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The ecology, epidemiology and virulence of Enterococcus

The ecology, epidemiology and virulence of Enterococcus Microbiology (2009), 155, 1749–1757 DOI 10.1099/mic.0.026385-0 The ecology, epidemiology and virulence of Review Enterococcus Katie Fisher and Carol Phillips Correspondence University of Northampton, School of Health, Park Campus, Boughton Green Road, Northampton Katie Fisher NN2 7AL, UK Katie.fisher@northampton.ac.uk Enterococci are Gram-positive, catalase-negative, non-spore-forming, facultative anaerobic bacteria, which usually inhabit the alimentary tract of humans in addition to being isolated from environmental and animal sources. They are able to survive a range of stresses and hostile environments, including those of extreme temperature (5–65 6C), pH (4.5”10.0) and high NaCl concentration, enabling them to colonize a wide range of niches. Virulence factors of enterococci include the extracellular protein Esp and aggregation substances (Agg), both of which aid in colonization of the host. The nosocomial pathogenicity of enterococci has emerged in recent years, as well as increasing resistance to glycopeptide antibiotics. Understanding the ecology, epidemiology and virulence of Enterococcus speciesisimportant for limiting urinary tract infections, hepatobiliary sepsis, endocarditis, surgical wound infection, bacteraemia and neonatal sepsis, and also stemming the further development of antibiotic resistance. Introduction Taxonomy For many years Enterococcus species were believed to be The genus Enterococcus consists of Gram-positive, catalase- harmless to humans and considered unimportant med- negative, non-spore-forming, facultative anaerobic bacteria ically. Because they produce bacteriocins, Enterococcus that can occur both as single cocci and in chains. species have been used widely over the last decade in the Enterococci belong to a group of organisms known as food industry as probiotics or as starter cultures (Foulquie lactic acid bacteria (LAB) that produce bacteriocins Moreno et al., 2006). Recently, enterococci have become (Health Protection Agency, 2005). The genera of LAB with one of the most common nosocomial pathogens, with which Enterococcus are grouped are identified by a low patients having a high mortality rate of up to 61 % (De G+C content of ,50 mol% (Klein et al., 1998). There are Fa´tima Silva Lopes et al., 2005). no phenotypic characteristics to distinguish Enterococcus species from other Gram-positive, catalase-negative cocci In 2005 there were 7066 reported cases of bacteraemia bacteria, so identification is usually established by reverse caused by Enterococcus species in the UK, an 8 % increase methodology (elimination of other species traits first). As a from 2004, with the Health Protection Agency (2007) genus Enterococcus has been recognized since 1899, when stating that ‘an increase in a bacteraemia causing pathogen Thiercelin identified it as an intestinal organism (Stiles & like this has not been observed for some time’. Twenty- Holzapfel, 1997); its taxonomy and ecology were reviewed eight per cent of all cases were antibiotic resistant (Health by Klein (2003). Many attempts have been made to Protection Agency, 2007). The risk of death from distinguish Enterococcus species from Streptococcus species. vancomycin-resistant enterococci (VRE) is 75 %, compared In 1937, Sherman classified Streptococcus species into four with 45 % for those infected with a susceptible strain subgroups: faecal streptococci (enterococci), dairy strep- (Bearman & Wenzel, 2005). These figures are mirrored in tococci, viridans group and pyogenous streptococci (Klein, the USA. Over a 15 year period there was a 20-fold increase 2003). Sherman noted that the enterococci subgroup in VRE associated with nosocomial infections reported to included the Lancefield group D streptococci and suggested CDC’s National Nosocomial Infections Surveillance that the latter could be differentiated by haemolytic and (NNIS) (National Nosocomial Infections Surveillance, proteolytic reactions, although this is inappropriate as 2004). haemolysis is determined by a plasmid (Stiles & Holzapfel, This dramatic increase in antibiotic resistance of 1997). Traditional methods such as biotyping, serotyping Enterococcus species worldwide highlights the need for a and phage typing left questions as to which of the greater understanding of this genus, including its ecology, Streptococcus species actually belonged to the genus epidemiology and virulence. Enterococcus (Saeedi et al., 2002). 026385 2009 SGM Printed in Great Britain 1749 K. Fisher and C. Phillips In 1984, through the use of DNA hybridization and 16S enzyme activities such as pyroglutamyl aminopeptidase rRNA sequencing, it was established that the species (PYRase) (Domig et al., 2003), growth at defined Streptococcus faecium and Streptococcus faecalis were temperatures and physiological characteristics is essential sufficiently distinct from the other streptococci to be in the identification of Enterococcus species (Shanks et al., 2006). designated another genus: Enterococcus (Foulquie Moreno et al., 2006). This means that the D group antigen is found The differences in the genomes of E. faecalis and E. faecium in both streptococci and enterococci. Nine species were were assessed in a study using competitive DNA hybrid- transferred from the Streptococcus groups and now ization (Shanks et al., 2006). E. faecalis-specific sequences Enterococcus includes 28 species (Foulquie Moreno et al., compared with those of E. faecium mainly encoded surface- 2006). The molecular data that were collected using 16S exposed proteins. Overall 6.4 % of the Enterococcus genome rRNA sequencing of Streptococcus enabled the construction is associated with cell-surface proteins and 22.6 % of the of an 16S rRNA-dendrogram showing the relationship differences between the two species are found in these between Streptococcus, Enterococcus and Lactococcus species genes. This variation is thought to have implications in the (Fig. 1). This method also allowed the grouping of species avoiding different host immune responses (Shanks Enterococcus species. The Enterococcus faecalis species group et al. 2006). includes E. faecalis, Enterococcus haemoperoxidus and Enterococcus moraviensis whilst the Enterococcus faecium species group includes E. faecium, Enterococcus durans, Physiology Enterococcus hirae, Enterococcus mundtii, Enterococcus Enterococcus species will grow at a range of temperatures porcinus and Enterococcus villorum (Klein, 2003). The from 5 to 50 C. The optimum, minimum and maximum discrimination of enterococci from streptococci is mainly temperatures, according to the Rosso model, are 42.7, 6.5 established by Lancefield group D antigen, as only and 47.8 C, respectively, on brain heart infusion (BHI) Streptococcus bovis, Streptococcus alactolyticus and agar in aerobic conditions (Van den Berghe et al., 2006), Streptococus equinus are serogroup D. These groups can although growth will also occur in anaerobic atmospheres be distinguished from Enterococcus species by the lack of (Domig et al., 2003). Both E. faecalis and E. faecium can growth in 6.5 % (w/v) sodium chloride at 10 C. It is survive heating at 60 C for 30 min, making Enterococcus harder to distinguish Enterococcus species from other cocci species distinguishable from other closely related genera that do not express the D group antigen such as such as Streptococcus (Foulquie Moreno et al., 2006). Pediococcus, Lactococcus or Tetragenococcus species because Trypticase soy agar or Columbia agar with 5 % (v/v) no other phenotypic differences have been reported that defibrinated sheep blood may be used to assess the allow distinction. Thus the use of fermentation patterns, haemolysis produced by enterococci. If human or horse blood is used, haemolysis is based on cytolysin activity and causes a b-haemolytic reaction (Domig et al., 2003). E. faecalis and E. faecium will grow in a wide range of pH (4.6–9.9), with the optimum being 7.5 (Van den Berghe et al., 2006). They will also tolerate and grow in the presence of 40 % (w/v) bile salts. E. faecalis is able to grow in 6.5 % NaCl and has a cation homeostasis which is thought to contribute to its resistance to pH, salt, metals and desiccation. When assessing growth of Enterococcus species using optical densities the most important variable of the growth conditions is pH, with temperature and salt concentration having a lesser effect (Gardin et al., 2001). During the lag phase, temperature is the most important factor influ- encing growth, with stationary-phase cells being the most resistant to heat (Gardin et al., 2001; Martinez et al., 2003). The resistance of E. faecalis to a range of pH values is thought to be due to its membrane durability and impermeabilty to acid and alkali, although some studies have suggested that it may be associated with membrane- bound H -ATPase activity (Nakajo et al., 2005). Temperature resistance is also associated with membrane structure and has been related to lipid and fatty acid content. The membrane has been demonstrated to be more Fig. 1. 