Antimicrobial synergy of Ilex paraguariensis St. Hil. leaf extracts against carbapenemase producing bacteria.

ONETTO, AL; STOCKMANNS, PE; NOVOSAK MG; DE LIMA, CJ; CORTESE, IJ; WINNIK, DL; OVIEDO, PN; LACZESKI, ME

Congreso; 32º Congresso Brasileiro de Microbiología -SBM; 2023

SBM

The increase in antimicrobial prescriptions and use has resulted in the emergence of antimicrobialresistance among pathogens. The World Health Organization has classified CarbapenemaseProducingEnterobacteriaceaeasahighpriorityforthedevelopmentofnewandeffectiveantibiotictreatmentsandcombinations.IlexparaguariensisSt.Hil.(yerbamate)isanautochthonousspeciesinArgentina, Brazil, Uruguay, and Paraguay. It has potential applications in the pharmaceuticalindustry, and multiple studies supporting its medicinal and antimicrobial effects. The double-discsynergy assay (according to the CLSI guidelines with modifications) was used for preliminarydetection of positive interactions between yerba mate extracts and commercial antibiotics againstKlebsiella pneumoniae ATCC BAA-2342 (resistant to carbapenem antibiotics), K. pneumoniae ATCC700603 (producing extended-spectrum β-lactamases), and three reference carbapenemaseproducingstrains:Providenciarettgeri(NDM,metallo-β-lactamase),Pseudomonasaeruginosa(IMP,metallo-β-lactamase)andP.aeruginosa(VIM,metallo-β-lactamase).Leavesandbranchesofyerbamateplants from Misiones, Argentina, were collected, scalded, and treated at differenttemperatures and times in a laboratory oven: untreated extract, 50°C for 30 min (A), 50°C for 60min (B), 80°C for 30 min (C), and 80°C for 60 min (D). The leaves were dried and crushed in a Wileyblade mill. Extracts were obtained by digestion in 96% hydroalcoholic solution, concentrated in arotary evaporator, and dried entirely at 35 ± 2ºC. Drug synergism between the extract andcommercial antibiotics was determined using the double-disc test and the disc diffusion method.Imipenem 10 ug, meropenem 10 ug, colistin 10 ug, tigecycline 15 ug, aztreonam 30 ug, ceftazidimeavibactam14 ug, fosfomycin 200ug, amikacin 30ug, minocycline 30ug, and ceftazidime 30ug(Britania S.A., Argentina) were used. The untreated extract exhibited synergism with ceftazidimeavibactam, colistin, phosphomycin, imipenem, and meropenem. Treatments A and D showed synergism with aztreonam and phosphomycin, whereas treatments B and C showed synergism with ceftazidima-avibactam and phosphomycin, against K. pneumoniae ATCC BAA-2342. Similarly, thiseffect was observed for treatment A with imipenem, treatment B with cefotaxime, and treatmentsA, B, and C with meropenem against K. pneumoniae ATCC 700603. Furthermore, drug synergismwas detected in all extracts and amikacin, ceftazidime, and colistin, treatment A with aztreonamand ceftazidime-avibactam, treatment B with ceftazidime-avibactam, fosfomycin and imipenem,and treatments C and D with imipenem against P. rettgeri. All extracts demonstrated drugsynergism with aztreonam, whereas the untreated extract showed activity with meropenem againstP. aeruginosa (IMP). Untreated extract and treatment B showed synergism with aztreonam, andtreatments A, B, and D showed synergism with ceftazidime against P. aeruginosa (VIM). The in vitro drug synergism suggests that ethanolic I. paraguariensis St. Hil. extracts constitute an outstandingsource of new antibacterial compounds, particularly in combination with commercially availableantibiotics, and further studies should be conducted to understand their mechanism of action.