Response Surface D-Optimal Design for Optimizing Fortimicins Production by Micromonospora olivasterospora and New Synergistic Fortimicin-A-Antibiotic Combinations

Fortimicins (FTMs) are fortamine-containing aminoglycoside antibiotics (AGAs) produced by M. olivasterospora DSM 43868 with excellent bactericidal activities against a wide range of Enterobacteriaceae and synergistic activity against multidrug-resistant (MDR) pathogens. Fortimicin-A (FTM-A), the most active member of FTMs, has the lowest susceptibility to inactivation by the aminoglycoside modifying enzymes (AMEs). Therefore, this study aimed to evaluate the antibacterial activity of FTM-A alone or in combination with other antibiotics against 18 non-clonal clinically relevant MDR Gram-positive and Gram-negative pathogens. This study also aimed to statistically optimize various environmental factors affecting its production using the response surface D-optimal design. Results showed that FTM-A/meropenem combination showed the highest synergistic bactericidal activity (61.1%) followed by its combination with cefotaxime and cefepime (38.8% each). However, FTM-A/gentamicin and FTM-A/doxycycline combinations showed mostly additive effects in 66.6% and 50% of the tested isolates, respectively. For FTM-A production optimization, maximum specific activity (µg/mg) to cell growth was achieved using aminoglycoside production medium followed by yeast extract-malt extract and M65 production medium. A D-optimal quadratic model consisting of 27 different media composition variations was used to predict an optimal composition for FTM-A production and verified experimentally. Lab verification of the model was carried out using HPLC analysis, resulting in a 10.5-fold increase in their production compared to the un-optimized conditions. The model revealed that the initial pH, incubation temperature, and incubation time significantly affected FTMs production (P-value  0.05). In conclusion, the D-optimal design resulted in an effective model and optimized FTMs production on the shake flask level. FTM-A combinations with meropenem, cefotaxime, cefepime, and gentamicin showed mostly synergistic/additive effects and are advised for clinical evaluation.