Keywords

1 Introduction

From the last three decades, Enterococcus spp. have emerged as one of the important etiological agents of hospital-associated infections in immunocompromised individuals and in patients with prolonged antibiotic treatment and hospital stay (Gilmore et al. 2013). The two species Enterococcus faecalis and E. faecium out of 50 known species of enterococci are particularly pathogenic to man; for example, E. faecalis accounts for 85–90%, and E. faecium accounts for 5–10% of all Enterococcal diseases (Maki and Agger 1988). Other species that can also be pathogenic include E. gallinarum, E. casseliflavus, E. durans, and E. mundtii. In the USA and Europe, enterococci have been reported to be the second and third leading cause of nosocomial infections (Sievert et al. 2013; Zarb et al. 2012). In India, enterococci accounted for 2.3–9.7% of all nosocomial infections (Sreeja et al. 2012; Chakraborty et al. 2015; Karmarkar et al. 2004). Enterococci have immense genome plasticity, are prolific colonizers, and have the ability to persist in hospital environment for a long time owing to their sturdy nature. The various clinical manifestations of Enterococcal infection are urinary tract infections, bacteremia, endocarditis, and intra-abdominal infections (Murray 1990). Apart from E. faecalis, the incidence of nosocomial E. faecium infections is also on the rise, primarily due to antibiotic resistance in E. faecium. The ability to cause infection in E. faecium is mainly attributed to the increasing resistance to antibiotics, whereas E. faecalis has been reported to exhibit innate ability to cause infections irrespective of the antibiotic resistance (Mundy et al. 2000). To better understand the molecular mechanisms involved in the pathogenicity of Enterococcus, the whole genome sequences of various pathogenic (Qin et al. 2012) and commensal (Brede et al. 2011) Enterococcus spp. have been published and compared.

2 General Characterization of Enterococci

Enterococci are Gram-positive, facultative anaerobic bacteria, which are ovoid in shape and may occur in diploids, in chains, or as single cells (Fig. 15.1). They are catalase- and cytochrome c oxidase-negative. According to the classification by Sherman (1937), the bacteria belonging to Enterococcus genera possess characteristic ability to grow at 10° and 45 °C, in media containing 6.5% NaCl, and do not produce gas from glucose (Schleifer and Kilpper-Bälz 1984). Hydrolysis of L-pyrrolidonyl-3-naphthylamide and group D antisera test is also one of the characteristic features of enterococci genera. The genera fall in low GC branch of bacteria, and G + C content ranges between 37 and 45 mol%. Species-level identification of Enterococcus genera is generally done by 16S rDNA sequencing or whole cell protein analysis. Till date approximately 50 species of Enterococcus have been reported. The prominent species present in the gut of animals and humans is E. faecium followed by E. faecalis (Silva et al. 2012).

Fig. 15.1
figure 1

(a) Photomicrograph showing Gram-positive coccus-shaped Enterococcal cells in the pulmonary tissue. (b) Scientific classification of Enterococcus genera

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3 Habitat

Enterococci are ubiquitous members of gastrointestinal tracts (GIT) of various organisms. In the 1960s and 1970s, enterococci were isolated from GIT of reptiles (85.7%), mammals (71.3%), insects (53%), and birds (31.8%) by Mundt et al. Enterococci have also been isolated from plants (Mundt 1963), soil (Mundt 1961), water, and fermented foods. It is postulated that enterococci may have been one of the earliest members of the GIT with their existence dating back from early Devonian period (Gilmore et al. 2013). Due to their role in highly evolved and extremely competitive environment of GIT, enterococci have well-adapted and reduced genomes ranging from 2.6 to 3.6 Mb. These bacteria are fastidious in nature and draw a number of vitamins and amino acids from their habitats (Niven and Sherman 1944). They have reduced their genome by eliminating genes necessary for the biosynthesis of amino acids and vitamins from simpler precursors.

4 Enterococcal Diseases

4.1 Endocarditis

Enterococcus spp. are known to cause endocarditis since 1899, when the first clinical description of the strain which was most certainly E. faecalis was published (MacCallum and Hastings 1899). Enterococci are the third main causative agents of infective endocarditis (IE) and prosthetic valve endocarditis worldwide after Streptococcus spp. and Staphylococcus spp. Worldwide approximately 10–15% IE cases are caused by enterococci (Murdoch et al. 2009). Frequency of Enterococcal IE is more in elderly debilitated patients with prior valvular damage, intracardiac devices, or a prosthetic valve. It occurs more in men than women (McDonald et al. 2005). Most common clinical findings of IE include subacute fever and the presence of a cardiac murmur. Almost half of the IE patients suffer from heart failure. Mortality rate in Enterococcal IE is significant at 9–15%, but it is lower than IE caused by other pathogens such as S. aureus (McDonald et al. 2005; Rice et al. 1991). The first report of IE by vancomycin-resistant E. faecium (VRE) appeared in the late 1990s (Vijayvargiya and Veis 1996). The risk of VRE IE increases in the patients with the history of hemodialysis and organ transplantation and presence of a central venous catheter (Stevens and Edmond 2005). The study on clinical outcomes of IE by E. faecalis and E. faecium showed high mortality rates and longer persistence of bacteremia in E. faecium IE patients (Forrest et al. 2011).