16S rRNA dendrogram of phylogenetic position of stable near the minimal temperature for growth, which is a Enterococcus species (adapted from Klein, 2003). specific mechanism associated with enterococci (Ivanov et 1750 Microbiology 155 Enterococcus ecology, epidemiology and virulence al., 1999). At higher temperatures enterococci are less cell. This occurs by the enzymic removal of an N-terminal resilient, with the membrane fatty acid content increasing leader peptide at a double glycine cleavage site, and export and the saturated fatty acid levels decreasing. The heat via a Sec-dependent pathway. Bacteriocins are cationic, resistance of enterococci is dependent not only on the amphiphilic proteins containing little or no cysteine, and temperature but also the phase of growth (Martinez et al., their structures usually occur as random coils under 2003). aqueous conditions (Garneau et al., 2002). Bacteriocin production is favoured in stressful growth conditions, When E. faecalis is grown at non-stress temperatures, which is thought to be due to lower growth rates, resulting subsequently cultured cells do not have the resilience to in better utilization of energy and greater availability of warm and cold environments that would occur if the first metabolites for the synthesis of bacteriocins. Under generation were grown at stressful temperatures (Ivanov optimal growth conditions and thus high growth rates et al., 1999). Three distinct temperature groups (10–13 C, there is a lack of amino acids available for bacteriocin u u 17–22 C and 42–47 C) have been established for E. faecalis production (Van den Berghe et al., 2006). Enterococcus at which permeability of the membrane to 3 % NaCl is species are known to produce a range of enterocins different. This has significant implications with regard to (Table 1) including enterocins A, B, I, L and P, which are biotechnology and food science (Ivanov et al., 1999). active against Listeria species, Clostridum species and The production of amines is also closely related to the Staphylococcus aureus (Campos et al., 2006). Most of the growth temperature and pH. The production of decarbox- bacteriocins produced by E. faecalis and E. faecium are ylases is optimum at acid pH, whereas biogenic amine identical to enterocins A and B first described from E. production by E. faecalis EF37 decreases at low pH. faecium CTC492 and E. faecium T136 (De Kwaadsteniet Temperature does not have a significant effect on amine et al., 2005). E. faecium RZS C5 is a natural cheese isolate, production itself, but the effect that temperature has on cell which is lacking in virulence factors and has antilisterial yield alters the quantity of amines being produced (Gardin properties (Leroy et al., 2003). Enterocin EJ97 from E. et al., 2001). Other products of Enterococcus species that are faecalis S-47 has also shown antilisterial properties, with 1.6 affected by pH are bacteriocins. log reduction after 6 h and complete inhibition after 24 h at a concentration of 20 AU ml (Garcia et al., 2004). The enterocin AS-48 (produced by E. faecalis A-48-32) inhibits Bacteriocins growth of Bacillus coagulans vegetative cells, at refrigeration and high temperatures, in canned fruits and vegetables Bacteriocins are ribosomally synthesized, extracellular released antimicrobial peptides that show activity against (Lucas et al., 2006). The bacteriocin activity against Gram- negative bacteria is unusual, but bacteriocin ST15 from E. closely related bacterial species. Four genes are required to mundtii has been shown to be effective against a range of produce bacteriocins: a structural gene encoding a prepeptide, a dedicated immunity gene, a dedicated ABC- Gram-positive and Gram-negative bacteria including Acinetobacter, Bacillus, Clostridium, Klebsiella, Lacto- immunity gene and a gene encoding the protein necessary bacillus and Pseudomonas (De Kwaadsteniet et al., 2005). for export of the bacteriocin. Bacteriocins are formed as prepeptides in the cell and mature during export from the The bactericidal effects of bacteriocins are thought to be Table 1. Bacteriocins produced by Enterococcus species Adapted from De Kwaadsteniet et al. (2005). Bacteriocin Produced by Isolated from Size (Da) Enterocins A and B E. faecium P21 Chorizo Enterocin EJ97 E. faecalis S-47 No name E. faecium A2000 Cheese Enterocin CRL35 E. faecium CRL35 Cheese 3500 Bacteriocin N15 E. faecium N15 Nuka 3000–5000 Enterocins A and B E. faecium WHE81 Cheese 4833 and 5462 No name E. faecium RZS C5; E. faecium DPC 1146 AS-48 E. faecalis subsp. liquefaciens S-48 Porcine intestinal tract Enterocin 012 E. gallinarum Duodenum of ostrich 3400 No name E. faecium CRL 1385 Free-range chicken Enterocin P E. faecium P13 Dry-fermented sausage Enterocins 1071A and 1071B E. faecalis BFE 1071 Faeces of minipigs 4285 and 3899 Mundticin ATO6 E. mundtii ATO6 Vegetables 4287 Mundticin KS E. mundtii NFRI 7393 Grass silage 4290 http://mic.sgmjournals.org 1751 K. Fisher and C. Phillips due to permeabilization of the cell membrane. The of E. faecalis whereas healthy individuals show only a 39– random-coiled peptides of the bacteriocins, on contact 40 % occurrence (Mutnick et al., 2003). Hospitalized with the cell membrane, form a helical structure, which patients may have a greater incidence of enterococcal incorporates into and spans the membrane, creating a pore. infection not only because of virulence, but because the This mode of action has been observed in enterocin P. The hospital itself is a hub. This is illustrated by a report for resulting pore causes leakage of K ions, dissipation of the Department of Health in the UK, which highlighted the membrane potential and inhibition of amino acid uptake. fact that enterococci may contaminate and survive around The cycle of ATP-driven K uptake by the cell and the patient for several days (Brown et al., 2006). bacteriocin-mediated release of K leads to cell death Enterococci also play a role in endodontic failure and are (Garneau et al., 2002; Hechard & Sahl, 2002). often isolated from the root canal system. The results of one study showed that out of 100 root-filled teeth with apical periodontitis, 69 % of the isolated bacteria were Ecology and epidemiology facultative and 50 % of those were enterococci (Dahlen et al., 2000). E. faecalis is responsible for 80–90 % of human The origins of Enterococcus species vary from envir- onmental to animal and human sources. As enterococci enterococcal endodontic infection and is usually the only Enterococcus species isolated from the obturated root canal are an essential part of the microflora of both humans and animals their distribution is very similar in these sources. E. (Love, 2001; Peciuliene et al., 2001). faecium and E. faecalis are the most common in the human gastrointestinal tract, E. faecium in production animals and Rates of infection E. mundtii and E. casseliflavus in plant sources (Klein, 2003). The numbers of E. faecalis in human faeces range Enterococcal infections include urinary tract infections, 5 7 4 from 10 to 10 per gram, and those of E. faecium from 10 hepatobiliary sepsis, endocarditis, surgical wound infec- to 10 per gram. The isolation of E. faecium and E. faecalis tion, bacteraemia and neonatal sepsis (Poh et al., 2006). In is less prevalent from livestock than from human faeces Europe, infection with Enterococcus species was considered (Franz et al., 1999). harmless to humans for a long time. However in the last decade enterococci have been reported as the second most Studies of the ecology and epidemiology of Enterococcus common cause of wound and urinary tract infection and have reported E. faecalis and E. faecium being regularly the third most common cause of bacteraemia (De Fa´tima isolated from cheese, fish, sausages, minced beef and pork Silva Lopes et al., 2005). In 2005 in the UK there were 7066 (Foulquie Moreno et al., 2006; Klein, 2003). Foods such as reported cases of Enterococcus bacteraemia, 63 % of these sausages and cheese that are of animal origin are often cases being due to E. faecalis and 28 % to E. faecium, both associated with contamination by Enterococcus species, as of which have increasing