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

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

Foz do Iguacu

Congreso; 32° Congresso Brasileiro de Microbiologia; 2023

Sociedade Brasileira de Microbiologia

The increase in antimicrobials prescription and use has resulted in the emergence of antimicrobial resistance amongst pathogens. The World Health Organization has classified Carbapenemase-Producing Enterobacteriaceae as the high priority for developing new and effective antibiotic treatments and combinations. Ilex paraguariensis St. Hil. (yerba mate) is an autochthonous species from Argentina, Brazil, Uruguay, and Paraguay. It has potential applications in the pharmaceutical industry, with multiple studies supporting its medicinal properties 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-30min (A), 50°C-60min (B), 80°C-30min (C), and 80°C-60min (D). Leaves were left to dry and crushed in a Wiley-type blade mill. Extracts were obtained by digestion in 96% hydroalcoholic solution, were concentrated in a rotary evaporator, and then dried entirely at 35 ± 2ºC. Drug synergism between extract and commercial antibiotics was determined by the double-disc test and 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 the commercial antibiotics tested. The untreated extract showed synergism with ceftazidime-avibactam, colistin, phosphomycin, imipenem, and meropenem, and treatments A and D showed synergism with aztreonam and phosphomycin, while treatments B and C showed synergism with ceftazidima-avibactam and phosphomycin against K. pneumoniae ATCC BAA-2342. Likewise, 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 with 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, while 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 with ceftazidime against P. aeruginosa (VIM). The in vitro drug synergism suggests that ethanolic I. paraguariensis extracts constitute an outstanding source for new antibacterial compounds, particularly in combination with commercially available antibiotics, and further studies should be carried out to understand their mechanism of action.