Chapter one
The Genus Enterococcus
The name "entérocoque" was first used by Thiercelin in a paper from France published in 1899 (Murray, 1990, Thiercelin, 1899). The same year, MacCallum and Hastings characterized a similar organism, now known to be Enterococcus faecalis, from a lethal case of endocarditis, thus providing a first detailed description of its pathogenic capabilities (Lebreton et al., 2014). But (Andrewes and Horder, 1906) preferred to know this new found bacteria as Streptococcus faecalis, as they saw that it is so characteristic of the human intestine that this term may justly be applied to it (Murray, 1990).
In the year 1937 Sherman proposed separation of streptococci into four groups: pyogenic, viridans, lactic, and enterococcus, based on hemolytic reaction, group carbohydrate antigens, and phenotypic tests (Facklam, 2002). He used the term ‘enterococcus’ to comprise streptococci of faecal origin which have reduction action, resist heat, tolerate bile, and ferment mannitol (Sherman, 1937).
Based on difference in morphology, biological, cultural and serological peculiarities, Kalina proposed that Streptococcus faecalis and Streptococcus faecium should be transferred to the genus “Enterococcus” (Kalina, 1970). Later, (Whittenbury, 1965) demonstrated distinction between the enterococci and Streptococcus bovis, Streptococcus equinus, and other streptococci by comparative biochemical studies, and immunological studies by (London et al., 1975) confirmed this. Nucleic acid studies, in particular, DNA-rRNA homology studies and comparative oligonucleotide cataloging of 16s rRNA have confirmed that Streptococcus faecalis and Streptococcus faecium are only distantly related to Streptococcus bovis and Streptococcus equinus (Kilpper-Bälz et al., 1982). This work was cited in Bergey's Manual of 1984 and considered to support fully the creation of a new genus to encompass the enterococcal group of organisms (Murray, 1990).
The genus belongs to the family Enterococcaceae (Ludwig et al., 2009), it is a member of human normal microbiota inhabiting lumen (Carroll et al., 2016) and may colonize skin as a member of the transient skin flora (Swartz, 2004). Enterococci are primarily of faecal origin and can be human pathogens, and have the potential for causing HAIs , so they are the most common pathogens found in blood after Staphylococcus spp. (Madigan et al., 2015).
As well as clinical specimens, Enterococci can be isolated from food-stuffs (Trivedi et al., 2011), different animals such as pigs (Fard et al., 2011), cattle (Zaheer et al., 2013), birds (Radhouani et al., 2012), fish (Petersen and Dalsgaard, 2003) plants (Micallef et al., 2013, Müller et al., 2001), untreated waters (Macedo et al., 2011) and soil (Micallef et al., 2013). This distribution may be explained by their persistence and resistance to growth-inhibiting factors such as acidity, salt, drying, heat and chemical agents (Lauková, 2012).
General and Diagnostic Characteristics of Enterococci
Members of Enterococcus spp. are gram-positive spherical bacteria that characteristically form pairs or chains during growth (Carroll et al., 2016). They form small, cream or white, smooth, entire; alpha-, beta-, or gamma hemolysis (i.e., no hemolysis around colony) on 5% Sheep Blood Agar (Tille, 2014).
Enterococci are usually non motile (Narayan et al., 2010), but strains of some species may be motile by scanty flagella (Ludwig et al., 2009). They are none-sporulating bacteria (Javed et al., 2011).
Enterococcus bacteria are facultative anaerobe (Yilema et al., 2017) chemo-organotrophic organisms of complex nutrient requirements, lactic acid fermenters, and generally (not reported in the lesser known species): resistant to 40% (v/v) bile, production of leucine arylamidase, hydrolysis of esculin, production of acid from N-acetylglucosamine, cellobiose, d-fructose, galactose, glucose, lactose, maltose, d-mannose, ribose (Ludwig et al., 2009) and PYR positive (Aslanzadeh, 2006). The following tests are mostly negative for Enterococci: catalase (Yilema et al., 2017), urease and alkaline phosphatase (Ludwig et al., 2009). They are able to grow in 6.5% NaCl, and 0.1% methylene blue milk and at pH 9.6 and the growth is performed at 10 and 45°C and can resist 30 min at 60°C (Manero and Blanch, 1999).