antibiotic resistance (Health they are able to survive the heating process. In one study in Protection Agency, 2007). In the USA approximately 12 % the UK, samples taken from urban sewage and from of the hospital-acquired infections are Enterococcus species. farmland using pig manure and crops generated from this E. faecalis is the most common species associated with land, were found to be 100 % positive for Enterococcus clinical infection while E. faecium poses the higher species In crops to which animal fertilizers were not antibiotic resistance threat (Giraffa, 2002). applied, the incidence of Enterococcus species was reduced to 33 % (Kuhn et al., 2003). A similar study in Germany isolated 416 strains of Enterococcus from 155 samples of Antibiotic resistance food of animal origin, 72 % of which were E. faecalis and The antibiotic resistance of Enterococcus is well documen- 13 % E. faecium (Peters et al., 2003). ted. Bacteria may show resistance to glycopeptides such as The distribution of Enterococcus species varies throughout vancomycin and teicoplanin, which are licensed in the UK, Europe. In Spain and the UK, E. faecalis and E. faecium are and to aminoglycosides (Kacmaz & Aksoy, 2005). the most commonly isolated species from both clinical and Antibiotic resistance has been of growing concern for a environmental sources. Sweden has a lower incidence of E. number of years. Vancomycin was first used in the clinical faecium and a higher isolation rate of E. hirae, whereas in arena in 1972 and the first vancomycin-resistant entero- Denmark E. hirae is the dominant species and is isolated cocci were recognized only 15 years later. NNIS reported an mainly from slaughtered animals (Kuhn et al., 2003). increase of 7.6 % in VRE between 1989 and 1993 (Metan et al., 2005). It has been reported that if glycopeptide- Clinical isolates of enterococci show a lower diversity than those obtained from the environment and other human resistant enterococci (GRE) are present in an infected patient rather than an antibiotic-susceptible strain, clinical sources, with E. faecalis being the dominant species (Kuhn et al., 2003). The reason for this lack of diversity may be treatment failure is increased by 20 % and mortality is linked with the virulence factors associated with this increased from 27 % to 52 % (Brown et al., 2006). When assessing the studies on enterococcal antibiotic resistance, species. The fact that Enterococcus species are opportunistic pathogens was highlighted by a study in Denmark which the pattern that is emerging is the possible occurrence of showed that hospitalized patients have a 57 % isolation rate multidrug resistant strains (Peters et al., 2003). 1752 Microbiology 155 Enterococcus ecology, epidemiology and virulence In both the Surveillance and Control of Pathogens of are present on the chromosome but can also be carried on Epidemiological Importance (SCOPE) and SENTRY a plasmid (Gilmore, 2002; Klare et al., 2003). Enterococcus (Antimicrobial Resistance Surveillance Program) data- species do not possess cytochrome enzymes and thus bases, figures show that, of enterococcal isolates from the cannot produce the energy required to take up antibiotics bloodstream, 2 % of E. faecalis and 60 % of E. faecium into the cell. This means they show resistance to isolates are resistant to vancomycin (Bearman & Wenzel, aminoglycosides at low levels (Klare et al., 2003). 2005). Resistance rates of Enterococcus species have reached Antibiotic resistance in Enterococcus species can be endemic or epidemic proportions in North America, with transferred by pheromone-mediated conjugative plasmids Europe having lower, but increasing, levels (Mutnick et al., or transposons. The resistance genes may be passed on not 2003). Enterococcal antibiotic resistance is not exclusive to only to antibiotic-susceptible enterococci, but also to other the clinical arena but is also prevalent in the food industry. pathogens (Giraffa, 2002). The presence of VRE in individuals who have been In contrast to Gram-negative conjugation systems, con- hospitalized, when they have not previously been in jugation of Enterococcus species does not require pili, and hospital or taken antibiotics, suggests that VRE may have involves a pheromone-induced system (Andrup & been contracted through the food chain. GRE may emerge Andersen, 1999). Bacteria containing conjugative plasmids in the food chain through use of avoparcin in animal feed respond to pheromones (plasmid specific) for genetic (Mannu et al., 2003). exchange; these bacteria generally have a narrow recipient Glycopeptide resistance in enterococci involves a two- range for conjugation, including only closely related component system where the cell wall composition is species. This lateral transfer of genetic elements leads to altered from the peptidoglycan precurser D-Ala-D-Ala rapid dissemination of antibiotic resistance. The plasmids (vancomycin-susceptible) to D-Ala-D-lactate (D-Lac). The occurring in Enterococcus species can also be transmission latter has 1000 times less affinity for vancomycin, while D- vehicles for transposons (Williams & Hergenrother, 2008). Ala-D-Ser has a sevenfold decrease in affinity for vanco- The most extensively investigated pheromone-inducible mycin, thus removing the susceptible target (Gilmore, plasmids in E. faecalis are pCF10, pAD1 and pPD1. In the 2002). The genes involved in this two-component system case of pAD1 the trans-acting regulatory protein encoded are vanS/vanR. The VanS sensor kinase is activated in by the traE gene is expressed (Folli et al., 2008). The response to vancomycin, resulting in the activation of D- transfer of these plasmids occurs in response to specific sex Lac or D-Ser peptidoglycan precursor and the repression of pheromone peptides secreted by plasmid-free recipient D-Ala-D-Ala (Stephenson & Hoch, 2002). To date six gene cells. Uptake of the exogenous pheromone by the donor clusters associated with glycopeptide resistance have been cell causes it to express proteins involved in the identified in Enterococcus species: vanA to vanG (Table 2). conjugation process. Production of aggregation substance The three main types of resistance are those encoded by the (Agg) on the donor cell surface facilitates contact with the vanC, vanA and vanB clusters. Intrinsic vanC resistance is recipient cell by binding to enterococcal binding substance specific to E. gallinarum, E. casseliflavus and E. flavescens, (EBS) displayed on the surface, resulting in conjugation and the vanC operon is chromosomally located and is not and the ability to pass antibiotic resistance on to the transferable. The vanA resistance operon comprises seven recipient cell (Clewell et al., 2002). The pAD1 plasmid has genes (vanH, vanA, vanX, vanR, vanS, vanY and vanZ) and also been shown to carry the Tn917 transposon associated is acquired through the Tn1546 transposon (Gilmore, with E. faecalis; conjugal transfer of Tn916 involves 2002). Over 100 enterococcal isolates from humans, excision of a circular intermediate that is transferred via animals and food have shown vanA resistance residing on a plasmid into the recipient cell where it inserts into the Tn1546 (Williams & Hergenrother, 2008). The transfer of recipient chromosome (Gilmore, 2002). Pheromones vanB (acquired) resistance occurs through the exchange of released for plasmids pCF10, pAD1 and pPD1 are pAD1 transposon Tn1547 and/or Tn5382. Both vanA and vanB or cCF10, cAD1 and cPD1 respectively (Folli et al., 2008). Table 2. Vancomycin resistance genotypes Adapted from Gilmore (2002). ”1 Genotype Vancomycin MIC (mgml ) Location Expression Precursor vanA 64–1000 Plasmid or chromosome Inducible D-Ala-D-Lac vanB 4–1000 Plasmid or chromosome Inducible D-Ala-D-Lac vanC 2–32 Chromosome Constitutive or inducible D-Ala-D-Ser vanD 64–168 Chromosome Constitutive D-Ala-D-Lac vanE 16 ? Inducible D-Ala-D-Ser vanG ,16 ? ? ? http://mic.sgmjournals.org 1753 K. Fisher and C. Phillips In E. faecalis the proteins RepA, RepB and RepC and the receiving plasmid transfer of the esp gene, were able to par locus are involved in the regulation of the pheromone- produce biofilms (Latasa et al., 2006). Twenty-one out of responding pAD1 replicon. The repA gene encodes a 28 clinical isolates of E. faecium were found to have replication initiator protein, while repB and repC are sequences that were specific for the esp gene. This goes involved in control of the replication frequency and some way to suggesting that the esp gene may be associated stability of the plasmid (Weaver et al., 2009). with pathogenicity, since the esp gene was absent from Enterococcus plasmids can also be utilized for the genetic