Paromomycin production from Streptomyces rimosus NRRL 2455: Statistical optimization and new synergistic antibiotic combinations against multidrug resistant pathogens

Abstract

Background: Response surface methodology (RSM) employing Box-Behnken design was used to optimize the environmental factors for the production of paromomycin, a 2 deoxystreptamine aminocyclitol aminoglycoside antibiotic, (2DOS-ACAGA) from Streptomyces (S.) rimosus NRRL 2455. Emergence of bacterial resistance caught our attention to consider the combination of antimicrobial agents. The effect of paromomycin combination with other antimicrobial agents was tested on some multiple drug resistant isolates. To the best of our knowledge, this is the first report on optimization of paromomycin production from S. rimosus NRRL 2455. A Quadratic model and response surface method were used by choosing three model factors; pH, incubation time and inoculum size. A total of 17 experiments were done and the response of each experiment was recorded. Concerning the effect of combining paromomycin with different antimicrobial agents, it was tested using the checkerboard assay against six multidrug resistant (MDR) pathogens including; Pseudomonas (P.) aeruginosa (2 isolates), Klebsiella (K.) pneumoniae, Escherichia (E.) coli, methicillin sensitive Staphylococcus aureus (MSSA) and methicillin resistant Staphylococcus aureus (MRSA). Paromomycin was tested in combination with ceftriaxone, ciprofloxacin, ampicillin/sulbactam, azithromycin, clindamycin and doxycycline. Results: The optimum conditions for paromomycin production were a pH of 6, an incubation time of 8.5 days and an inoculum size of 5.5% v/v using the optimized media (soybean meal 30 g/L, NH 4 CL 4 g/L, CaCO 3 5 g/L and glycerol 40 ml/L), 28 °C incubation temperature, and 200 rpm agitation rate that resulted in 14 fold increase in paromomycin production as compared to preliminary fermentation level using the basal medium. The tested antibiotic combinations showed either synergistic effect on paromomycin activity on most of the tested MDR pathogens (45.83%), additive effect in 41.67% or indifferent effect in 12.5%. Conclusion: RSM using multifactorial design was a helpful and a reliable method for paromomycin production. Paromomycin combination with ceftriaxone, ciprofloxacin, ampicillin/sulbactam, azithromycin, clindamycin or doxycycline showed mostly synergistic effect on certain selected clinically important MDR pathogens.