The inhibitory effects of essential oil constituents against germination, outgrowth and vegetative growth of spores of Clostridium perfringens type A in laboratory medium and chicken meat

Highlights

• Cinnamaldehyde, eugenol and carvacrol inhibited C. perfringens spore germination.

• Cinnamaldehyde, eugenol, carvacrol and AITC blocked spore outgrowth in medium.

• All 4 tested EOCs arrested C. perfringens vegetative growth in rich medium.

• Among 4 EOCs, only AITC effectively inhibited the growth of spores in chicken meat.

Abstract

C. perfringens type A is the causative agent of C. perfringens type A food poisoning (FP) and non-food-borne (NFB) human gastrointestinal diseases. Due to its ability to form highly heat-resistant spores, it is of great interest to develop strategies alternative to thermal processing to inactivate C. perfrinegens. Thus, in this study we evaluated the inhibitory effects of essential oil constituents (EOCs) (cinnamaldehyde, eugenol, allyl isothiocyanate (AITC), and carvacrol) against germination, outgrowth and vegetative growth of spores of C. perfringens FP and NFB disease isolates in laboratory medium and chicken meat. The cinnamaldehyde, eugenol and carvacrol, but not AITC, all at 0.05–0.1%, inhibited the germination of spores of all tested C. perfringens isolates in Tripticase-glucose-yeast extract (TGY) medium. Furthermore, all tested EOCs at 0.05–0.1% arrested the outgrowth and vegetative growth of C. perfringens spores in TGY, with AITC and carvacrol being the most effective. However, among four tested EOCs, only AITC (at 0.5%–2.0%) was able to inhibit the growth of C. perfringens spores in chicken meat and no such inhibitory effect was observed even with a 10-fold higher concentration (5%) of carvacrol. In conclusion, our current work identified AITC as an effective EOC to control spores and vegetative cells of C. perfringens isolates in laboratory medium and chicken meat. Further studies on evaluating the effectiveness of different combination of EOCs against C. perfringens spore growth in different meat products should establish an effective use of EOCs to control the risk of C. perfringens-mediated illnesses.

Introduction

Clostridium perfringens is a gram-positive, rod-shaped, anaerobic, endospore forming bacterium, which is a causative agent of histotoxic and gastrointestinal (GI) diseases in human and animals (McClane et al., 2013, Petit et al., 1999). C. perfringens is classified into 5 types (A-E) and these types are established based on the production of four major toxins (alpha, beta, epsilon, and iota) (McClane et al., 2013, Petit et al., 1999). A small group (∼5%) of type A isolates, that produces C. perfringens enterotoxin (CPE), cause C. perfringens type A food poisoning (FP) and nonfood-borne (NFB) human GI illnesses such as antibiotic-associated diarrhea and sporadic diarrhea (Sarker et al., 1999). Previous studies have shown that C. perfringens isolates associated with FP typically carry the cpe on their chromosome (referred to in this study as FP isolates), whereas NFB human GI illnesses are linked to isolates carrying cpe on their large plasmid (referred to in this study as NFB isolates) (Collie and McClane, 1998, Cornillot et al., 1995, Sarker et al., 2000, Sparks et al., 2001). However, recent studies suggested that isolates carrying plasmid borne cpe also can be a causative agent for FP (Lahti et al., 2008, Lindström et al., 2011, Xiao et al., 2012).