dairy isolates (Mannu et al., 2003). E. faecium strains that exchange of virulence factors. carry the gene esp have higher conjugation rates than fm strains that do not possess this gene. They also demonstrate higher resistance to ampicillin, ciprofloxacin and imipe- Virulence nem (Billstro¨m et al., 2008). Enterococcus species with the highest virulence are medical The ability of enterococci to produce biofilms is fun- isolates, followed by food isolates and then starter strains damental in causing endodontic and urinary tract infec- (Busani et al., 2004; Ben Omar et al., 2004). Many factors tions, as well as endocarditis. The formation of pili by determine the virulence of Enterococcus species, for enterococci is necessary for biofilm formation, the gene example (1) ability to colonize the gastrointestinal tract, cluster associated with this being ebp (endocarditis- and which is the normal habitat; (2) ability to adhere to a range biofilm-associated pili). The ebp operon consists of ebpA, of extracellular matrix proteins, including thrombospon- ebpB ebpC and an associated srtC (encoding sortase C) gene din, lactoferrin and vitronectin; and (3) ability to adhere to (Singh et al., 2007). A non-piliated mutant of E. faecalis urinary tract epithelia, oral cavity epithelia and human was unable to produce a biofilm (Budzik & Schneewind, embryo kidney cells. Most infection is thought to be 2006). Enterococcal pili are heterotrimetric and the pilus endogenous, by translocation of the bacteria through the shaft contains two minor pilins. A feature of Gram-positive epithelial cells of the intestine, which then cause infection pili is that a specific sortase is dedicated to their assembly via lymph nodes and thus spread to other cells within the (Mandlik et al., 2008). The pili are constructed by cross- body (Franz et al., 1999). The aggregation substance (Agg) linking of multiple classes of precursor proteins that are on the surface of E. faecalis, has been shown in vivo to form assigned by sortases, which covalently anchor proteins with large aggregates and hence may contribute to pathogenesis. a C-terminal pilin-associated motif to the peptidoglycan The presence of Agg increases the hydrophobicity of the (Nallapareddy et al., 2006). E. faecalis contains two classes enterococcal cell surface. This induces localization of of sortase: sortase A links most proteins with a C-terminal cholesterol to the phagosomes and is thought to delay or sortase motif to cell wall peptidoglycan, while sortase C is prevent fusion with lysosomal vesicles (Eaton & Gasson, designated Bps (biofilm and pilus-associated sortase) and 2002). Agg is a pheromone-inducible surface glycoprotein links the pilin subunits. and mediates aggregate formation during conjugation, thus aiding in plasmid transfer as well as adhesion to an array of Secreted virulence factors of Enterococcus species also have a function in pathogenesis. Cytolysin (also called haemo- eukaryotic surfaces (Koch et al., 2004). Pulsed-field gel lysin) is a bacterial toxin, the genes for the production of electrophoresis analysis of clinical isolates of E. faecalis showed that the gene encoding Agg was not present in E. which are located on pheromone-responsive plasmids (Koch et al., 2004). Cytolysin has b-haemolytic properties faecium isolates (Ha¨llgren et al., 2008). Another cell-surface protein present in E. faecalis is Ace (adhesion of collagen in humans and is bactericidal against other Gram-positive bacteria. The cylL group of genes are the non-regulatory from E. faecalis). This is a collagen-binding protein, belonging to the microbial surface components recognizing genes of the cytolysin operons (Ha¨llgren et al., 2008), and adhesive matrix molecules (MSCRAMM) family. Ace may higher incidences of these genes occur in clinical isolates (33 %, compared to 6 % in food isolates) (Semedo et al., play a role in the pathogenesis of endocarditis (Koch et al., 2004). 2003). Cytolysin is regulated by a quorum-sensing mechanism involving a two-component system. Extracellular surface protein (Esp) is a cell-wall-associated protein first described in Enterococcus species by Shankar et A group of hydrolytic enzymes including hyaluronidases, al. (1999). The esp gene consists of 5622 bp and is found at gelatinase and serine protease are involved in the virulence high frequency in infection-derived isolates. It is thought to of Enterococcus species, although their precise roles are yet promote adhesion, colonization and evasion of the to be clearly understood (Semedo et al., 2003). Hyaluronidase acts on hyaluronic acid and is a degradative immune system, and to play some role in antibiotic resistance (Foulquie Moreno et al., 2006). Esp also enzyme which is associated with tissue damage. contributes to enterococcal biofilm formation, which could Hyaluronidase depolymerizes the mucopolysaccharide lead to resistance to environmental stresses, and adhesion moiety of connective tissue, thus facilitating spread of to eukaryotic cells such as those of the urinary tract enterococci as well as their toxins through host tissue (Kayaoglu & Orstavik, 2004). Hyaluronidase is encoded by (Borgmann et al., 2004). Studies have shown that disruption of the esp gene impairs the ability of E. faecalis the chromosomal hyl gene. One study showed that, out of to form biofilms. Esp-negative E. faecalis strains, after 26 vancomycin-resistant E. faecium clinical isolates, seven 1754 Microbiology 155 Enterococcus ecology, epidemiology and virulence (27 %) carried the hyl gene, but it was found in only 14 % thought mainly to be part of the human endogenous non- of faecal isolates (Vankerckhoven et al., 2004). pathogenic microflora (Franz et al., 1999). Recently, enterococci have become one of the most common The main role of both gelatinase and serine protease in nosocomial pathogens, giving a high mortality rate of up enterococcal pathogenesis is thought to be in providing to 61 % (De Fa´tima Silva Lopes et al., 2005). The ability of nutrients to the bacteria by degrading host tissue, although Enterococcus species to survive a range of adverse environ- they also have some function in biofilm formation (Gilmore, ments (Van den Berghe et al., 2006) allows multiple routes 2002; Mohamed & Huang, 2007). Gelatinase (GelE) is an of cross-contamination of enterococci in causing human extracellular zinc metallo-endopeptidase secreted by E. disease, including those from food, environmental and faecalis (Koch et al., 2004). It is able to hydrolyse gelatin, hospital sources. Overall, greater understanding of the casein, haemoglobin and other bioactive peptides. The gene ability of Enterococcus species to survive stresses, of (gelE) encoding GelE is located on the chromosome and is virulence traits, and especially of increasing antibiotic regulated in a cell-density-dependent manner. Another gene resistance, is needed in order to fully appreciate the sprE, coding for a serine protease, is located directly complexity of Enterococcus species in causing disease. downstream from and is cotranscribed with gelE (De Fa´tima Silva Lopes et al., 2006). Transcription of gelE and sprE is regulated in a growth-phase-dependent fashion by References the quorum-sensing system encoded by the fsr (faecal Alksne, L. E. & Projan, S. J. (2000). Bacterial virulence as a target for streptococci regulator) locus (Sifri et al., 2002). antimicrobial chemotherapy. Curr Opin Biotechnol 11, 625–636. Quorum sensing occurs when a bacterial population Andrup, L. & Andersen, K. (1999). A comparison of the kinetics of produces a signal via an autoinducing peptide (AIP), plasmid transfer in the conjugation systems encoded by the F plasmid regulated by a two-component system. AIP then accumu- from Escherichia coli and plasmid pCF10 from Entercoccus faecalis. lates in the environment by increased expression of the Microbiology 145, 2001–2009. communication signal, or by increased numbers of cells Bearman, G. M. L. & Wenzel, R. P. (2005). Bacteraemias: a leading producing the signal. Once the AIP reaches a threshold cause of death. Arch Med Res 36, 646–659. concentration, it interacts with a cell-surface receptor or re- Ben Omar, N., Castro, A., Lucas, R., Abriouel, H., Yousif, N. M., Franz, enters the cell and causes a cascade of transcriptional C. M., Holzapfel, W. H., Pe ´ rez-Pulido, R., Martı´nez-Can ˜ amero, M. & Ga ´ lvez, A. (2004). Functional and safety aspects of enterococci isolated regulation (Alksne & Projan, 2000; Gobbetti et al., 2007). from different Spanish foods. Syst Appl Microbiol 27, 118–130. The fsr locus contains the fsrA, fsrB and fsrC genes. The fsrA gene is monocistronically transcribed into a response Billstro ¨ m, H., Lund, B., Sullivan, A. & Nord, C. E. (2008). Virulence and antimicrobial resistance in clinical Enterococcus faecium. Int J regulator, and fsrB and fsrC, encoding a processing enzyme Antimicrob Agents 32, 374–377. and a sensor kinase respectively, are co-transcribed Borgmann, S., Niklas, D. M., Klare, I., Zabel, L. T., Buchenau, P., (Podbielski & Kreikemeyer, 2004). FsrB liberates gelatinase Autenrieth, I. B. & Heeg, P. (2004). Two episodes of vancomycin- biosynthesis activating pheromone (GBAP) peptide, and resistant Enterococcus faecium outbreaks caused by two genetically with the accumulation of GBAP a transition from different clones in a newborn intensive care unit. Int J Hyg Environ exponential to stationary phase occurs and gelE and sprE Health 207, 386–389. are induced. It has been shown that in E. faecalis when Brown, D. F. J., Brown, N. M., Cookson, B. 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The ecology, epidemiology and virulence of Enterococcus

