6 Enterococcus and its Association with Foodborne Illness Simona F. Oprea and Marcus J. Zervos 1. INTRODUCTION Enterococci are an important group of bacteria and their interaction with humans is complex. On one hand, enterococci are part of the normal flora of humans and animals, and some of their strains are used for the manufacturing of foods or as probiotics, whereas others are known to cause serious diseases in humans. With the emergence of enterococci as the third most common cause of nosocomial blood-stream infections (1) as well as the alarming rise in enterococci resistance to multiple antimicrobials, more concentrated effort has been invested in the better understanding of this versatile microorganism. Although they are not classic foodborne pathogens, enterococci have earned their own place, in this book, as organisms that can be acquired from food. They have been associated with foodborne outbreaks because of their presence in foods and their capacity to carry and disseminate resistance genes through the food chain. The role of enterococci in disease raises concerns regarding their use in foods or as probiotics, whereas the imminent crisis of emergent antimicrobial resistance leads to measures that are urgently needed for the judicious use of antimicrobials in agriculture and human medicine. 2. CLASSIFICATION AND IDENTIFICATION Considered for a long time a major division of the genus Streptococcus, enterococci have undergone considerable changes in taxonomy in the last decades, including the recognition of Enterococcus as a separate genus in 1984 (2,3). Enterococci are catalase-negative Gram-positive cocci that occur singly, in pairs, or in short chains. They are facultative anaerobes and very tolerant to extreme temp- eratures, salinity, and pH; thus, they grow in 6.5% NaCl broth at pH 9.6 and at temperatures ranging from 10 to 45°C, with optimum growth at 35°C (2). Enterococci hydrolyze esculin in the presence of 40% bile-salts. Most enterococci hydrolyze L-pyrrolidonyl-G-naphtylamide (PYR) and all strains hydrolyze leucine-G-naphtylamide by producing leucine aminopeptidase (LAPase). Some species, such as E. gallinarum, are motile. From: Infectious Disease: Foodborne Diseases Edited by: S. Simjee © Humana Press Inc., Totowa, NJ 157 158 Oprea and Zervos Current criteria for the inclusion into the Enterococcus genus are a combination of DNA–DNA reassociation values, 16S rRNA gene sequencing, whole-cell protein analysis, and conventional phenotypic tests (2–4). Enterococci belong to the clostridial subdivision of Gram-positive bacteria, together with the other genera of lactic-acid bacteria: Aerococcus, Carnobacterium, Globicatella, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus, and Weissella (5). So far 23 distinct Enterococcus species have been recognized (3) but new species continue to be identified (6). They have been separated into five groups on the basis of acid formation in mannitol and sorbose broths, and hydrolysis of arginine (3). Group I consists of Enterococcus avium, Enterococcus malodoratus, Enterococcus raffinosus, Enterococcus pseudoavium, Enterococcus saccharolyticus, Enterococcus pallens, and Enterococcus gilvus. They form acid in both mannitol and sorbose broths but do not hydrolyze arginine. Group II consists of Enterococcus faecalis, Enterococcus faecium, Enterococcus casseliflavus, Enterococcus mundtii, and Enterococcus gallinarum. These five species form acid in mannitol but not sorbose broth and hydrolyze arginine. Group III includes Enterococcus durans, Enterococcus porcinus, Enterococcus ratti, Enterococcus hirae, Enterococcus dispar, and mannitol-negative variants of E. faecalis and E. faecium. The enterococci in Group IV do not produce acid in mannitol or sorbose broths and do not hydrolyze arginine. They are Enterococcus asini, Enterococcus sulfureus, and Enterococcus cecorum. Finally, Group V consists of the variant strains of Enterococcus casseliflavus, Enterococcus gallinarum, and Enterococcus faecalis that fail to hydrolyze arginine (3). Three new species of Enterococcus were recently identified from human clinical sources by the analysis of whole-cell protein profiles and DNA–DNA reassociation experiments, in conjunction with conventional physiological tests. These new enterococcal species, provisionally designated Enterococcus sp. nov. CDC PNS-E1, Enterococcus sp. nov. CDC PNS-E2, and Enterococcus sp. nov. CDC PNS-E3, resemble the physio- logical groups I, II and IV; two were isolated from human blood and one from human brain tissue. Resistance to vancomycin was detected in one of the strains, which is harboring the vanA resistance gene (6). The species most broadly distributed in nature are E. faecalis and E. faecium. Identification of the different Enterococcus species can be done by conventional physiological tests, by commercial systems, or by molecular methods. Correct species identification is important for appropriate antimicrobial treatment, for epidemiologic surveillance, and for the selection of starter strains and labeling of the product to which the starter is added. 