Interactions of Foodborne Pathogens with Free-living Protozoa: Potential Consequences for Food Safety Mario J.M. Vaerewijck, Julie Bar´e, Ellen Lambrecht, Koen Sabbe, and Kurt Houf Abstract: Free-living protozoa (FLP) are ubiquitous in natural ecosystems where they play an important role in the reduction of bacterial biomass and the regeneration of nutrients. However, it has been shown that some species such as Acanthamoeba castellanii, Acanthamoeba polyphaga,andTetrahymena pyriformis can act as hosts of pathogenic bacteria. There is a growing concern that FLP might contribute to the maintenance of bacterial pathogens in the environment. In addition to survival and/or replication of bacterial pathogens in FLP, resistance to antimicrobial agents and increased virulence of bacteria after passage through protozoa have been reported. This review presents an overview of FLP in food-associated environments and on foods, and discusses bacterial interactions with FLP, with focus on the foodborne pathogens Campylobacter jejuni, Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes. The consequences of these microbial interactions to food safety are evaluated. Keywords: Acanthamoeba, bacteria–FLP interactions, food safety, foodborne pathogens, free-living protozoa (FLP), Tetrahymena Introduction 1978) and Legionella pneumophila (Rowbotham 1980) in amebae. Food safety is a major subject of public concern. Food com- In the meantime, intraprotozoan survival and replication is well panies have to implement food safety systems such as GMP and documented for L. pneumophila, the causal agent of Legionnaires’ HACCP to meet the legal requirement for safe food and to gain disease (a severe pneumonia which can be lethal) and Pontiac consumer confidence. Such systems are based on the control by re- fever (a milder respiratory illness without pneumonia). L. pneu- duction or, preferably, elimination of hazardous physical, chemical, mophila survives in biofilms but is also able to multiply in FLP and biological agents before or during food processing. However, such as the amebae Acanthamoeba castellanii and Vermamoeba vermi- the presence or even persistence of microbes, including foodborne formis (= formerly Hartmannella vermiformis) (Abu Kwaik and oth- pathogens, in food processing industries cannot always completely ers 1998; Taylor and others 2009; Hilbi and others 2011; Richards be excluded. Moreover, microorganisms often develop dense and and others 2013). L. pneumophila and amebae are both found in complex communities despite hostile treatments, such as cleaning natural and man-made systems such as drinking water supplies, and sanitizing, and can survive environmental stresses such as low whirlpools, cooling towers, and air conditioning devices. People temperatures and desiccation. Important microbial response mech- become sick by inhalation of tiny water droplets containing free L. anisms to survive these adverse conditions are induction of cold- pneumophila bacteria or legionellae-laden vesicles expelled by ame- shock proteins, altered membrane compositions (Beales 2004), and bae. Noteworthy, legionellae inside amebae are protected against growth in biofilms, which act protectively against antimicrobial antimicrobial agents and are actively spread by motile protozoans. agents (Bridier and others 2011). Several foodborne and waterborne pathogens have been shown to During the last 4 decades, increasing attention has been paid benefit from FLP in a similar way. Consequently, FLP are now not to the interactions of pathogenic bacteria with free-living proto- merely considered as predators of bacteria but their potential role zoa (FLP) (amebae, flagellates, and ciliates) and to the potential in survival and protection of pathogenic bacteria is increasingly role of FLP in the maintenance of pathogens in the environment. recognized. FLP, which act as hosts of (foodborne) pathogens, are The first studies reported observations on engulfment and re- considered as “reservoirs” for these bacteria (Brown and Barker tention of mycobacteria (Jadin 1975; Krishna Prasad and Gupta 1999). L. pneumophila developed an escape mechanism to avoid digestion by amebae and applies several steps of this pathway in human alveolar macrophages, which suggests that amebae may MS 20140351 Submitted 3/3/2014, Accepted 18/5/2014. Authors Vaerewijck, Bare,´ Lambrecht, and Houf are with Dept. of Veterinary Public Health and Food also serve as “biological gyms” (Harb and others 2000), “evolu- Safety, Ghent Univ., Belgium Author Sabbe is with Laboratory of Protistology and tionary cribs” (Greub and Raoult 2004), or “training grounds” Aquatic Ecology, Dept. of Biology, Ghent Univ., Belgium. Direct inquiries to author (Molmeret and others 2005). Finally, in some cases