Downloaded from www.microbiologyresearch.org by IP: 23.20.70.183 On: Wed, 07 Sep 2016 02:11:41 Attenuation of Pseudomonas aeruginosa virulence by medicinal plants in a Caenorhabditis elegans model system Allison Adonizio, 1 Sixto M. Leal, Jr, 1 Frederick M. Ausubel 3 and Kalai Mathee 1,2 Correspondence Kalai Mathee Kalai.Mathee@fiu.edu 1 Department of Biological Sciences, College of Arts and Sciences, Florida International University, Miami, FL 33199, USA 2 Department of Molecular Microbiology and Immunology, College of Medicine, Florida International University, Miami, FL 33199, USA 3 Department of Genetics, Harvard Medical School, and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA Received 7 December 2007 Accepted 19 March 2008 Expression of a myriad of virulence factors and innate antibiotic resistance enables the opportunistic human pathogen Pseudomonas aeruginosa to create intractable infections. Using a nematode model, we screened for novel inhibitors of this pathogen. Aqueous extracts of three plants, Conocarpus erectus, Callistemon viminalis and Bucida buceras, were examined for their effects on P. aeruginosa killing of the nematode Caenorhabditis elegans. The results were evaluated in toxin-based and infection-based assays using P. aeruginosa strains PAO1 and PA14. The tested plant extracts prevented mortality via gut infection in approximately 60 % of the worms and caused a 50–90 % reduction in death from toxin production. All extracts inhibited nematode death by P. aeruginosa without host toxicity, indicating their potential for further development as anti-infectives. INTRODUCTION Pseudomonas aeruginosa is one of the leading pathogens among patients suffering from cystic fibrosis, diffused pan- bronchitis and chronic obstructive pulmonary disease (Hoiby, 1994; Lieberman, 2003; Registry, 2005). In addition, it remains one of the major causes of nosocomial infections (National Nosocomial Infections Surveillance System, 2004). The success of this organism is attributed to numerous virulence factors (Smith & Iglewski, 2003b; Tang et al., 1996), its ability to form biofilms (Costerton et al., 1995) and innate antibiotic resistance (De Kievit et al., 2001; Fisher et al., 2005). Conventional anti-pseudomonal treatment includes ele- vated doses of b-lactam, fluoroquinolone or aminoglyco- side antibiotics (Hauser & Sriram, 2005). However, these drugs possess a high degree of toxicity, and mucoid strains of P. aeruginosa are rarely eradicated by these treatments (Hauser & Sriram, 2005; Pedersen, 1992). The failure of existing antibiotics to control infection makes it crucial to find alternatives to currently available drugs. Since pathogenicity in many bacteria is regulated by quorum sensing (QS), or cell-to-cell communication, inhibition of this system can cause attenuation of virulence and protect against infection (Hentzer & Givskov, 2003; Juhas et al., 2005; Smith & Iglewski, 2003a). Plants have evolved numerous chemical strategies for deterring pathogen attack, including the production of bactericidal and anti-infective compounds, leading to their use as medicines (reviewed by Lewis & Ausubel, 2006). In our previous work, we demonstrated that a number of medicinal plants exhibit anti-QS activity (Adonizio et al., 2006). Extracts of these plants were later shown to have an effect on virulence factor production, biofilm formation, QS gene expression and autoinducer production in P. aeruginosa (Adonizio et al., 2008). In this study, we assessed the ability of three plant extracts to attenuate P. aeruginosa killing of the nematode Caenorhabditis elegans. Caenorhabditis elegans is well established as a pertinent and practical model for studying bacterial virulence (Darby et al., 1999; Tan & Ausubel, 2000), as a number of P. aeruginosa factors important in the killing of Caenorhabditis elegans are also relevant to mammalian systems (Rahme et al., 1995; Tan et al., 1999a). ‘Fast killing’ of Caenorhabditis elegans by P. aeruginosa strain PA14 (on rich media) is mediated through the production of virulence factors such as phenazines, whereas ‘slow killing’ (on minimal media) occurs via ingestion of the bacteria and subsequent infection (Mahajan-Miklos et al., 1999; Abbreviation: QS, quorum sensing. Journal of Medical Microbiology (2008), 57, 809–813 DOI 10.1099/jmm.0.47802-0 47802 G 2008 SGM Printed in Great Britain 809