Microbiology: The genus Enterococcus: friend, foe or just passing by?

Taxonomy and classification of microorganisms are based on criteria that do not always, if ever, fit with the complexity of the microbial world. Commensal, starter, probiotic, pathogen? Since the early days of Pasteur and Koch, this approach is not anymore applicable for most of the major foodborne and/or waterborne microorganisms. In the past few years, due to the evolution of regulation and need for safety demonstration, microbiologists face the new challenge of understanding and demonstrating the mechanism of action (most commonly pleiotropic) of one organism in order to assess its potential application. Among the most debated genera, one might like to see how those consequences apply to the benefit / risk assessment of Enterococcus spp., formerly Streptococcus spp. Lancefield Group D or faecal Streptococci (for microbes also, sometimes the name does not help).

Enterococci form part of the lactic acid bacteria (LAB), of importance in foods. For decades, they were considered for their role in the ripening and aroma development of certain traditional cheeses and sausages, while they can also be implicated in spoilage of foods and are commonly monitored as hygiene indicators in other type of food matrices. Enterococci have also been studied and used as probiotic strains. Most recently, they have emerged as nosocomial pathogens of concern due to their antibiotic resistance and their potential to cause bacteraemia, endocarditis and other infections. On top of this, even molecular investigations do not always help to differentiate ‘good’ Enterococci from ‘bad’ ones. Enterococcus spp.: friend or foe?

The genus Enterococcus comprises a wide variety from pathogenic, commensal and beneficial gram-positive bacteria. These microorganisms are common constituents of fermented products such as traditionally manufactured cheeses and dry sausages. Enterococci are most commonly used to extend the shelf life and improve the hygienic safety of foodstuffs because they produce bacteriocins which have a potential in bio-preservation and in the enhancement of the hygienic quality of food products^12,9.

Enterococci have emerged in recent years as serious nosocomial pathogens, with vancomycin-resistant strains raising major issues for public health⁸.

Whether beneficial organisms or opportunistic pathogens, Enterococci have been studied more intensively in the recent years to discriminate between food-grade and pathogenic strains³.

Taxonomy

The genus Enterococcus consists of Gram-positive, catalase negative, non-spore-forming, facultative anaerobic bacteria that occur both as single cocci and in chains. Enterococci belong to the group of lactic acid bacteria (LAB).

This genus was initially described in 1899 by Thiercelin and Jouhaud as an intestinal organism. Closely related to the family Streptococcaceae, the family Enterococcaceae with the sole genus Enterococcus has only been recently described in 1984, almost one century after the first isolation. It comprises currently over 30 species (Taxonomic Outline of Archea and Bacteria, http://www.taxonomicoutline.org/), of which E. faecalis and E. faecium are the most documented for their various implications, deleterious or beneficial.

Current roles of other Enterococci in fermented foods are currently being investigated; E. durans e.g. has been found to play a role in dairy product and sourdough¹. Enterococcci are sometimes still referred to as faecal Streptococci, their former name suggested by Sherman in 1937 which does not take into consideration the wide distribution of these organisms.

Ecology of Enterococcus spp.

Enterococci are ubiquitous in many different habitats such as soil, surface waters, ocean water, sewage, on plants and in the gastrointestinal tract of animals and humans. Enterococcus spp. will grow at a range of temperatures from 5 to 50°C. The optimum, minimum and maximum temperatures, according to the Rosso model (Cardinal model), are 42.7, 6.5 and 47.8°C, respectively in laboratory media in aerobic conditions. Growth will also occur in anaerobic atmosphere. E. faecalis and E. faecium will grow in a wide range of pH (4,6 – 9,9), with an optimum around 7.5. They grow in the presence of up to 40 per cent bile salts, therefore have a high prevalence in mammalian guts. On top of being thermophilic, Enterococci are considered to be among the most vegetative thermoresistant bacteria, both E. faecalis and E. faecium surviving a high water activity heat treatment of 60°C for 30 minutes.

The production of amines through decarboxylation is also closely related to the growth temperature and pH (see undesirables properties part). Enterococci often occur in foods of animal origin such as meats, fermented and cooked meats, as well as cheese and dairy products. They play a role in the fermentation process of indigenous fermented foods, either as starters or non-starters lactic acid bacteria.

The wide ecological potential of enterococci with respect to salt and pH tolerance results in the growth and occurrence at high population level in fermented foods, showing beneficial technological properties such as their positive impact on ripening and aroma production of fermented meats and aroma production in cheeses through their proteolytic and lipolytic activities.

Most recently, their ability to form biofilm and vancomycin resistance of a growing number of isolates is a major topic of concern for general public health due to constant increase of report of nosocomial infections in the past 20 years.

Positive properties of Enterococci

Enterococcus spp. represent one of the autochthonous lactic acid bacteria species that can be found in high numbers in various type of food products, where they contribute to flavour development due to glycolytic, proteolytic and lipolytic activities.