Enterococci can be grown on standard laboratory media such as 5% Sheep Blood and Chocolate Agars and on Gram-positive selective media such as CNA (Columbia Agar with colistin and nalidixic acid) media PEA (phenylethyl-alcohol agar) BEA (Bile esculin agar) (Tille, 2014) but cannot be grown on media like XLD (Xylose-Lysine–Deoxycholate), SS (Salmonella-Shigella) agar, DCA (Deoxycholate–citrate agar) and HEA (Hektoen enteric agar) (Vandepitte et al., 2003).
1.1. Classification of the Genus Enterococcus
The taxonomy of Enterococcus spp. was established based upon molecular tools, such as: polymerase chain reaction (PCR), nucleotide sequencing, restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), denaturing gradient gel electrophoresis (DGGE) (Ong et al., 2014).
Table Error! No text of specified style in document. 1: Modern Classification of the Genus Enterococcus (rearranged from: (Ludwig et al., 2009) and (Madigan et al., 2015))
Domain Bacteria
Phylum Firmicutes
Class Bacilli
Order Lactobacillales
Family Enterococcaceae
Genus Enterococcus
Species of Enterococcus
Fifty five species had been classified under the genus Enterococcus as listed in the internet site LPSN (List of Procaryotic Names with standing in nomenclature), the names are mentioned below alphabetically:
Enterococcus alcedinis, E. aquimarinus, E. asini, E. avium, E. bulliens, E. caccae, E. camelliae, E. canintestini, E. canis, E. casseliflavus, E. cecorum, E. columbae, E. devriesei, E. diestrammenae, E. dispar, E. durans, E. faecalis, E. faecium, E. flavescens, E. gallinarum, E. gilvus, E. haemoperoxidus, E. hermanniensis, E. hirae, E. italicus, E. lactis, E. malodoratus, E. moraviensis, E. mundtii, E. olivae, E. pallens, E. phoeniculicola, E. plantarum, E. porcinus, E. pseudoavium, E. quebecensis, E. raffinosus, E. ratti, E. rivorum, E. rotai, E. saccharolyticus, E. saccharominimus, E. saigonensis, E. seriolicida, E. silesiacus, E. solitarius, E. sulfureus, E. termitis, E. thailandicus, E. ureasiticus, E. ureilyticus, E. viikkiensis, E. villorum, E. xiangfangensis (Euzèby, 2017).
Enterococcus of Medical Importance
Enterococcus species were believed to be harmless to humans and considered unimportant medically for many years, but recently, enterococci have become one of the most common nosocomial pathogens (Fisher and Phillips, 2009). But have been recognized as being potentially pathogenic for humans since the turn of the last century .Two species: E. faecalis and E. faecium, are the causative agent of many important infections to human (Sava et al., 2010).
Diseases caused by Enterococcus sp.
The two species cause many diseases for human including: periodontitis, peri-implantitis, pharyngitis, otitis, mastoidites, meningitis, endocarditis, urogenital tract infections and even septic conditions (Karayasheva and Radeva, 2017).
Lactic acid bacteria (LAB)
The term Lactic Acid Bacteria refers to bacteria producing lactic acid as one of the main products of fermentation of carbohydrates. Although they are different in shape, cocci and rods, they belong to the Phylum Firmicues, Class Bacilli, and order Bacillilaes and different families (Wright and Axelsson, 2014). LAB are increasingly considered as beneficial microorganisms; some strains are even thought to be health promoting (probiotic)
Table Error! No text of specified style in document. 2: Common genera of LAB (WRIGHT AND AXELSSON, 2014)
Family Genera Shape
Aerococcaceae Aerococcus Cocci
Carnobacteriaceae Carnobacterium Rods
Enterococcaceae Enterococcus Cocci
Vagococcus Cocci
Tetragenococcus Cocci
Lactobacillaceae Lactobacillus Rods
Pediococcus Cocci
Leuconostocaceae Leuconostoc Cocci
Oenococcus Cocci
Weissella Cocci
Streptococcaceae Lactococcus Cocci
Streptococcus Cocci
Enterococcus faecalis
Virulence Factors
Enterococcus species isolated from medical specimens have the highest virulence, followed by food isolates and then starter strains (Fisher and Phillips, 2009).
Virulence factors associated with pathogenesis of enterococcal infections may be divided into: secreted factors, surface-located proteins and cell-wall polysaccharides (Nannini and Murray, 2006). Each of which is discussed below:
The aggregation substance (Agg)
ANDREWES, F. & HORDER, T. 1906. THE STUDY OF THE STREPTOCOCCI PATHOGENIC IN MAN. The Lancet, 168, 1621-1622.
