An Optimized Checkerboard Method for Phage-Antibiotic Synergy Detection.

Phage-antibiotic synergy is a promising therapeutic strategy, but there is no reliable method for synergism estimation. Although the time-kill curve assay is a gold standard, the method is not appropriate for fast and extensive screening of the synergy. The aim is to optimize the checkerboard method to determine phage-chemical agent interactions, to check its applicability by the time-kill curve method, and to examine whether the synergy can be obtained with both simultaneous and successive applications of these agents. In addition, the aim is to determine interactions of the Pseudomonas phage JG024 with ciprofloxacin, gentamicin, or ceftriaxone, as well as the Staphylococcus phage MSA6 and SES43300 with ciprofloxacin, gentamicin, and oxacillin. The results show that the optimized checkerboard method is reliable and that results correspond to those obtained by the time-kill curve. The synergy is detected with the phage JG024 and ciprofloxacin or ceftriaxone against Pseudomonas aeruginosa, and the phage SES43300 with ciprofloxacin against MRSA. The synergy was obtained after simultaneous applications, and in the case of P. aeruginosa, after application of the second agent with delay of one hour, indicating that simultaneous application is the best mode of synergy exploitation for therapy. The checkerboard method can be used for thorough clinical studies on synergy and in the future for personalized therapy when infections are caused by multiple resistant bacteria.

Synergistic activity of bile salts and their derivatives in combination with conventional antimicrobial agents against Acinetobacter baumannii.

ETHNOPHARMACOLOGICAL RELEVANCE: Bile traditionally was used in wound healing, having erodent, antioxidant and antimicrobial potential. Acinetobacter baumannii is a frequent etiological agent of wound infections, exhibiting high level of resistance to conventional antibiotics. AIM OF THE STUDY: To determine the effect of selected bile acid sodium salts and their 3-dehydro (i.e. 3-oxo) derivatives, as well as their combinations with commercial antibiotics against A. baumanniia, to confirm bile ethnopharmacological application in wound healing from aspect of microbiology. MATERIALS AND METHODS: The sensitivity of reference and multidrug resistant (MDR) A. baumannii strains to bile salts, their derivatives and conventional antibiotics were examined by a microtiter plate method. The interaction of bile salts/derivatives and antibiotics was examined by a checkerboard method and time kill curve method. The interaction of bile salts with ciprofloxacin in terms of micelles formation was examined by DOSY NMR technique. RESULTS: The bile salts sodium deoxycholate (Na-DCA) and sodium chenodeoxycholate (Na-CDCA), as well as their derivatives sodium 3-dehydro-deoxycholate (Na-3DH-DCA) and sodium 3-dehydro-chenodeoxycholate (Na-3DH-CDCA), potentiate antibiotic activity and resensitize A. baumannii. The bile salts and their derivatives enhance A. baumannii sensitivity to antibiotics, particularly those that should penetrate cell to exhibit activity. The sodium salts of bile acid derivatives, namely Na-3DH-DCA and Na-3DH-CDCA, showed synergy against both reference and MDR strain in combination with ciprofloxacin or gentamicin, while synergy with gentamicin was obtained in all combinations, regardless of bile salt type and bacterial strains. The synergy with Na-3DH-CDCA was further confirmed by the time-kill curve method, as bacterial number decreased after 12 h. NMR experiment revealed that this bile salt derivative and ciprofloxacin form co-aggregates when bile salts concentration was higher than critical micelle concentrations (CMC), which indicate the possibility that bile salts enhance ciprofloxacin cell penetration by membrane destabilization, contributing to the synergy. CONCLUSION: The synergistic interactions between bile salts or derivatives with ciprofloxacin and particularly gentamicin represent a promising strategy for the treatment of A. baumannii wound infections.

Anti-Acinetobacter baumannii activity of selected phytochemicals alone, in binary combinations and in combinations with conventional antibiotics.

Acinetobacter baumannii is one of the emerging multidrug- and pandrug-resistant pathogens. The aim of the study was to determine anti-A. baumannii activity of selected terpenes, terpenoids and phenylpropanoids alone, in binary combinations, and in combinations with conventional antibiotics using microdilution-checkerboard and time-kill curve method. The most effective were terpenoids carvacrol (7.0-28.0 µg mL-1) and thymol (22.0-76.0 µg mL-1), as well as phenylpropanoid compound eugenol (90.5-304.0 µg mL-1), with the active concentrations were comparable to antibiotics. The binary combinations showed additive or indifferent effects. The combination gentamicin-carvacrol was synergistic only against reference strain (FICI = 0.50), while other combinations were additive. The best bacteriostatic activity showed carvacrol, thymol or eugenol in combination with ciprofloxacin (FICI range 0.11-0.50) against both the reference and multidrug-resistant strains. The synergistic effect was further confirmed by time kill curve method and obtained after only 15 h. The results indicate a new possible therapeutic strategy against multiple resistant A. baumannii.