Siderophore production by Enterobacter kobei an environmental isolate of selected Gram-negative bacteria

Antimicrobial resistance presents a significant challenge, emphasising the urgency for novel antibiotic development. A promising strategy involves utilising bacterial nutrient-import carriers, such as siderophore-dependent iron uptake pathways, for antibiotic delivery. This study explored the aquatic environment for siderophore-producing bacteria, predicting the low iron concentration and complex iron structures with organic ligands are special features of the aquatic ecology thereby a potential source for exploring siderophore-producing bacteria. Screening 110 bacterial isolates with Chrome Azurol S (CAS) assay identified 18 isolates further narrowed down to four isolates for in-depth analysis following 16S rRNA-based taxonomical identification.

Optimisation conditions evaluating different media, pH, temperature, cell density, incubation time, agitation, and iron content, as well as bacterial iron-reserves were meticulously assessed to determine the parameters that resulted in the highest siderophore production. Compared to other isolates in the cohort, the top-siderophore-producing strain, Enterobacter kobei (G59) was identified using whole genome sequencing (WGS). Further analysis revealed E. kobei (G59) harbours biosynthetic genes for both hydroxamate (aerobactin) and catecholate (enterobactin) siderophore. Advanced techniques, including high-performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS), confirmed aerobactin production, while enterobactin presence was confirmed through alternative methods such as thin-layer chromatography (TLC) and biochemical assays. Gene expression profiling via qPCR provided insights into siderophore biosynthesis regulation, particularly for the entA gene (enterobactin biosynthesis gene), under varying time intervals and iron levels. Comparative genetics analysis of E. coli and E. kobei (G59) uncovered evolutionary links in their siderophore production pathways, highlighting their shared use of enterobactin biosynthesis genes and receptors.

Notably, the study employed siderophore produced by E. kobei (G59) as an exogenous-siderophore source for an entA deleted E. coli mutant (ΔentA), which cannot produce the key precursor, 2,3-dihydroxybenzoic acid (DHB). Results showed that under iron-deficient conditions, the ΔentA strain could utilise non-self-siderophores from E. kobei (G59) for iron acquisition. This finding suggests potential therapeutic applications of using siderophore-mediated iron transport systems to enhance antibiotic delivery and efficacy.