Both FP and NFB isolates can produce metabolically dormant spores that are highly resistant to various stresses related to food preservation approaches (Li and McClane, 2006a, Li and McClane, 2006b, Paredes-Sabja et al., 2007). The remaining viable spores then germinate, outgrow and multiply in food products to a high level and cause disease after entering into GI tract through consumption of contaminated foods (McClane et al., 2013, Paredes-Sabja et al., 2008). Thus, one of the main goals of the food industry is to develop bacterial spore-inactivation strategies alternative to heat processing technologies, that meet consumer demand for ensured food safety, extended shelf life, and enhanced food quality. Previous studies in our laboratory demonstrated the inhibitory effects of polyphosphates, nisin, sorbate, benzoate, and chitosan against C. perfringens FP and NFB isolates (Akhtar et al., 2008, Alnoman et al., 2015, Alnoman et al., 2017, Udompijitkul et al., 2012). In laboratory conditions, these antimicrobial agents exhibited slight inhibition on spore germination and significant inhibition on spore outgrowth and vegetative growth of C. perfringens isolates. However, in meat model system, only polyphosphates and chitosan exhibited inhibitory activity against spore germination and outgrowth of C. perfringens isolates (Akhtar et al., 2008, Alnoman et al., 2017).

Alternative technologies also consider the use of natural antimicrobial compounds in foods such as spices and herbs (Sabah et al., 2003, Thippareddi et al., 2003, Valenzuela-Martinez et al., 2010). Essential oil constituents (EOCs) are natural, aromatic and volatile liquids extracted from plant material such as flowers, roots, seeds, leaves, peel and whole plant (Hyldgaard et al., 2012, Kuorwel et al., 2011). These are important for plant defense and considered to be secondary metabolites (Herman et al., 2013). Many EOCs have been accepted by the European Commission as flavoring agents in food products such as linalool, thymol, eugenol, carvone, cinnamaldehyde, vanillin, carvacrol, citral, and limonene. Also, the United States Food and Drug Administration (FDA) considers these EOCs and others such as, clove, oregano, thyme, nutmeg, basel, mustard, and cinnamon as 'generally recognized as safe (GRAS)' (Hyldgaard et al., 2012). Extensive research has been reported on the use of essential oils as antimicrobial agents against different spoilage bacteria (Burt, 2004, Oussalah et al., 2006, Smith-Palmer et al., 1998, Ultee et al., 2002) and their inhibitory activity has been assigned to a number of substituted aromatic molecules such as cinnamaldehyde, eugenol, allyl isothiocyanate (AITC), and carvacrol (Burt, 2004, Cha and Chinnan, 2004, Gutierrez et al., 2008, Kuorwel et al., 2011, Natrajan and Sheldon, 2000). Cinnamaldehyde, eugenol and AITC have been reported to inhibit the growth of Clostridium botulinum, Staphylococcus aureus, Escherichia coli 0157:H7 and Salmonella enterica serovar Typhimurium (Blaszyk and Holley, 1998, Burt, 2004, Cosentino et al., 1999). Carvacrol has been well examined for its antimicrobial activity against many food-borne pathogens such as E. coli, Listeria monocytogenes, Bacillus cereus, Salmonella spp, and Lactobacillus sakei (Nair et al., 2014, Natrajan and Sheldon, 2000, Smith-Palmer et al., 1998). Although two studies have been conducted on evaluating the inhibitory effects of EOC against growth of C. perfringens spores during abusive chilling of cooked ground beef and turkey (Junja et al., 2006, Junja and Friedman, 2007), each of those studies was limited to C. perfringens type A FP laboratory strains. Therefore, a detailed study on the effect of EOCs against a collection of enterotoxigenic C. perfringens type A FP and NFB clinical isolates both in laboratory conditions and in meat products is warranted. Moreover, the fundamental knowledge of whether EOCs inhibits initiation of spore germination or prevents outgrowth of germinated spores remains unclear.

The objectives of this study were to evaluate (1) the inhibitory effects of EOC (cinnamaldehyde, eugenol, AITC, and carvacrol) against spore germination, outgrowth and vegetative growth of C. perfringens FP and NFB isolates in laboratory medium, and (2) the effectiveness of EOC against growth of C. perfringens spores in cooked chicken meat during storage at extremely abusive conditions. We found that while all tested EOCs were effective against spore outgrowth and vegetative growth of C. perfringens in laboratory medium, only AITC could control the growth of C. perfringens in cooked chicken meat during storage at abusive conditions.