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Microbiology (2009), 155, 1749–1757 DOI 10.1099/mic.0.026385-0 The ecology, epidemiology and virulence of Review Enterococcus Katie Fisher and Carol Phillips Correspondence University of Northampton, School of Health, Park Campus, Boughton Green Road, Northampton Katie Fisher NN2 7AL, UK Katie.fisher@northampton.ac.uk Enterococci are Gram-positive, catalase-negative, non-spore-forming, facultative anaerobic bacteria, which usually inhabit the alimentary tract of humans in addition to being isolated from environmental and animal sources. They are able to survive a range of stresses and hostile environments, including those of extreme temperature (5–65 6C), pH (4.5”10.0) and high NaCl concentration, enabling them to colonize a wide range of niches. Virulence factors of enterococci include the extracellular protein Esp and aggregation substances (Agg), both of which aid in colonization of the host. The nosocomial pathogenicity of enterococci has emerged in recent years, as well as increasing resistance to glycopeptide antibiotics. Understanding the ecology, epidemiology and virulence of Enterococcus speciesisimportant for limiting urinary tract infections, hepatobiliary sepsis, endocarditis, surgical wound infection, bacteraemia and neonatal sepsis, and also stemming the further development of antibiotic resistance. Introduction Taxonomy For many years Enterococcus species were believed to be The genus Enterococcus consists of Gram-positive, catalase- harmless to humans and considered unimportant med- negative, non-spore-forming, facultative anaerobic bacteria ically. Because they produce bacteriocins, Enterococcus that can occur both as single cocci and in chains. species have been used widely over the last decade in the Enterococci belong to a group of organisms known as food industry as probiotics or as starter cultures (Foulquie lactic acid bacteria (LAB) that produce bacteriocins Moreno et al., 2006). Recently, enterococci have become (Health Protection Agency, 2005). The genera of LAB with one of the most common nosocomial pathogens, with which Enterococcus are grouped are identified by a low patients having a high mortality rate of up to 61 % (De G+C content of ,50 mol% (Klein et al., 1998). There are Fa´tima Silva Lopes et al., 2005). no phenotypic characteristics to distinguish Enterococcus species from other Gram-positive, catalase-negative cocci In 2005 there were 7066 reported cases of bacteraemia bacteria, so identification is usually established by reverse caused by Enterococcus species in the UK, an 8 % increase methodology (elimination of other species traits first). As a from 2004, with the Health Protection Agency (2007) genus Enterococcus has been recognized since 1899, when stating that ‘an increase in a bacteraemia causing pathogen Thiercelin identified it as an intestinal organism (Stiles & like this has not been observed for some time’. Twenty- Holzapfel, 1997); its taxonomy and ecology were reviewed eight per cent of all cases were antibiotic resistant (Health by Klein (2003). Many attempts have been made to Protection Agency, 2007). The risk of death from distinguish Enterococcus species from Streptococcus species. vancomycin-resistant enterococci (VRE) is 75 %, compared In 1937, Sherman classified Streptococcus species into four with 45 % for those infected with a susceptible strain subgroups: faecal streptococci (enterococci), dairy strep- (Bearman & Wenzel, 2005). These figures are mirrored in tococci, viridans group and pyogenous streptococci (Klein, the USA. Over a 15 year period there was a 20-fold increase 2003). Sherman noted that the enterococci subgroup in VRE associated with nosocomial infections reported to included the Lancefield group D streptococci and suggested CDC’s National Nosocomial Infections Surveillance that the latter could be differentiated by haemolytic and (NNIS) (National Nosocomial Infections Surveillance, proteolytic reactions, although this is inappropriate as 2004). haemolysis is determined by a plasmid (Stiles & Holzapfel, This dramatic increase in antibiotic resistance of 1997). Traditional methods such as biotyping, serotyping Enterococcus species worldwide highlights the need for a and phage typing left questions as to which of the greater understanding of this genus, including its ecology, Streptococcus species actually belonged to the genus epidemiology and virulence. Enterococcus (Saeedi et al., 2002). 026385 2009 SGM Printed in Great Britain 1749 K. Fisher and C. Phillips In 1984, through the use of DNA hybridization and 16S enzyme activities such as pyroglutamyl aminopeptidase rRNA sequencing, it was established that the species (PYRase) (Domig et al., 2003), growth at defined Streptococcus faecium and Streptococcus faecalis were temperatures and physiological characteristics is essential sufficiently distinct from the other streptococci to be in the identification of Enterococcus species (Shanks et al., 2006). designated another genus: Enterococcus (Foulquie Moreno et al., 2006). This means that the D group antigen is found The differences in the genomes of E. faecalis and E. faecium in both streptococci and enterococci. Nine species were were assessed in a study using competitive DNA hybrid- transferred from the Streptococcus groups and now ization (Shanks et al., 2006). E. faecalis-specific sequences Enterococcus includes 28 species (Foulquie Moreno et al., compared with those of E. faecium mainly encoded surface- 2006). The molecular data that were collected using 16S exposed proteins. Overall 6.4 % of the Enterococcus genome rRNA sequencing of Streptococcus enabled the construction is associated with cell-surface proteins and 22.6 % of the of an 16S rRNA-dendrogram showing the relationship differences between the two species are found in these between Streptococcus, Enterococcus and Lactococcus species genes. This variation is thought to have implications in the (Fig. 1). This method also allowed the grouping of species avoiding different host immune responses (Shanks Enterococcus species. The Enterococcus faecalis species group et al. 2006). includes E. faecalis, Enterococcus haemoperoxidus and Enterococcus moraviensis whilst the Enterococcus faecium species group includes E. faecium, Enterococcus durans, Physiology Enterococcus hirae, Enterococcus mundtii, Enterococcus Enterococcus species will grow at a range of temperatures porcinus and Enterococcus villorum (Klein, 2003). The from 5 to 50 C. The optimum, minimum and maximum discrimination of enterococci from streptococci is mainly temperatures, according to the Rosso model, are 42.7, 6.5 established by Lancefield group D antigen, as only and 47.8 C, respectively, on brain heart infusion (BHI) Streptococcus bovis, Streptococcus alactolyticus and agar in aerobic conditions (Van den Berghe et al., 2006), Streptococus equinus are serogroup D. These groups can although growth will also occur in anaerobic atmospheres be distinguished from Enterococcus species by the lack of (Domig et al., 2003). Both E. faecalis and E. faecium can growth in 6.5 % (w/v) sodium chloride at 10 C. It is survive heating at 60 C for 30 min, making Enterococcus harder to distinguish Enterococcus species from other cocci species distinguishable from other closely related genera that do not express the D group antigen such as such as Streptococcus (Foulquie Moreno et al., 2006). Pediococcus, Lactococcus or Tetragenococcus species because Trypticase soy agar or Columbia agar with 5 % (v/v) no other phenotypic differences have been reported that defibrinated sheep blood may be used to assess the allow distinction. Thus the