ASLANZADEH, J. 2006. Biochemical Profile-Based Microbial Identification Systems. In: TANG, Y.-W. & STRATTON, C. (eds.) Advanced Techniques in Diagnostic Microbiology. New York, USA: Springer Science+Business Media, LLC.
CARROLL, K. C., HOBDEN, J. A., MILLER, S., MORSE, S. A., MIETZNER, T. A., DETRICK, B., MITCHELL, T. G., MCKERROW, J. H. & SAKANARI, J. A. 2016. Jawetz, Melnick, & Adelberg’s Medical Microbiology, McGraw-Hill Education.
EUZÈBY, J. P. 2017. All names cited in the List of Prokaryotic names with Standing in Nomenclature: List D – L [Online]. Available: http://www.bacterio.net/-allnamesdl.html#r [Accessed Jan, 5 2018].
FACKLAM, R. 2002. What Happened to the Streptococci: Overview of Taxonomic and Nomenclature Changes. Clinical Microbiology Reviews, 15, 613-630.
FARD, R. M. N., HEUZENROEDER, M. W. & BARTON, M. D. 2011. Antimicrobial and heavy metal resistance in commensal enterococci isolated from pigs. Veterinary microbiology, 148, 276-282.
FISHER, K. & PHILLIPS, C. 2009. The ecology, epidemiology and virulence of Enterococcus. Microbiology, 155, 1749-57.
JAVED, A., MASUD, T., UL AIN, Q., IMRAN, M. & MAQSOOD, S. 2011. Enterocins of Enterococcus faecium, emerging natural food preservatives. Annals of microbiology, 61, 699-708.
KALINA, A. P. 1970. The Taxonomy and Nomenclature of Enterococci. Int. J. Sys. Bacteriol., 20, 185-189.
KARAYASHEVA, D. & RADEVA, E. 2017. Importance of Enterococci (Enterococcus faecalis) for Dental Medicine ? Microbiological Characterization, Prevalence and Resistance. International Journal of Science and Research (IJSR), 6, 1970-1973.
KILPPER-BÄLZ, R., FISCHER, G. & SCHLEIFER, K. H. 1982. Nucleic Acid Hybridization of Group N and Group D Streptococci. Curr. Microbiol. , 7, 245-250.
LAUKOVÁ, A. 2012. Potential Application of Probiotic, Enterocin-Producing Enterococci and their Enteocin. In: LAHTINEN, S., OUWEHAND, A. C., SALMINEN, S. & WRIGHT, A. V. (eds.) Lactic Acid Bacteria: Microbiological and Functional Aspects, Fourth Edition. 4th ed.: CRC Press.
LEBRETON, F., WILLEMS, R. J. L. & GILMORE, M. S. 2014. Enterococcus Diversity, Origins in Nature, and Gut Colonization. In: MICHAEL S GILMORE, D. B. C., YASUYOSHI IKE, NATHAN SHANKAR (ed.) Enterococci.. From Commensals to Leading Causes of Drug Resistant Infection. Boston: Massachusetts Eye and Ear Infirmary.
LONDON, J., CHACE, N. M. & KLINE, K. 1975. Aldolase of Lactic Acid Bacteria: Immunological Relationships Among Aldolases of Streptococci and Gram-Positive Nonsporeforming Anaerobes. Int. J. Sys. Bacteriol., 25, 114-123.
LUDWIG, W., SCHLEIFER, K.-H. & WHITMAN, W. B. 2009. Family IV. Enterococcaceae fam. nov. In: VOS, P. D., GARRITY, G. M., JONES, D., KRIEG, N. R., LUDWIG, W., RAINEY, F. A., SCHLEIFER, K.-H. & WHITMAN, W. B. (eds.) Bergey's Manual of Systematic Bacteriology, Volume Three: The Firmicutes. Springer.
MACEDO, A. S., FREITAS, A. R., ABREU, C., MACHADO, E., PEIXE, L., SOUSA, J. C. & NOVAIS, C. 2011. Characterization of antibiotic resistant enterococci isolated from untreated waters for human consumption in Portugal. International journal of food microbiology, 145, 315-319.
MADIGAN, M. T., MARTINKO, J. M., BENDER, K. S., BUCKLEY, D. H. & STAHL, D. A. 2015. Brocks Biology of Microorganisms, Pearson Education, Inc.
MANERO, A. & BLANCH, A. R. 1999. Identification of Enterococcus spp. with a biochemical key. Appl Environ Microbiol, 65, 4425-30.