4.2 Urinary Tract Infections (UTIs)

Enterococcus spp. are the third most common pathogen isolated from catheter-associated UTIs in hospitalized patients causing approximately 15% of the UTI cases in ICU patients. UTIs occur most frequently in elderly men. Vancomycin resistance was found to be 81% in E. faecium and 6% in E. faecalis urinary isolates in catheter-associated UTI (Hidron et al. 2008)

4.3 Bacteremia

At present, enterococci are the second leading causative agent of healthcare-related bacteremia (Hidron et al. 2008). The most common route of Enterococcal bloodstream infection (EBSI) is genitourinary tract and endovascular, intra-abdominal, or soft tissue infections. Other factors like old age, liver disease, male gender, renal impairment, hematologic transplant, diabetes, prior treatment with antibiotics, and malignancy have also been associated with EBSI (Noskin et al. 1995; Gray et al. 1994). In a study on patients diagnosed with acute leukemia, it was demonstrated that the risk of EBSI increases with increase in duration of hospital stay and with administrations of carbapenes and corticosteroids, diarrhea, and severe neutropenia (Ford et al. 2015). Twenty-five percent of EBSI cases have tendency to be polymicrobial in nature (Billington et al. 2014). Mortality rate in polymicrobial EBSI is approximately two times higher than monomicrobial EBSI (McKenzie 2006). Almost 9.5% and 82.6% E. faecalis and E. faecium isolated from blood stream infections are vancomycin resistant (Lautenbach et al. 1999). Unlike S. aureus-associated bacteremia, Enterococcal bacteremia rarely causes metastatic abscesses or seeds distant organs. Some studies have reported more mortality rate in E. faecium caused by bacteremia than that caused by E. faecalis (Noskin et al. 1995). Death rates can be up to 75% in patients with serious health conditions like diabetes mellitus, transplantation, heart diseases, or malignancy.

4.4 Intra-abdominal Infections

Intra-abdominal infections (IAIs) include various pathological conditions, such as fecal peritonitis, uncomplicated appendicitis, perforated viscus, and postoperative complications (Menichetti and Sganga 2009). In a 14-year-long study on recurrent and relapsing peritonitis in patients undergoing peritoneal dialysis, enterococci were found to be common causative agents of recurrent peritonitis (Szeto et al. 2009). The data collected at 68 medical centers demonstrated that enterococci were the most commonly isolated Gram-positive bacteria from IAI patients and constituted almost 12.9% of the isolates out of which 9.2% were E. faecalis and 3.7% were E. faecium (Sartelli et al. 2014). Further, it was reported that the increased intrinsic and acquired resistance in enterococci pose great challenges in the effective treatment of the diseases.

5 Nosocomial Transmission

Nosocomial Enterococcal infections are caused by increased colonization of GIT by resistant enterococci. Prolonged exposure of hospitalized patients to antibiotics alters gut microbiota which decreases the prevalence of Gram-negative microbes in the GIT, thus facilitating colonization by resistant Gram-positive bacteria such as enterococci. The primary mode of transmission of resistant enterococci in hospital setting is through the hands of healthcare workers (Hayden 2000). Studies have shown that enterococci have the ability to persist on the hands for approximately 1 h and for 4 months on inanimate surfaces, which thus act as reservoirs for transmission in the absence of regular sanitization (Kramer et al. 2006). Medical equipments like thermometers, stethoscopes, blood pressure cuffs, intravenous fluid pumps, bed rails, gowns, bedside tables, urinals, bedpans, and bed linens are readily contaminated with high densities of VRE (Bonilla et al. 1997; Hota 2004). Other factors posing VRE colonization risk include duration of patient’s stay in hospital or intensive care unit and patient’s proximity to other VRE-colonized patients (Fig. 15.2; Tornieporth et al. 1996). Strict surveillance of health workers is required to curb the spread of nosocomial Enterococcal infections. Further, various precautions, such as proper decontamination of inanimate surfaces, use of gloves, hand sanitization, etc., should be strictly followed.

Fig. 15.2
figure 2

Factors affecting the transmission of vancomycin-resistant enterococci (VRE) in nosocomial settings. The close proximity to VRE-infected patient passes on the contamination to hospital staff or hospital equipments and finally to the immunocompromised patient

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6 Virulence Determinants of Enterococci

Virulence of enterococci is a multifactorial process, with the participation of several genes and their products. The virulence genes are mostly known to be present on the genome in special regions which are termed pathogenicity islands (PAI). However, some virulence factors may be present on plasmids also. E. faecalis are known to carry more numbers of virulence factors as compared to E. faecium.

