Article pubs.acs.org/jnp
Serrulatane Diterpenoid from Eremophila neglecta Exhibits Bacterial Biofilm Dispersion and Inhibits Release of Pro-inflammatory Cytokines from Activated Macrophages Htwe H. Mon,†,‡ Susan N. Christo,†,§ Chi P. Ndi,† Marek Jasieniak,⊥ Heather Rickard,† John D. Hayball,†,§ Hans J. Griesser,‡,⊥ and Susan J. Semple*,† †
Sansom Institute for Health Research, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia ‡ Wound Management Innovation Cooperative Research Centre, Toowong, QLD 4066, Australia § Experimental Therapeutics Laboratory, Hanson Institute, Adelaide, SA 5000, Australia ⊥ Future Industries Institute, University of South Australia, Mawson Lakes, SA 5095, Australia S Supporting Information *
ABSTRACT: The purpose of this study was to assess the biofilm-removing efficacy and inflammatory activity of a serrulatane diterpenoid, 8-hydroxyserrulat-14-en-19-oic acid (1), isolated from the Australian medicinal plant Eremophila neglecta. Biofilm breakup activity of compound 1 on established Staphylococcus epidermidis and Staphylococcus aureus biofilms was compared to the antiseptic chlorhexidine and antibiotic levofloxacin. In a time-course study, 1 was deposited onto polypropylene mesh to mimic a wound dressing and tested for biofilm removal. The ex-vivo cytotoxicity and effect on lipopolysaccharide-induced pro-inflammatory cytokine release were studied in mouse primary bone-marrow-derived macrophage (BMDM) cells. Compound 1 was effective in dispersing 12 h pre-established biofilms with a 7 log10 reduction of viable bacterial cell counts, but was less active against 24 h biofilms (approximately 2 log10 reduction). Compound-loaded mesh showed dosagedependent biofilm-removing capability. In addition, compound 1 displayed a significant inhibitory effect on tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) secretion from BMDM cells, but interleukin-1 beta (IL-1β) secretion was not significant. The compound was not cytotoxic to BMDM cells at concentrations effective in removing biofilm and lowering cytokine release. These findings highlight the potential of this serrulatane diterpenoid to be further developed for applications in wound management.
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production of an exopolysaccharide matrix by the bacteria. This matrix has distinct physical and chemical characteristics, acting as a diffusion barrier or binding directly to antimicrobial agents, thereby preventing their access to bacterial cells within a biofilm.9−14 Another evolving idea includes the emergence of a biofilm-specific phenotype that results in the expression of active mechanisms, for example, multidrug efflux pumps, to combat the effect of antibiotic agents.15 Thus, the challenge remains to develop alternative strategies that take into account the features that make biofilms difficult to combat. Biofilms also complicate the wound-healing process by leading to prolonged inflammation at the wound site. Here, macrophages play a central role.16 The wide array of macrophage phenotypes and functionalities within the wound environment may contribute to its progression,17 for example,
he focus of treatments for infected skin wounds is to kill the infectious microorganisms present in the wound and to remove any dead tissue that may provide a favorable environment for microbial growth.1 Inflammation plays a crucial role in healing, and without inflammation it would be impossible for the wound to heal.2 Release of various cytokines and chemokines coordinates the attraction of macrophages to the wound area to remove dead tissue and to prevent bacterial infection.3,4 It has also been hypothesized that insufficiency in the initial immune response can allow bacteria to establish a biofilm community in the wound.5 Biofilms are difficult to remove, being less susceptible to host defenses and antibacterial agents. Evidence suggests that once biofilms are formed, the bacteria in the biofilm structure are from 10 to 1000 times more resistant to antimicrobial agents and are the root of persistent infections.6−8 The exact reasons that biofilm bacteria are less susceptible to host defenses and antibacterial treatments known to kill their planktonic counterparts remain unclear. A possible mechanism for antibiotic resistance in biofilms is the © XXXX American Chemical Society and American Society of Pharmacognosy
Received: September 16, 2015