3. RESERVOIRS Enterococci have the capacity to grow and survive in very harsh environments and thus occupy a variety of ecological niches. They can be found in soil, water, food, plants, animals (including mammals, birds, and insects), and humans. The major natural habitat of these organisms appears to be the gastrointestinal tract of humans and other animals were they make up a significant portion of the normal gut flora (4). With the increase in antimicrobial-resistant enterococci worldwide and their occasional implication in serious human diseases, there has been considerable interest in identifying the reservoirs of these organisms and of antimicrobial resistance genes. Enterococcus and Foodborne Illness 159 3.1. Nonhuman Reservoirs 3.1.1. In Mammals, Birds, and Insects Enterococci are a natural part of the intestinal flora in most mammals and birds. Insects and reptiles were also found to harbor these organisms. Some of the Enterococcus species are host-specific, while others are more broadly distributed. Exemples of host-specific enterococci are E. columbae, specific for pigeons, and E. asini, found so far only in donkeys. Several enterococcal species, although identifiable in a number of hosts, appear to have some host-specific properties (7). Few occasions, similar E. faecium strains have been found in animals and humans, and in some of these cases strains were epidemiologically related, originating from the farmer and one of his animals (7). Of importance, all of these studies were done on glycopeptide-resistant E. faecium. The most often encountered enterococcal species in the intestines of farm animals are E. faecalis, E. faecium, E. hirae, and E. durans. In chickens, E. faecalis, found early in life, is later replaced by E. faecium, and then by E. cecorum. Other species occasionally isolated from chickens are E. casseliflavus, E. gallinarum, and E. mundtii. 3.1.2. In Soil, Plants, and Water The presence of enterococci in these sources is usually a result of fecal contamination. E. faecium and E. faecalis have been the species most commonly recovered from water. E. casseliflavus, E. mundtii, and E. sulfureus appear to be plant-associated (7). 3.1.3. In Foods Enterococci are used as starter and probiotic cultures in foods, and they occur as natural food contaminants (8). They have been ascribed to both beneficial and detrimental roles in foods. In processed meats, enterococci can survive heat processing and cause spoilage, whereas in certain cheeses they contribute to ripening and development of flavor (5). Because of their heat-resistance, enterococci are often the only surviving bacte- rium (other than spore-formers) in heat-treated, semipreserved, nonsterile foods such as processed meats (9). Some enterococci produce bacteriocins (called enterocins) that exert anti-Listeria activity (5) and are also able to inhibit or kill other enterococci, clostridia, bacilli, and staphylococci. On the other hand, enterococci can cause food intoxication through the production of biogenic amines and can serve as a reservoir for virulence traits and antimicrobial resistance (10,11). E. faecalis and E. faecium, the strains most commonly associated with human infection, are also the strains most commonly found in foods or used as starter cultures. 3.1.4. Probiotics in Animal Feeds Enterococci are used as probiotics to improve the microbial balance of the intestine and to treat gastroenteritis in humans and animals (8). Their effects when used as pro- biotics are unclear, but studies have focused on growth-promoting of food animals and diminishing E. coli weaning diarrhea (7). The identification of common antimicrobial resistance determinants and antimicrobial resistant enterococcal isolates in humans, food, and food-producing animals, has led to concern about the safety of use of certain enterococcal strains as probiotics (5,12). 160 Oprea and Zervos 3.2. Human Reservoir Enterococci make up approx 1% of the normal intestinal flora of humans (13), being the predominant Gram-positive cocci in stool with concentrations ranging from 105 to 107 CFU/g in feces (14). E. faecalis is normally found in the stool of 90–100% of animals and humans, whereas E. faecium is found in 25%. Small num- bers of enterococci also occur in oropharyngeal secretions, the urogenital tract, and on the skin, especially in the perineal area (2,4). The prevalence of the different