MICALLEF, S. A., GOLDSTEIN, R. E. R., GEORGE, A., EWING, L., TALL, B. D., BOYER, M. S., JOSEPH, S. W. & SAPKOTA, A. R. 2013. Diversity, distribution and antibiotic resistance of Enterococcus spp. recovered from tomatoes, leaves, water and soil on US Mid-Atlantic farms. Food microbiology, 36, 465-474.
MÜLLER, T., ULRICH, A., OTT, E. M. & MÜLLER, M. 2001. Identification of plant‐associated enterococci. Journal of Applied Microbiology, 91, 268-278.
MURRAY, B. E. 1990. The life and times of the Enterococcus. Clin Microbiol Rev, 3, 46-65.
NANNINI, E. C. & MURRAY, B. E. 2006. Enterococcus spp. In: GILLESPIE, S. H. & HAWKEY, P. M. (eds.) Principles and Practice of Clinical Bacteriology. John Wiley and Sons Ltd.
NARAYAN, S. S., JALGAONKAR, S., SHAHANI, S. & KULKARNI, V. N. 2010. Probiotics: current trends in the treatment of diarrhoea. Hong Kong Med J, 16, 213-218.
ONG, Y. Y., TAN, W. S., MOHAMAD, R., SIEO, C. C. & TEY, B. T. 2014. Biochemical and molecular identification of Enterococcus spp. from red pitaya. Process Biochemistry, 49, 563-568.
PETERSEN, A. & DALSGAARD, A. 2003. Species composition and antimicrobial resistance genes of Enterococcus spp., isolated from integrated and traditional fish farms in Thailand. Environmental Microbiology, 5, 395-402.
RADHOUANI, H., POETA, P., GONCALVES, A., PACHECO, R., SARGO, R. & IGREJAS, G. 2012. Wild birds as biological indicators of environmental pollution: antimicrobial resistance patterns of Escherichia coli and enterococci isolated from common buzzards (Buteo buteo). Journal of medical microbiology, 61, 837-843.
SAVA, I. G., HEIKENS, E. & HUEBNER, J. 2010. Pathogenesis and immunity in enterococcal infections. Eur. Soci. Clin. Microbiol. Inf. Dis., 533–540.
SHERMAN, J. M. 1937. The Enterococci and Related Streptococci. J. Bacteriol., 35.
SWARTZ, M. N. 2004. Skin microbiology. In: SCHAECHTER, M. (ed.) The Desk Encyclopedia of Microbiology. San Diego, California, USA: Elsevier.
THIERCELIN, M. 1899. Sur un diplocoque saprophyte de l'intestin susceptible de devenir pathogene. CR Soc Biol, 5, 269-71.
TILLE, P. M. 2014. BAILEY & SCOTT’S DIAGNOSTIC MICROBIOLOGY, Elsevier Inc.
TRIVEDI, K., CUPAKOVA, S. & KARPISKOVA, R. 2011. Virulence factors and antibiotic resistance in enterococci isolated from food-stuffs. Veterinarni Medicina, 56, 352-357.
VANDEPITTE, J., VERHAEGEN, J., ENGBAEK, K., ROHNER, P., PIOT, P. & HEUCK, C. C. 2003. Basic laboratory procedures in clinical bacteriology, Geneva, World Health Organization.
WHITTENBURY, R. 1965. The Differentiation of Streptococcus faecalis and S. faecium. J. Gen. Microbiol., 279-287.
WRIGHT, A. V. & AXELSSON, L. 2014. Lactic Acid Bacteria: An Introduction. In: HOLZAPFEL, W. H. & WOOD, B. J. B. (eds.) Lactic Acid Bacteria: Biodiversity and Taxonomy. Wiley.
YILEMA, A., MOGES, F., TADELE, S., ENDRIS, M., KASSU, A., ABEBE, W. & AYALEW, G. 2017. Isolation of enterococci, their antimicrobial susceptibility patterns and associated factors among patients attending at the University of Gondar Teaching Hospital. BMC Infect Dis, 17, 276.
ZAHEER, R., COOK, S., KLIMA, C., STANFORD, K., ALEXANDER, T., TOPP, E., READ, R. & MCALLISTER, T. 2013. Effect of subtherapeutic vs. therapeutic administration of macrolides on antimicrobial resistance in Mannheimia haemolytica and enterococci isolated from beef cattle. Frontiers in microbiology, 4, 133.