FLP func- Houf (E-mail: [email protected]). tion as “Trojan horses” where the protozoan “horse” may bring C 2014 Institute of Food Technologists® r 924 ComprehensiveReviewsinFoodScienceandFoodSafety Vol.13,2014 doi: 10.1111/1541-4337.12100 Interactions of foodborne pathogens with FLP . internalized bacteria within the human “Troy,” enabling bacte- Gutierrez´ and others 2001; Hausmann and others 2003). Cysts ria to pass the 1st line of the human defense system (Barker and are nonmotile and generally smaller than trophozoites and usually Brown 1994; Greub and Raoult 2004). Because FLP can facil- have a thick, often double- or multilayered wall (Corliss 2001). itate the survival, growth, and dispersal of bacterial pathogens, The cyst wall is composed of proteins, glycoproteins, and carbo- host–microbe interactions, which benefit bacterial pathogens, are hydrates such as cellulose or chitin (Corliss 2001). The cyst shape increasingly considered as a potential health risk (Thomas and may vary (for example, spherical, ovoid, pyriform) and several Ashbolt 2011). cysts have an outer surface ornamentation (Corliss 2001), which The aim of this review is to provide an overview of the cur- can aid for dispersal. Cysts can survive harsh conditions and, for rent knowledge on interactions of foodborne pathogens with example, cysts of Acanthamoeba spp. resist gamma and UV irradi- FLP. It starts with a brief description of FLP, followed by an ation (Aksozek and others 2002), heat (Storey and others 2004) overview of FLP presence in food-related environments and and disinfectants (Kilvington and Price 1990; Storey and others on food. The possible modes of interactions between bacteria 2004; Coulon and others 2010), and they can remain viable for and FLP are summarized, and an overview of known interac- many years (Mazur and others 1995; Sriram and others 2008). tions of foodborne pathogens with FLP is presented. The in- Excystment is the transformation from cyst to trophozoite and traprotozoan survival of 4 major bacterial foodborne pathogens is triggered by favorable conditions or chemicals (Hausmann and (Campylobacter jejuni, Salmonella spp., Escherichia coli O157:H7, others 2003). and Listeria monocytogenes) is discussed in detail. Where appropri- Based on morphology and locomotion, FLP are divided into ate, reference is made to other, well-characterized bacteria–FLP the amebae, the flagellates, and the ciliates (Figure 1). This division interactions (Legionella–Acanthamoeba, Legionella–Te tra hy m e na ,and is used very often for convenience, but it is strongly discouraged Mycobacterium–Acanthamoeba) to highlight important cellular and by protistologists because it does not take into account the phy- ecological features. Finally, the consequences of these microbial logenetic relationships between these organisms (Adl and others interactions to food safety are evaluated. 2005). However, for the sake of simplicity and to present a general overview, the FLP-morphogroups amebae, flagellates, and cili- Free-Living Protozoa ates will be used throughout this review. In the rest of the text, Definition and morphology only organisms nonparasitic to humans (“free-living”) are cov- While there is no unambiguous definition of “protozoa”, a ered. Indeed, each morphogroup contains a few human parasites general agreement is to consider protozoa as heterotrophic pro- such as the flagellate parasites Giardia duodenalis (causing giardia- tists. Protists are defined by Adl and others (2005) as “eukaryotes sis) and Trypanosoma brucei (causative agent of sleeping sickness), with a unicellular level of organization, without cell differentiation the ameba Entamoeba histolytica (amebic dysentery), and the ciliate into tissues”. FLP comprise protists which do not have an obligate Balantidium coli (balantidiosis). Obviously, other obligate parasitic parasitic life cycle, although some species such as A. castellanii, protozoa, such as Plasmodium spp. (causative agents of malaria) and Balamuthia mandrillaris,andNaegleria fowleri occasionally cause foodborne pathogens such as Cryptosporidium parvum, Cyclospora infections in humans (Marciano-Cabral and Cabral 2003; Khan cayetanensis, Sarcocystis hominis,andToxoplasma gondii, all organisms 2006; Visvesvara and others 2007). classified in the past in the Sporozoa, will also not be discussed in FLP are single-celled microorganisms. Two life stages are usually this review. distinguished: a trophozoite (vegetative cell) and a cyst (also: rest- Amebae are characterized by the possession of pseudopodia ing or dormant cyst). However, not all species produce cysts. The (Greek: false feet), which are transient cytoplasmic extensions of trophozoite is the life stage in which the cell feeds and multiplies. the cell.