use of fermentation patterns, haemolysis produced by enterococci. If human or horse blood is used, haemolysis is based on cytolysin activity and causes a b-haemolytic reaction (Domig et al., 2003). E. faecalis and E. faecium will grow in a wide range of pH (4.6–9.9), with the optimum being 7.5 (Van den Berghe et al., 2006). They will also tolerate and grow in the presence of 40 % (w/v) bile salts. E. faecalis is able to grow in 6.5 % NaCl and has a cation homeostasis which is thought to contribute to its resistance to pH, salt, metals and desiccation. When assessing growth of Enterococcus species using optical densities the most important variable of the growth conditions is pH, with temperature and salt concentration having a lesser effect (Gardin et al., 2001). During the lag phase, temperature is the most important factor influ- encing growth, with stationary-phase cells being the most resistant to heat (Gardin et al., 2001; Martinez et al., 2003). The resistance of E. faecalis to a range of pH values is thought to be due to its membrane durability and impermeabilty to acid and alkali, although some studies have suggested that it may be associated with membrane- bound H -ATPase activity (Nakajo et al., 2005). Temperature resistance is also associated with membrane structure and has been related to lipid and fatty acid content. The membrane has been demonstrated to be more Fig. 1. 16S rRNA dendrogram of phylogenetic position of stable near the minimal temperature for growth, which is a Enterococcus species (adapted from Klein, 2003). specific mechanism associated with enterococci (Ivanov et 1750 Microbiology 155 Enterococcus ecology, epidemiology and virulence al., 1999). At higher temperatures enterococci are less cell. This occurs by the enzymic removal of an N-terminal resilient, with the membrane fatty acid content increasing leader peptide at a double glycine cleavage site, and export and the saturated fatty acid levels decreasing. The heat via a Sec-dependent pathway. Bacteriocins are cationic, resistance of enterococci is dependent not only on the amphiphilic proteins containing little or no cysteine, and temperature but also the phase of growth (Martinez et al., their structures usually occur as random coils under 2003). aqueous conditions (Garneau et al., 2002). Bacteriocin production is favoured in stressful growth conditions, When E. faecalis is grown at non-stress temperatures, which is thought to be due to lower growth rates, resulting subsequently cultured cells do not have the resilience to in better utilization of energy and greater availability of warm and cold environments that would occur if the first metabolites for the synthesis of bacteriocins. Under generation were grown at stressful temperatures (Ivanov optimal growth conditions and thus high growth rates et al., 1999). Three distinct temperature groups (10–13 C, there is a lack of amino acids available for bacteriocin u u 17–22 C and 42–47 C) have been established for E. faecalis production (Van den Berghe et al., 2006). Enterococcus at which permeability of the membrane to 3 % NaCl is species are known to produce a range of enterocins different. This has significant implications with regard to (Table 1) including enterocins A, B, I, L and P, which are biotechnology and food science (Ivanov et al., 1999). active against Listeria species, Clostridum species and The production of amines is also closely related to the Staphylococcus aureus (Campos et al., 2006). Most of the growth temperature and pH. The production of decarbox- bacteriocins produced by E. faecalis and E. faecium are ylases is optimum at acid pH, whereas biogenic amine identical to enterocins A and B first described from E. production by E. faecalis EF37 decreases at low pH. faecium CTC492 and E. faecium T136 (De Kwaadsteniet Temperature does not have a significant effect on amine et al., 2005). E. faecium RZS C5 is a natural cheese isolate, production itself, but the effect that temperature has on cell which is lacking in virulence factors and has antilisterial yield alters the quantity of amines being produced (Gardin properties (Leroy et al., 2003). Enterocin EJ97 from E. et al., 2001). Other products of Enterococcus species that are faecalis S-47 has also shown antilisterial properties, with 1.6 affected by pH are bacteriocins. log reduction after 6 h and complete inhibition after 24 h at a concentration of 20 AU ml (Garcia et al., 2004). The enterocin AS-48 (produced by E. faecalis A-48-32) inhibits Bacteriocins growth of Bacillus coagulans vegetative cells, at refrigeration and high temperatures, in canned fruits and vegetables Bacteriocins are ribosomally synthesized, extracellular released antimicrobial peptides that show activity against (Lucas et al., 2006). The bacteriocin activity against Gram- negative bacteria is unusual, but bacteriocin ST15 from E. closely related bacterial species. Four genes are required to mundtii has been shown to be effective against a range of produce bacteriocins: a structural gene encoding a prepeptide, a dedicated immunity gene, a dedicated ABC- Gram-positive and Gram-negative bacteria including Acinetobacter, Bacillus, Clostridium, Klebsiella, Lacto- immunity gene and a gene encoding the protein necessary bacillus and Pseudomonas (De Kwaadsteniet et al., 2005). for export of the bacteriocin. Bacteriocins are formed as prepeptides in the cell and mature during export from the The bactericidal effects of bacteriocins are thought to be Table 1. Bacteriocins produced by Enterococcus species Adapted from De Kwaadsteniet et al. (2005). Bacteriocin Produced by Isolated from Size (Da) Enterocins A and B E. faecium P21 Chorizo Enterocin EJ97 E. faecalis S-47 No name E. faecium A2000 Cheese Enterocin CRL35 E. faecium CRL35 Cheese 3500 Bacteriocin N15 E. faecium N15 Nuka 3000–5000 Enterocins A and B E. faecium WHE81 Cheese 4833 and 5462 No name E. faecium RZS C5; E. faecium DPC 1146 AS-48 E. faecalis subsp. liquefaciens S-48 Porcine intestinal tract Enterocin 012 E. gallinarum Duodenum of ostrich 3400 No name E. faecium CRL 1385 Free-range chicken Enterocin P E. faecium P13 Dry-fermented sausage Enterocins 1071A and 1071B E. faecalis BFE 1071 Faeces of minipigs 4285 and 3899 Mundticin ATO6 E. mundtii ATO6 Vegetables 4287 Mundticin KS E. mundtii NFRI 7393 Grass silage 4290 http://mic.sgmjournals.org 1751 K. Fisher and C. Phillips due to permeabilization of the cell membrane. The of E. faecalis whereas healthy individuals show only a 39– random-coiled peptides of the bacteriocins, on contact 40 % occurrence (Mutnick et al., 2003). Hospitalized with the cell membrane, form a helical structure, which patients may have a greater incidence of enterococcal incorporates into and spans the membrane, creating a pore. infection not only because of virulence, but because the This mode of action has been observed in enterocin P. The hospital itself is a hub. This is illustrated by a report for resulting pore causes leakage of K ions, dissipation of the Department of Health in the UK, which highlighted the membrane potential and inhibition of amino acid uptake. fact that enterococci may contaminate and survive around The cycle of ATP-driven K uptake by the cell and the patient for several days (Brown et al., 2006). bacteriocin-mediated release of K leads to cell death Enterococci also play a role in endodontic failure and are (Garneau et al., 2002; Hechard & Sahl, 2002). often isolated from the root canal system. The results of one study showed that out of 100 root-filled teeth with apical periodontitis, 69 % of the isolated bacteria were Ecology and epidemiology facultative and 50 % of those were enterococci (Dahlen et al., 2000). E. faecalis is responsible for 80–90 % of human The origins of Enterococcus species vary from envir- onmental to animal and human sources. As enterococci enterococcal endodontic infection and is usually the only Enterococcus species isolated from the obturated root canal are an essential part of the microflora of both humans and animals their distribution is very similar in these sources. E. (Love, 2001; Peciuliene et al., 2001). faecium and E. faecalis are the most common in the human gastrointestinal tract, E. faecium in production animals and Rates of infection E. mundtii and E. casseliflavus in plant sources (Klein, 2003). The numbers of E. faecalis in human faeces range Enterococcal infections include urinary tract infections, 5 7 4 from 10 to 10 per gram, and those of E. faecium from 10 hepatobiliary sepsis, endocarditis, surgical wound infec- to 10 per gram. The isolation of E. faecium and E. faecalis tion, bacteraemia and neonatal sepsis (Poh et al., 2006). In is less