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DOI: 10.1021/acs.jnatprod.5b00833 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
increased proportions of the pro-inflammatory or “M1” phenotype that may perpetuate the inflammatory response.18 The major player in this inflammatory phase is the release of cytokine molecules whose roles are to regulate and resolve the inflammation.19 Clinical strategies to improve wound healing include early use of antibiotics directed at planktonic forms of bacteria and to prevent or break up biofilms, enabling clearance by the host immune system.5 Strategies directed at preventing prolonged inflammatory responses may also be employed.5 Ideally, a better alternative to antibiotics would be a novel antibacterial that both disrupts biofilm, making it more amenable to removal, and also reduces the excessive inflammatory response. Plant compounds have been a focus of investigations to find new antimicrobial and antibiofilm drugs.20,21 They could possibly overcome the problems of using current antibiotics in wounds where resistance may develop.5,22 While a number of plant-derived antimicrobials have been found to inhibit bacterial biofilm formation, there are fewer examples that can remove biofilm once it is already established.20,21 Compounds that can remove existing biofilms are of interest because in many clinical settings therapeutic interventions would be commenced after a biofilm has already formed.20 Some plant species in the genus Eremophila (Scrophulariaceae) have been traditionally used by Aboriginal people in both coastal and central Australia.23 Several species in this genus have been used for the treatment of sores, wounds, and other symptoms indicative of bacterial infection and inflammation.23−25 Previous studies have identified a range of serrulatane diterpenoids as antibacterial compounds in Eremophila species.26−29 One of these serrulatanes, 8-hydroxyserrulat-14en-19-oic acid (1),30 was found previously by our group to be a major component responsible for activity of an extract of Eremophila neglecta J. Black (Scrophulariaceae) against Grampositive bacteria.30 A subsequent study by Nowakowska et al.27 suggested that compound 1 exhibited effects on bacterial membranes and inhibition of bacterial macromolecular biosynthesis. It was also shown that the compound decreased the rate of biofilm growth of adherent Staphylococcus epidermidis.27 Serrulatane diterpenes found in Eremophila species are structurally related to a group of potent anti-inflammatory compounds, pseudopterosins, isolated from Pseudopterogorgia species (sea whips).28,29 It has been speculated that serrulatanes may also have an anti-inflammatory activity.24 In fact, two serrulatane compounds from E. sturtii have been reported to exhibit some moderate inhibitory activity against the inflammation pathway enzymes cyclooxygenase (COX)-1 and -2.31 However, no published study has examined the effects of serrulatane diterpenoids in a cell-based model of inflammation relevant to wound healing including cytokine release. As part of investigations to develop new therapies to combat biofilm infections in wounds, we sought to further understand the impact of compound 1 on different stages of bacterial biofilm formation. We also investigated the effects of the compound on pro-inflammatory cytokine release and cytotoxicity in a cellular model relevant to wound healing.
determined using selected reference Gram-positive and Gramnegative bacteria (Table 1). All MIC values for reference GramTable 1. Antimicrobial Activity of 1 from Eremophila neglectaa microorganism Staphylococcus aureus (ATCC 25923) sensitivity testing control Staphylococcus aureus (ATCC 29213) sensitivity testing control Staphylococcus epidermidis (ATCC 35984) biofilm forming strain Staphylococcus aureus (ATCC 35556) biofilm forming strain Pseudomonas aeruginosa (ATCC 27853) sensitivity testing control Escherichia coli (ATCC 25922) sensitivity testing control
Gram-staining characteristics
1 MIC (MBC), μM
Gram-positive
41 (82)
Gram-positive
82 (82)
Gram-positive
82 (82)
Gram-positive
41 (41)
Gram-negative
NA
Gram-negative
NA
a
Values shown are MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration, in μM. NA: not active at maximum concentration tested (658 μM). MICs of levofloxacin (positive control) against the reference Gram-positive bacterial strains were
