Antibacterial efficacy of Rosmarinus officinalis phytochemicals against nosocomial multidrug-resistant bacteria grown in planktonic culture and biofilm

S. MORENO; E.M. GALVÁN; N. VÁZQUEZ; G. FIORILLI; P.A. CÁCERES GUIDO

The Battle Against Microbial Pathogens: Basic Science, Technological Advances and Educational Programs

A. Méndez-Vilas. Formatex Research Centre

Lugar: Badajoz; Año: 2015; p. 3 - 8

Phytochemicals with anti-infective properties can be of great significance in therapeutic treatments [1]. Most studies have been focused on the antimicrobial properties of plant compounds against planktonic cells, but there are limited reports evaluating their efficacy against biofilms that are more resistant to antimicrobial agents [2]. We reported previously the antibacterial activity of phytochemicals of Rosmarinus officinalis (culinary herb known as rosemary) [3,4,5]. Between them, carnosic acid (CA) which is the main diterpene of leaves, and the main constituent of rosemary essential oils, 1,8 cineole. These compounds inhibited important human pathogens as Staphylococcus aureus and Klebsiella pneumoniae, respectively, [6]. Here, we challenge these phytochemicals against planktonic and biofilm cells of nosocomial S. aureus methicillin resistant (MRSA) and carbapenemase-producing Klebsiella pneumoniae. Susceptible control strains were also tested. The antibacterial activity was study by the broth microdilution technique. The SYTOX green assay, a fluorescent nucleic acid stain that does not penetrate living cells, was used to assess the integrity of the bacterial plasma membranes. Biofilms were developed in vitro and evaluated for susceptibility to phytochemicals using the microtiter-plate test with the crystal violet assay. Different initial inoculum, incubation times and overall medium compositions which have a great impact on biofilm development, were evaluated. Results showed that CA was active against planktonic cells of nosocomial MRSA strains (minimal inhibition concentration, MIC=24-32 µg/ml), while 1,8-cineole inhibited planktonic nosocomial multidrug resistant-K. pneumoniae at 1 to 4% (v/v). A strong permeabilization effect on the plasma membrane of both non-resistant and multidrug-resistant K. pneumoniae was found using the SYTOX green assay after treatment with 1,8-cineole starting from 0.25% (v/v) for 24 h.In order to study the effect of phytochemicals on biofilm disruption, the amount of biofilm synthesized by each strain was assessed. Our data indicate that K. pneumoniae strains were higher biofilm producer in M9 minimal medium. Interestingly, a clear anti-biofilm effects were observed after 24 h-treatment with 1,8-cineole in a concentration-dependent manner (0.003 to 4%, v/v) against both susceptible and multidrug-resistant K. pneumoniae strains. In comparison, significant amount of staphylococcal biofilms were detected only in rich medium as Mueller Hinton (MH) and Tryptic Soy Broth (TSB) for the susceptible S. aureus ATCC 25923. Other data showed that several nosocomial SAMR isolates harbouring at least 6 antibiotic resistance genes were unable to synthesize significant amount of biofilm under the same conditions. In conclusion: our in vitro findings suggest that the rosemary phytochemicals studied here may be useful for the prevention and/or treatment of health care-associated MRSA and K. pneumoniae infections. In-vivo studies using animal models and clinical trials are needed to further evaluate the potential of these compounds as antibacterial agents.Keywords: antibacterial activity; Rosmarinus officinalis; Staphylococcus aureus; Klebsiella pneumoniae; biofilmReferences[1] Daglia M. Polyphenols as antimicrobial agents. Curr Opin Biotechnol, 2012. 23(2):174-81.[2] Malic S, Jordan RP, Waters MG, Stickler DJ, Williams DW. Biocide activity against urinary catheter pathogens. Antimicrobial Agent and Chemoterapy, 2014; 58: 1192-1194. [3] Moreno S, Scheyer T, Romano CS, Vojnov AA. Antimicrobial and antioxydant activities of argentinean Rosmarinus officinalis L. extracts. Free Radical Research, 2006; 40(2): 223-231.[4] Ojeda-Sana AM, van Baren CM, Elechosa MA, Juárez MA, Moreno S. New insights into antibacterial and antioxidant activities of rosemary essential oils and their main components. Food Control, 2013 ; 31: 189-195.[5] Ojeda-Sana AM, Repetto V, Moreno S. Carnosic acid is an efflux pumps modulator by dissipation of the membrane potential in Enterococcus faecalis and Staphylococcus aureus. World J Microbiol Biotechnology, 2013. 29: 137?144.[6] World health Organization, 2014. Antimicrobial resistance. Global report on surveillance. Available from: http://www.who.int/drugresistance/documents/surveillancereport/en/.