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

ONETTO, ANDREA LILIANA; NOVOSAK, MARINA GISEL; DE LIMA, CARLOS JAVIER; CORTESE, ILIANA JULIETA; WINNIK, DANIANA; STOCKMANNS, PATRICIA ELIZABETH; OVIEDO, PATRICIA NOEMÍ; LACZESKI, MARGARITA ESTHER

Congreso; 32º Congresso Brasileiro de Microbiologia; 2023

The increase in antimicrobial prescriptions and use has resulted in the emergence of antimicrobial resistance among pathogens. The World Health Organization has classified Carbapenemase-Producing Enterobacteriaceae as a high priority for the development of new and effective antibiotic treatments and combinations. Ilex paraguariensis St. Hil. (yerba mate) is an autochthonous species in Argentina, Brazil, Uruguay, and Paraguay. It has potential applications in the pharmaceutical industry, and multiple studies supporting its medicinal and antimicrobial effects. The double-disc synergy assay (according to the CLSI guidelines with modifications) was used for preliminary detection of positive interactions between yerba mate extracts and commercial antibiotics against Klebsiella pneumoniae ATCC BAA-2342 (resistant to carbapenem antibiotics), K. pneumoniae ATCC 700603 (producing extended-spectrum β-lactamases), and three reference carbapenemase-producing strains: Providencia rettgeri (NDM, metallo-β-lactamase), Pseudomonas aeruginosa (IMP, metallo-β-lactamase) and P. aeruginosa (VIM, metallo-β-lactamase). Leaves and branches of yerba mate plants from Misiones, Argentina, were collected, scalded, and treated at different temperatures and times in a laboratory oven: untreated extract, 50°C for 30 min (A), 50°C for 60 min (B), 80°C for 30 min (C), and 80°C for 60 min (D). The leaves were dried and crushed in a Wiley blade mill. Extracts were obtained by digestion in 96% hydroalcoholic solution, concentrated in a rotary evaporator, and dried entirely at 35 ± 2ºC. Drug synergism between the extract and commercial 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, ceftazidime-avibactam 14 ug, fosfomycin 200ug, amikacin 30ug, minocycline 30ug, and ceftazidime 30ug (Britania S.A., Argentina) were used. The untreated extract exhibited synergism with ceftazidime-avibactam, 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, this effect was observed for treatment A with imipenem, treatment B with cefotaxime, and treatments A, B, and C with meropenem against K. pneumoniae ATCC 700603. Furthermore, drug synergism was detected in all extracts and amikacin, ceftazidime, and colistin, treatment A with aztreonam and ceftazidime-avibactam, treatment B with ceftazidime-avibactam, fosfomycin and imipenem, and treatments C and D with imipenem against P. rettgeri. All extracts demonstrated drug synergism with aztreonam, whereas the untreated extract showed activity with meropenem against P. aeruginosa (IMP). Untreated extract and treatment B showed synergism with aztreonam, and treatments A, B, and D showed synergism with ceftazidime against P. aeruginosa (VIM). The in vitrodrug synergism suggests that ethanolic I. paraguariensis St. Hil. extracts constitute an outstanding source of new antibacterial compounds, particularly in combination with commercially available antibiotics, and further studies should be conducted to understand their mechanism of action.