Enhanced Inhibition of Listeria monocytogenes by a Combination of Cold Pressed Terpeneless Valencia Orange Oil and Antibiotics

Introduction

L isteria monocytogenes is a foodborne pathogen that causes a rare but serious illness, with case fatality rates for immunocompromised patients ranging from 20% to 30% (Ramaswamy et al., 2007). Listeriosis is typically treated with β-lactam antibiotics, such as ampicillin and penicillin, but treatment is not always effective (Bortolussi, 2008; Lungu et al., 2011). Furthermore, the long incubation period and nonspecific symptoms of infection make listeriosis challenging to diagnose, particularly in countries where listeriosis is not nationally notifiable and/or where its incidence is not well understood (Bortolussi, 2008; Huang et al., 2010; Laciar et al., 2011).

Antimicrobial resistance in human clinical strains is still rare (Hansen et al., 2005), but resistant strains have been found with increasing frequency in animals (Srinivasan et al., 2005). This finding is a cause for concern and suggests that resistance in clinical human isolates may emerge in the near future.

Therefore, this study sought ways to enhance effectiveness of conventional listeriosis treatments through combination with plant essential oils, specifically cold pressed terpeneless Valencia orange oil (CPTVO). Sensitization of pathogens to antibiotic treatments through use of compounds found in plant essential oils has proven effective for Staphylococcus aureus and Escherichia coli (Brehm-Stecher and Johnson, 2003). CPTVO has been shown to be effective against L. monocytogenes alone and in combination with organic acids and nisin (Friedly et al., 2009; Shannon et al., 2011). Here, CPTVO was tested in combination with the antibiotics penicillin and chloramphenicol, selected to represent two different mechanisms of action.

Methods

Commercially available CPTVO (Orange CP Val Terp. FAB 968611) was obtained from Firmenich (Lakeland, FL) and stored per manufacturer's directions at 4°C prior to use. L. monocytogenes cultures were obtained from the University of Arkansas Center for Food Safety culture collection (Table 1). Chloramphenicol susceptibility test disks (5 and 30 μg) and penicillin sensitivity disks (2 and 10 units) were obtained from VWR International (Radnor, PA). L. monocytogenes strains were grown statically in brain heart infusion (BHI; EMB Chemicals, Gibbstown, NJ) broth at 37°C for 18 h and used in a standard Kirby Bauer method (NCCLS, 2000). Mueller Hinton agar plates (Becton Dickinson, Sparks, MD) were allowed to dry for 30 min before L. monocytogenes cultures were inoculated onto individual plates to produce a lawn. Sterile tweezers were used to place the sterile 6-mm antibiotic discs in the center of the agar in each Petri dish. For combination treatments, 10 μL of CPTVO was added aseptically to the antibiotic disc. Blank sterile discs were used for the negative control (10 μL of sterile deionized water) and for CPTVO alone. Treatments were allowed to diffuse at room temperature for 30 min. After 48 h of incubation at 37°C, the diameter of the zones of inhibition (in mm) was measured. Data represent an average of 10 biological replicates.

Results and Discussion

In combination with CPTVO, both penicillin and chloramphenicol produced zones of inhibition that were greater than for either antibiotic alone (Tables 2 and 3). Individually, penicillin alone produced zones of inhibition ranging from <6 to 13.4 mm for 2 units, and from 16 to 19.5 mm for 10 units. In combination with CPTVO, 2 units of penicillin produced zones of inhibition of from 20.2 to 25.3 mm, while 10 units produced zones of inhibition of from 21.9 to 28 mm. Chloramphenicol at 5 μg made zones ranging from <6 to 6.9 mm, while at 30 μg the range was from 10.8 to 15.9 mm. In combination with CPTVO, 5 μg of chloramphicol produced a zone of inhibition in the range of from 22 to 26.6 mm, while 30 μg had zones in the range of from 19.5 to 23.9 mm. Therefore, CPTVO increased the effectiveness of both antibiotics against L. monocytogenes. This result was intriguing as it was initially suspected that increased inhibition would be observed for penicillin plus CPTVO (versus CPTVO plus chloramphenicol), because penicillin is a β-lactam (which destabilizes the cell by inhibiting the formation of peptidoglycan cross-links in the cell wall) (Strominger et al., 1959) and its mode of action is similar to the proposed mechanism of action for plant essential oils (i.e., destabilization of the cell via interference with the cell membrane) (Burt, 2004). In contrast, chloramphenicol's mechanism of action is inhibition of protein synthesis (Wisseman et al., 1954), which was not hypothesized to reveal a synergistic relationship when combined with cell membrane targeting CPTVO. However, it may be that CPTVO was able to sensitize L. monocytogenes to chloramphenicol, as sensitization has been observed in other CPTVO combination treatments (Shannon et al., 2011). In conclusion, the combination of conventional antibiotics with CPTVO increases their ability to inhibit L. monocytogenes. With further development, this approach to inhibiting L. monocytogenes may have the potential to improve the standard treatments for combating this pathogen.