prevalent from livestock than from human faeces Europe, infection with Enterococcus species was considered (Franz et al., 1999). harmless to humans for a long time. However in the last decade enterococci have been reported as the second most Studies of the ecology and epidemiology of Enterococcus common cause of wound and urinary tract infection and have reported E. faecalis and E. faecium being regularly the third most common cause of bacteraemia (De Fa´tima isolated from cheese, fish, sausages, minced beef and pork Silva Lopes et al., 2005). In 2005 in the UK there were 7066 (Foulquie Moreno et al., 2006; Klein, 2003). Foods such as reported cases of Enterococcus bacteraemia, 63 % of these sausages and cheese that are of animal origin are often cases being due to E. faecalis and 28 % to E. faecium, both associated with contamination by Enterococcus species, as of which have increasing antibiotic resistance (Health they are able to survive the heating process. In one study in Protection Agency, 2007). In the USA approximately 12 % the UK, samples taken from urban sewage and from of the hospital-acquired infections are Enterococcus species. farmland using pig manure and crops generated from this E. faecalis is the most common species associated with land, were found to be 100 % positive for Enterococcus clinical infection while E. faecium poses the higher species In crops to which animal fertilizers were not antibiotic resistance threat (Giraffa, 2002). applied, the incidence of Enterococcus species was reduced to 33 % (Kuhn et al., 2003). A similar study in Germany isolated 416 strains of Enterococcus from 155 samples of Antibiotic resistance food of animal origin, 72 % of which were E. faecalis and The antibiotic resistance of Enterococcus is well documen- 13 % E. faecium (Peters et al., 2003). ted. Bacteria may show resistance to glycopeptides such as The distribution of Enterococcus species varies throughout vancomycin and teicoplanin, which are licensed in the UK, Europe. In Spain and the UK, E. faecalis and E. faecium are and to aminoglycosides (Kacmaz & Aksoy, 2005). the most commonly isolated species from both clinical and Antibiotic resistance has been of growing concern for a environmental sources. Sweden has a lower incidence of E. number of years. Vancomycin was first used in the clinical faecium and a higher isolation rate of E. hirae, whereas in arena in 1972 and the first vancomycin-resistant entero- Denmark E. hirae is the dominant species and is isolated cocci were recognized only 15 years later. NNIS reported an mainly from slaughtered animals (Kuhn et al., 2003). increase of 7.6 % in VRE between 1989 and 1993 (Metan et al., 2005). It has been reported that if glycopeptide- Clinical isolates of enterococci show a lower diversity than those obtained from the environment and other human resistant enterococci (GRE) are present in an infected patient rather than an antibiotic-susceptible strain, clinical sources, with E. faecalis being the dominant species (Kuhn et al., 2003). The reason for this lack of diversity may be treatment failure is increased by 20 % and mortality is linked with the virulence factors associated with this increased from 27 % to 52 % (Brown et al., 2006). When assessing the studies on enterococcal antibiotic resistance, species. The fact that Enterococcus species are opportunistic pathogens was highlighted by a study in Denmark which the pattern that is emerging is the possible occurrence of showed that hospitalized patients have a 57 % isolation rate multidrug resistant strains (Peters et al., 2003). 1752 Microbiology 155 Enterococcus ecology, epidemiology and virulence In both the Surveillance and Control of Pathogens of are present on the chromosome but can also be carried on Epidemiological Importance (SCOPE) and SENTRY a plasmid (Gilmore, 2002; Klare et al., 2003). Enterococcus (Antimicrobial Resistance Surveillance Program) data- species do not possess cytochrome enzymes and thus bases, figures show that, of enterococcal isolates from the cannot produce the energy required to take up antibiotics bloodstream, 2 % of E. faecalis and 60 % of E. faecium into the cell. This means they show resistance to isolates are resistant to vancomycin (Bearman & Wenzel, aminoglycosides at low levels (Klare et al., 2003). 2005). Resistance rates of Enterococcus species have reached Antibiotic resistance in Enterococcus species can be endemic or epidemic proportions in North America, with transferred by pheromone-mediated conjugative plasmids Europe having lower, but increasing, levels (Mutnick et al., or transposons. The resistance genes may be passed on not 2003). Enterococcal antibiotic resistance is not exclusive to only to antibiotic-susceptible enterococci, but also to other the clinical arena but is also prevalent in the food industry. pathogens (Giraffa, 2002). The presence of VRE in individuals who have been In contrast to Gram-negative conjugation systems, con- hospitalized, when they have not previously been in jugation of Enterococcus species does not require pili, and hospital or taken antibiotics, suggests that VRE may have involves a pheromone-induced system (Andrup & been contracted through the food chain. GRE may emerge Andersen, 1999). Bacteria containing conjugative plasmids in the food chain through use of avoparcin in animal feed respond to pheromones (plasmid specific) for genetic (Mannu et al., 2003). exchange; these bacteria generally have a narrow recipient Glycopeptide resistance in enterococci involves a two- range for conjugation, including only closely related component system where the cell wall composition is species. This lateral transfer of genetic elements leads to altered from the peptidoglycan precurser D-Ala-D-Ala rapid dissemination of antibiotic resistance. The plasmids (vancomycin-susceptible) to D-Ala-D-lactate (D-Lac). The occurring in Enterococcus species can also be transmission latter has 1000 times less affinity for vancomycin, while D- vehicles for transposons (Williams & Hergenrother, 2008). Ala-D-Ser has a sevenfold decrease in affinity for vanco- The most extensively investigated pheromone-inducible mycin, thus removing the susceptible target (Gilmore, plasmids in E. faecalis are pCF10, pAD1 and pPD1. In the 2002). The genes involved in this two-component system case of pAD1 the trans-acting regulatory protein encoded are vanS/vanR. The VanS sensor kinase is activated in by the traE gene is expressed (Folli et al., 2008). The response to vancomycin, resulting in the activation of D- transfer of these plasmids occurs in response to specific sex Lac or D-Ser peptidoglycan precursor and the repression of pheromone peptides secreted by plasmid-free recipient D-Ala-D-Ala (Stephenson & Hoch, 2002). To date six gene cells. Uptake of the exogenous pheromone by the donor clusters associated with glycopeptide resistance have been cell causes it to express proteins involved in the identified in Enterococcus species: vanA to vanG (Table 2). conjugation process. Production of aggregation substance The three main types of resistance are those encoded by the (Agg) on the donor cell surface facilitates contact with the vanC, vanA and vanB clusters. Intrinsic vanC resistance is recipient cell by binding to enterococcal binding substance specific to E. gallinarum, E. casseliflavus and E. flavescens, (EBS) displayed on the surface, resulting in conjugation and the vanC operon is chromosomally located and is not and the ability to pass antibiotic resistance on to the transferable. The vanA resistance operon comprises seven recipient cell (Clewell et al., 2002). The pAD1 plasmid has genes (vanH, vanA, vanX, vanR, vanS, vanY and vanZ) and also been shown to carry the Tn917 transposon associated is acquired through the Tn1546 transposon (Gilmore, with E. faecalis; conjugal transfer of Tn916 involves 2002). Over 100 enterococcal isolates from humans, excision of a circular intermediate that is transferred via animals and food have shown vanA resistance residing on a plasmid into the recipient cell where it inserts into the Tn1546 (Williams & Hergenrother, 2008). The transfer of recipient chromosome (Gilmore, 2002). Pheromones vanB (acquired) resistance occurs through the exchange of released for plasmids pCF10, pAD1 and pPD1 are pAD1 transposon Tn1547 and/or Tn5382. Both vanA and vanB or cCF10, cAD1 and cPD1 respectively (Folli et al., 2008). Table 2. Vancomycin resistance genotypes Adapted from Gilmore (2002). ”1 Genotype Vancomycin MIC (mgml ) Location Expression Precursor vanA 64–1000 Plasmid or chromosome Inducible D-Ala-D-Lac vanB 4–1000 Plasmid or chromosome Inducible D-Ala-D-Lac vanC 2–32 Chromosome