E. faecalis strains are generally more active than other enterococci strains. Citrate metabolism contributes to the sensorial characteristics of fermented foods by the production of diacetyl, acetaldehyde, acetoin, and 2,3-butanediol which have very distinct aroma properties and significantly influence the quality of fresh cheese, fermented milk, cream and butter. Carbon dioxide formation contributes to the texture of fermented products. Despite a weak lipolytic activity, they contribute through this pathway to the development of special flavours and texture of cheeses during manufacturing and ripening.

Enterococcus spp. can also be directly marketed as a probiotic product^6,7. The best known and probably best researched probiotic Enterococcus strain is E. faecium SF68¹¹. The most interesting (or at least historically documented) property of Enterococcus spp. however lies in the abilities to produce bacteriocin and play an interesting role in food biopreservation. A large number of enterocins has been studied since 1955, when the first bacteriocin-like substance was reported.

Bacteriocin-producing enterococci have been extensively studied and were already isolated from different sources, with the proposed applications as food preservative culture, for pathogen control or as a potential inhibitor of biofilm formation. Enterocins are found within the classes I, IIa, IIc, and III bacteriocins. The bacteriocins produced by E. faecalis strains are referred to as¹¹:

• Cytolysin (bacteriocin/hemolysin) from E. faecalis DS16
• Cyclic peptide antibiotic AS-48 (enterocin AS-48) from E. faecalis S-48
• Bacteriocin 31 from E. faecalis YI17
• Enterocin 1071A and enterocin 1071B from E. faecalis BEF 1071.

Undesirable properties of Enterococci

Every coins as two sides, and so does the foodborne Enterococci. Despite their health-promoting effects and their technological properties, they may also possess detrimental characteristics such as antibiotic resistance and virulence factors.

None of the current guidelines for establishing a clear separation between pathogenic species from non-pathogenic clearly do apply for Enterococci, either Codex² or Molecular Approach^4,5. A strain level approach, as proposed in the FAO/WHO guidelines^6,7 and the QPS demonstration by EFSA¹⁴, provide a rational approach to assess the undesirable properties of Enterococcus as following:

• Antibiotic resistance: the intrinsic resistance of Enterococci and the increase use of antibiotics in past years has grown their prevalence as nosocomial pathogens, mostly from the acquired and transferable resistance to vancomycin (VanA and VanB)
• Virulence factors associated with colonisation and invasion of host tissue also play a major role in the pathogenicity of Enterococci (Aggregation substance, Gelatinase, various adhesion factors). Pili, binding proteins to the extracellular matrix and ability to form biofilms are a major cause of concern for the outcome of endocarditis in hospital care settings
• Nosocomial opportunistic infection: the combination of antibiotic resistance, ecological persistence and ability to grow in the mammalian host make the Enterococci pathogen of concern for patients in intensive care units with underlying diseases with a wide range of hospital acquired infection. Enterococcal infections include urinary tract infections, hepatobiliary sepsis, endocarditis, surgical wound infection, bacteraemia and neonatal sepsis
• Deleterious metabolic activities: as starters, Enterococci are also a source of biogenic amines such as histamine and tyramine through decarboxylation of histidine and tyrosine that can cause food intolerance¹⁷.

A specific assessment is therefore to be conducted for the intended use of Enterococcus spp. in the food chain^10,15,16, as a probiotic food product^6,7 or a starter¹³.

Enterococci as indicator organism for water quality

Although Enterococci have a long history of use in fermented food products, the only ISO standard for enumeration of ‘intestinal enterococci’, ISO 7899 (Part 1 – Most Probable Number MPN and Part 2 – Filtration) is under responsibility of the technical committee #147 water quality. As for water quality, faecal contamination is a major issue, and faecal bacteria are monitored on a regular basis. Those indicator bacteria are the Gram negative total and faecal coliforms, Escherichia coli, and the Gram positive Enterococci, due to their occurrence in the digestive systems of humans and other warm-blooded animals and their ability to survive high salt concentration. Enterococci are therefore also used within the food industry as a hygiene criterion for the water used as an ingredient, yet also in low moisture food to verify the risk of any water leakage and contamination.

Conclusion

The classification of microorganisms in either a pathogenic or beneficial category is a Manichean approach which does unfortunately not apply to the complexity of microbial ecology and interaction of microbes with their different niches, the human intestinal tract just being one among numerous one. Then again, what about other major microorganisms of concern for the food industry? For full grade pathogens such as Listeria monocytogenes and Salmonella spp., the deal is set. What about Bacillus subtilis? It can be either ranked as probiotic food product (Natto), starter in indigenous fermented foods (Cocoa), major spoilage of concern (Dairy) or (mild) pathogenic. Bacillus subtilis: friend or foe? (This works with a lot of other species and genera, mostly Gram positive).

