances exhibited lysis holes on a rough surface and release of anamorphic material. Therefore, xanthyletin could be considered a promising alternative agent for treating cutaneous pythiosis in the near future.