We reviewed the licensed antifungal drugs and summarized their mechanisms of action, pharmacological profiles, and susceptibility to specific fungi. Approved antimycotics inhibit 1,3-β-d-glucan synthase, lanosterol 14-α-demethylase, protein, and deoxyribonucleic acid biosynthesis, or sequestrate ergosterol. Their most severe side effects are hepatotoxicity, nephrotoxicity, and myelotoxicity. Whereas triazoles exhibit the most significant drug–drug interactions, echinocandins exhibit almost none. The antifungal resistance may be developed across most pathogens and includes drug target overexpression, efflux pump activation, and amino acid substitution. The experimental antifungal drugs in clinical trials are also reviewed. Siderophores in the Trojan horse approach or the application of siderophore biosynthesis enzyme inhibitors represent the most promising emerging antifungal therapies.

In acutely ill patients, particularly in intensive care units or in mixed infections, time to a microbe-specific diagnosis is critical to a successful outcome of therapy. We report the application of evolving technologies involving mass spectrometry to diagnose and monitor a patient’s course. As proof of this concept, we studied five patients and used two rat models of mono-infection and coinfection. We report the noninvasive combined monitoring of Aspergillus fumigatus and Pseudomonas aeruginosa infection. The invasive coinfection was detected by monitoring the fungal triacetylfusarinine C and ferricrocin siderophore levels and the bacterial metabolites pyoverdin E, pyochelin, and 2-heptyl-4-quinolone, studied in the urine, endotracheal aspirate, or breath condensate. The coinfection was monitored by mass spectrometry followed by isotopic data filtering. In the rat infection model, detection indicated 100-fold more siderophores in urine compared to sera, indicating the diagnostic potential of urine sampling. The tools utilized in our studies can now be examined in large clinical series, where we could expect the accuracy and speed of diagnosis to be competitive with conventional methods and provide advantages in unraveling the complexities of mixed infections.

The noninvasive differentiation of acute pyelonephritis (AP) and complicated urinary tract infections (cUTI), from acute cystitis (AC), represents an important diagnostic challenge in urology and nephrology. In a study of 95 adults, urine from patients with AP (n = 17), cUTI (n = 6), AC (n = 28), and controls (n = 44) was examined using a multidisciplinary approach: enzyme-linked immunosorbent assays for quantitation of acute phase proteins ceruloplasmin (Cp), pentraxin-3 (Ptx3), and lipocalin-2 (Lcn2); inductively coupled plasma mass spectrometry for determination of total copper (Cu), zinc (Zn), and iron (Fe) concentrations; and liquid chromatography coupled to electrospray ionization MS (LC-MS) for identification and quantitation of bacterial metallophores. Markedly elevated levels of human Cp, Ptx3, and Lcn2, all normalized to urine creatinine (Cr), in AC, cUTI, and AP patients compared to controls identified ongoing urinary tract infection without further differentiation between AC and AP. On the contrary, total urine copper normalized to Cr (Cu/Cr index) significantly differentiated AP from AC (p = 0.0209), as well as from the controls. Bacterial metallophores aerobactin and yersiniabactin, but not enterobactin, were indicators of AP, cUTI, and AC caused by Enterobacteriaceae. Importantly, the newly proposed combined test based on the quantitation of Lcn2 normalized to Cr (Lcn2/Cr) and Cu/Cr could noninvasively differentiate AP and cUTIs from AC with a diagnostic accuracy of 78% sensitivity and 65% specificity. The combination of characteristic elemental and molecular biomarkers may represent a future research direction with the potential to improve diagnostics of UTIs.

Scedosporium apiospermum and Lomentospora prolificans secrete siderophores (iron scavengers) during hyphal proliferation. Siderophores are virulence factors and potential clinical biomarkers of invasive scedosporiosis and lomentosporiosis. Both strains secreted a uniform spectrum of siderophores, including coprogen B (CopB), N ^α-methyl-coprogen B, dimethyl-coprogen, and ferricrocin, with N ^α-methyl-coprogen B being the fastest secreted and most abundant coprogen. Under iron and zinc restriction, reflecting a nutrient-limited host environment, L. prolificans secreted 45 times more CopB than did S. apiospermum, presumably contributing to its higher virulence. This robust mobilization of CopB was further enhanced by zinc surplus. Additionally, two novel cyclic peptides, Scedocyclin A and B, were characterized inScedosporium boydii using the de novo sequencing tool CycloBranch. Utilizing matrix-assisted laser desorption/ionization, the portfolio of coprogens detected had limits of detection and quantitation of 4.9 and 14.6 fmol/spot in complex matrices, respectively, making them strong candidates for the next-generation, routine diagnosis of invasive scedosporiosis and lomentosporiosis through the Biotyper siderotyping.

Experimental bacterial meningitis is often analyzed as microbial burden and inflammation, but the tissue-level organization of host-pathogen interplay remains poorly resolved. Here, we use multimodal imaging to map Escherichia coli meningoencephalitis as a spatially organized process across infected rat brain tissue and cerebrospinal fluid (CSF). A low-dose intracerebral inoculum expands within 24 h into an anatomically structured infection involving ventricular, meningeal, and perivascular interfaces. Scanning electron microscopy reveals biofilm-like multicellular E. coli aggregates embedded in extracellular material, consistent with neutrophil extracellular traps. MALDI mass spectrometry imaging detects aerobactin and salmochelin-derived metabolites, whereas intact enterobactin is not detected despite favorable analytical sensitivity. These bacterial iron-acquisition signals partially overlap with calprotectin proteoforms, defining ventricular and periventricular metal-conflict microenvironments. Spatial peptidomics identifies infection-enriched antimicrobial territories dominated by rat neutrophil peptides RatNP-2, RatNP-3, and RatNP-4. Endogenous proenkephalin-derived neuropeptides are reduced in basal ganglia regions. Finally, cerebrospinal fluid captures both bacterial siderophores and host antimicrobial peptides with lipocalin-2 protein, linking tissue-resolved host-pathogen chemistry to a proximal diagnostic fluid. Together, these data show that experimental E. coli meningoencephalitis is a spatially organized host-pathogen process. Bacterial communities, nutritional immunity, antimicrobial peptides, and neuropeptide networks occupy distinct but connected CNS niches, components of which are recoverable in CSF.