References

1. Bourdichon, F., Casaregola, S., Farrokh, C., Frisvad, J.C., Gerds, M.L., Hammes, W.P., Harnett, J., Huys, G., Laulund, S., Ouwehand, A., Powell, I.B., Prajapati, J.B., Seto, Y., Ter Schure, E., Van Boven, A., Vankerckhoven, V., Zgoda, A., Tuijtelaars, S., Hansen, E.B., 2012. Food fermentations: microorganisms with technological beneficial use. Int. J. Food Microbiol. 154(3) pp87-97
2. Codex Alimentarius, 1999. Principles and guidelines for the conduct of microbiological risk assessment. CAC/GL-30 (1999), available at: http:// www.codexalimentarius.net/input/download/standards/…/CXG_030e.pdf‎
3. EFSA Panel on Biological Hazards (BIOHAZ), 2012. Scientific Opinion on the maintenance of the list of QPS biological agents intentionally added to food and feed (2012 update). EFSA Journal 10(12):3020. [84 pp.]
4. Falkow, S., 1998. Molecular Koch’s postulates applied to microbial pathogenicity. Rev Infect Dis. 10 Suppl 2:S274-6
5. Falkow, S., 2004. Molecular Koch’s postulates applied to bacterial pathogenicity–a personal recollection 15 years later. Nat Rev Microbiol. 2(1):67-72
6. FAO and WHO, 2001. Report on Joint FAO/WHO expert consultation on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria. Córdoba, Argentina, 1-4 October 2001
7. FAO and WHO, 2002. Guidelines for the evaluation of probiotics in Food. Joint FAO/WHO Workgroup on Drafting Guidelines for the Evaluation of Probiotics in Food. London, Ontario, Canada, April 30 and May 1, 2002
8. Fisher, K., Phillips, C., 2009. The ecology, epidemiology and virulence of Enterococcus. Microbiology 155 pp1749-57
9. Foulquié Moreno, M.R., Sarantinopoulos, P., Tsakalidou, E., De Vuyst, L., 2006. The role and application of enterococci in food and health. Int. J. Food Microbiol. 106 pp1-24
10. Franz, C.M., Stiles, M.E., Schleifer, K.H., Holzapfel, W.H., 2003. Enterococci in foods–a conundrum for food safety. Int J Food Microbiol.88(2-3):105-22
11. Franz, C.M.A.P., Huch, M., Abriouel, H., Holzapfel, W., Galvez, A., 2011. Enterococci as probiotics and their implications in food safety. Int. J. Food Microbiol. 151 pp125-40
12. Giraffa, 2002. Enterococci from foods. FEMS Micro. Rev. 26 pp163-171
13. Latorre-Moratalla, M.L., Bover-Cid, S., Veciana-Nogués, M.T., Vidal-Carou, M.C., 2012. Control of biogenic amines in fermented sausages: role of starter cultures. Front Microbiol. 3:169
14. Leuschner, R.-G.K., Robinson, T., Hugas, M., Cocconcelli, P.S., Richard-Forget, F., Klein, G., Licht, T.R., Nguyen-The, C., Querol, A., Richardson, M., Suarez, J.E., Vlak, J.M., von Wright, A., 2010. Qualified presumption of safety (QPS): a generic risk assessment approach for biological agents notified to the European Food Safety Authority (EFSA). Trends Food Sci. Technol. 21 pp425-435
15. Magnússon, S.H., Gunnlaugsdóttir, H., Van Loveren, H., Holm, F., Kalogeras, N., Leino, O., Luteijn, J.M., Odekerken, G., Pohjola, M.V., Tijhuis, M.J., Tuomisto, J.T:, Ueland, O., White, B.C., Verhagen, H., 2012. State of the art in benefit-risk analysis: food microbiology. Food Chem. Toxicol. 50(1) pp33-9
16. Ogier, J.C., Serror, P., 2008. Safety assessment of dairy microorganisms: the Enterococcus genus. Int. J. Food Microbiol. pp126 291-301
17. Spano, G., Russo, P. Lonvaud-Funel, A., Lucas, P., Alexandre, H., Grandvalet, C., Coton, E., Coton, M., Barnavon, L., Bach, B., Rattray, F., Bunte, A., Magni, C., Ladero, V., Alvarez, M., Fernández, M., Lopez, P., de Palencia, P.F., Corbi, A., Trip, H., Lolkema, J.S., 2010. Biogenic amines in fermented foods. Eur J Clin Nutr. 2010 Nov;64 Suppl 3:S95-100

Further reading

• International Journal of Food Microbiology Special Issue – Volume 88, Issues 2–3, Pages 103-346 (1 December 2003)
• Enterococci in Foods. Functional and Safety Aspects, Edited by Professor Wilhelm Holzapfel
• http://www.sciencedirect.com/science/journal/01681605/88/2-3
• Enterococcus and Safety. Semedo Lemsadek, Barreto-Crepso, Tenreiro, 2011. Nova Science Publishers, p534.
• http://www.ncbi.nlm.nih.gov/nlmcatalog/101562270
• EFSA Biohazard Panel – Qualified Presumption of Safety
• http://www.efsa.europa.eu/en/topics/topic/qps.htm