Constitutive or inducible D-Ala-D-Ser vanD 64–168 Chromosome Constitutive D-Ala-D-Lac vanE 16 ? Inducible D-Ala-D-Ser vanG ,16 ? ? ? http://mic.sgmjournals.org 1753 K. Fisher and C. Phillips In E. faecalis the proteins RepA, RepB and RepC and the receiving plasmid transfer of the esp gene, were able to par locus are involved in the regulation of the pheromone- produce biofilms (Latasa et al., 2006). Twenty-one out of responding pAD1 replicon. The repA gene encodes a 28 clinical isolates of E. faecium were found to have replication initiator protein, while repB and repC are sequences that were specific for the esp gene. This goes involved in control of the replication frequency and some way to suggesting that the esp gene may be associated stability of the plasmid (Weaver et al., 2009). with pathogenicity, since the esp gene was absent from Enterococcus plasmids can also be utilized for the genetic dairy isolates (Mannu et al., 2003). E. faecium strains that exchange of virulence factors. carry the gene esp have higher conjugation rates than fm strains that do not possess this gene. They also demonstrate higher resistance to ampicillin, ciprofloxacin and imipe- Virulence nem (Billstro¨m et al., 2008). Enterococcus species with the highest virulence are medical The ability of enterococci to produce biofilms is fun- isolates, followed by food isolates and then starter strains damental in causing endodontic and urinary tract infec- (Busani et al., 2004; Ben Omar et al., 2004). Many factors tions, as well as endocarditis. The formation of pili by determine the virulence of Enterococcus species, for enterococci is necessary for biofilm formation, the gene example (1) ability to colonize the gastrointestinal tract, cluster associated with this being ebp (endocarditis- and which is the normal habitat; (2) ability to adhere to a range biofilm-associated pili). The ebp operon consists of ebpA, of extracellular matrix proteins, including thrombospon- ebpB ebpC and an associated srtC (encoding sortase C) gene din, lactoferrin and vitronectin; and (3) ability to adhere to (Singh et al., 2007). A non-piliated mutant of E. faecalis urinary tract epithelia, oral cavity epithelia and human was unable to produce a biofilm (Budzik & Schneewind, embryo kidney cells. Most infection is thought to be 2006). Enterococcal pili are heterotrimetric and the pilus endogenous, by translocation of the bacteria through the shaft contains two minor pilins. A feature of Gram-positive epithelial cells of the intestine, which then cause infection pili is that a specific sortase is dedicated to their assembly via lymph nodes and thus spread to other cells within the (Mandlik et al., 2008). The pili are constructed by cross- body (Franz et al., 1999). The aggregation substance (Agg) linking of multiple classes of precursor proteins that are on the surface of E. faecalis, has been shown in vivo to form assigned by sortases, which covalently anchor proteins with large aggregates and hence may contribute to pathogenesis. a C-terminal pilin-associated motif to the peptidoglycan The presence of Agg increases the hydrophobicity of the (Nallapareddy et al., 2006). E. faecalis contains two classes enterococcal cell surface. This induces localization of of sortase: sortase A links most proteins with a C-terminal cholesterol to the phagosomes and is thought to delay or sortase motif to cell wall peptidoglycan, while sortase C is prevent fusion with lysosomal vesicles (Eaton & Gasson, designated Bps (biofilm and pilus-associated sortase) and 2002). Agg is a pheromone-inducible surface glycoprotein links the pilin subunits. and mediates aggregate formation during conjugation, thus aiding in plasmid transfer as well as adhesion to an array of Secreted virulence factors of Enterococcus species also have a function in pathogenesis. Cytolysin (also called haemo- eukaryotic surfaces (Koch et al., 2004). Pulsed-field gel lysin) is a bacterial toxin, the genes for the production of electrophoresis analysis of clinical isolates of E. faecalis showed that the gene encoding Agg was not present in E. which are located on pheromone-responsive plasmids (Koch et al., 2004). Cytolysin has b-haemolytic properties faecium isolates (Ha¨llgren et al., 2008). Another cell-surface protein present in E. faecalis is Ace (adhesion of collagen in humans and is bactericidal against other Gram-positive bacteria. The cylL group of genes are the non-regulatory from E. faecalis). This is a collagen-binding protein, belonging to the microbial surface components recognizing genes of the cytolysin operons (Ha¨llgren et al., 2008), and adhesive matrix molecules (MSCRAMM) family. Ace may higher incidences of these genes occur in clinical isolates (33 %, compared to 6 % in food isolates) (Semedo et al., play a role in the pathogenesis of endocarditis (Koch et al., 2004). 2003). Cytolysin is regulated by a quorum-sensing mechanism involving a two-component system. Extracellular surface protein (Esp) is a cell-wall-associated protein first described in Enterococcus species by Shankar et A group of hydrolytic enzymes including hyaluronidases, al. (1999). The esp gene consists of 5622 bp and is found at gelatinase and serine protease are involved in the virulence high frequency in infection-derived isolates. It is thought to of Enterococcus species, although their precise roles are yet promote adhesion, colonization and evasion of the to be clearly understood (Semedo et al., 2003). Hyaluronidase acts on hyaluronic acid and is a degradative immune system, and to play some role in antibiotic resistance (Foulquie Moreno et al., 2006). Esp also enzyme which is associated with tissue damage. contributes to enterococcal biofilm formation, which could Hyaluronidase depolymerizes the mucopolysaccharide lead to resistance to environmental stresses, and adhesion moiety of connective tissue, thus facilitating spread of to eukaryotic cells such as those of the urinary tract enterococci as well as their toxins through host tissue (Kayaoglu & Orstavik, 2004). Hyaluronidase is encoded by (Borgmann et al., 2004). Studies have shown that disruption of the esp gene impairs the ability of E. faecalis the chromosomal hyl gene. One study showed that, out of to form biofilms. Esp-negative E. faecalis strains, after 26 vancomycin-resistant E. faecium clinical isolates, seven 1754 Microbiology 155 Enterococcus ecology, epidemiology and virulence (27 %) carried the hyl gene, but it was found in only 14 % thought mainly to be part of the human endogenous non- of faecal isolates (Vankerckhoven et al., 2004). pathogenic microflora (Franz et al., 1999). Recently, enterococci have become one of the most common The main role of both gelatinase and serine protease in nosocomial pathogens, giving a high mortality rate of up enterococcal pathogenesis is thought to be in providing to 61 % (De Fa´tima Silva Lopes et al., 2005). The ability of nutrients to the bacteria by degrading host tissue, although Enterococcus species to survive a range of adverse environ- they also have some function in biofilm formation (Gilmore, ments (Van den Berghe et al., 2006) allows multiple routes 2002; Mohamed & Huang, 2007). Gelatinase (GelE) is an of cross-contamination of enterococci in causing human extracellular zinc metallo-endopeptidase secreted by E. disease, including those from food, environmental and faecalis (Koch et al., 2004). It is able to hydrolyse gelatin, hospital sources. Overall, greater understanding of the casein, haemoglobin and other bioactive peptides. The gene ability of Enterococcus species to survive stresses, of (gelE) encoding GelE is located on the chromosome and is virulence traits, and especially of increasing antibiotic regulated in a cell-density-dependent manner. Another gene resistance, is needed in order to fully appreciate the sprE, coding for a serine protease, is located directly complexity of Enterococcus species in causing disease. downstream from and is cotranscribed with gelE (De Fa´tima Silva Lopes et al., 2006). Transcription of gelE and sprE is regulated in a growth-phase-dependent fashion by References the quorum-sensing system encoded by the fsr (faecal Alksne, L. E. & Projan, S. J. (2000). Bacterial virulence as a target for streptococci regulator) locus (Sifri et al., 2002). antimicrobial chemotherapy. Curr Opin Biotechnol 11, 625–636. Quorum sensing occurs when a bacterial population Andrup, L. & Andersen, K. (1999). A comparison of the kinetics of produces a signal via an autoinducing peptide (AIP), plasmid transfer in the conjugation systems encoded by the F plasmid regulated by a two-component system. 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