7 Secreted Virulence Factors

7.1 Cytolysin

Cytolysin toxin belongs to the class of lantibiotic group of bacteriocins. Cytolysin production can be chromosomally encoded by PAI or encoded by a pheromone-responsive plasmid pAD1 (Ike et al. 1990). Cytolysin consists of two components CylLL and CylLS which are secreted as pro-peptides from the cell. These pro-peptides are proteolytically activated by CylM, CylB, and CylA.

Almost 30% of E. faecalis strains produce cytolysin, and its production has been associated with increased toxicity of Enterococcal infections. Cytolysin can lyse bacterial cells, mammalian erythrocytes, and other eukaryotic cells (Cox et al. 2005). In a rabbit endocarditis model, aggregation substance and cytolysin-positive strains caused 55% mortality as compared to 15% with only aggregation substance-positive strains (Chow et al. 1993).

7.2 Gelatinase

Gelatinase (Gel) is an extracellularly secreted Zn-metalloprotease which hydrolyzes casein, collagen, fibrinogen, complement components (C) 3 and C3a, and hemoglobin. Gel has been implicated as virulence factor by animal models and epidemiological studies. Gel expression is induced by fsr quorum-sensing system in response to high cell density (Qin et al. 2000). It mediates virulence by degrading host tissue and modulating host’s immune response (Park et al. 2008). Gel can activate autolysin which is a peptidoglycan-degrading enzyme and leads to release of DNA and has a role in biofilm formation (Thomas et al. 2009).

8 Cell Surface-Associated Virulence Determinants

8.1 Aggregation Substance

Adherence to host tissue is important for pathogens to cause infection. In enterococci, aggregation substance (AS) and extracellular surface protein (esp) have been reported to play a role in adherence (Waters et al. 2004). In E. faecalis, AS is encoded by pheromone-responsive plasmid in response to pheromone (Clewell 1993). AS mediates aggregation of Enterococcal cells and facilitates transfer of plasmid during conjugation. It also has been reported to promote internalization of Enterococcal cells by intestinal cells, adhesion to renal tubular cells, and survival of E. faecalis cells inside polymorphonuclear neutrophils (Olmested et al. 1994; Kreft et al. 1992). In endocarditis model, destruction of pulmonary and myocardial tissue was found to be caused due to AS. Moreover, AS has been reported to mediate bacterial aggregation on cardiac valve resulting in increased pathogenesis in endocarditis (Schlievert et al. 1998).

8.2 Enterococcal Surface Protein

Esp is a large molecular weight protein expressed on the surface of E. faecalis. Esp is mainly associated with biofilm formation (Heikens et al. 2007) and plays a role in biofilm-associated infections such as UTI (Shankar et al. 2001), bacteremia, and endocarditis (Heikens et al. 2001) as demonstrated by studies with an esp deletion mutant. The esp gene located on PAI encodes a very large LPXTG-motif cell wall-anchored protein in both E. faecalis and E. faecium. It is widely present in E. faecalis strains, but E. faecium esp is predominantly found in hospital-associated isolates, suggesting its role in virulence.

8.3 Adhesion to Collagen Proteins

The ability of E. faecalis to adhere to extracellular matrix proteins like collagen, fibrinogen, laminin, lactoferrin, fibronectin, thrombospondin, and vitronectin has been reported in many studies (Rich et al. 1999; Nallapareddy and Murray 2008). The search for the gene encoding these adhesion molecules led to the discovery of Ace, an adhesin to collagen of E. faecalis. Most of these studies also found that only few isolates of E. faecalis exhibit adherence to extracellular matrix proteins under laboratory conditions (Woodford et al. 2001). Further experiments have found that most clinical isolates of E. faecalis do not express adherence phenotypes constitutively, but expression of these phenotypes is elicited under stress conditions or in the presence of host-derived factors (Nallapareddy and Murray 2008). The role of Ace was studied in experimental endocarditis rat model. Immunization with recombinant anti-Ace antibodies decreased endocarditis development (Singh et al. 2010).

Adherence studies showed that like E. faecalis, many isolates of E. faecium can bind to collagen following growth in brain-heart media, and the protein responsible is a cell wall-anchored Acm (adhesin to collagen of E. faecium; Nallapareddy et al. 2003). Collagen adherence of clinical isolates of E. faecium was found to be significantly more than the isolates from animal, community, or fecal origin, thus suggesting that it is an important factor responsible for infection-causing ability of E. faecium. Incorporation of acm gene in acm-negative mutants resulted in the expression of adherence phenotype. In E. faecium endocarditis mice model, acm mutant was less likely to develop endocarditis; thus it has been shown to be antigenic (Nallapareddy et al. 2008).

8.4 Endocarditis- and Biofilm-Associated Pili (ebp)

The ebp are surface-associated filamentous structures in E. faecalis. It plays a role in biofilm formation by binding to abiotic surfaces and adhering to platelets (Nallapareddy et al. 2011a). In animal models, it contributes to tissue colonization in IE and UTI (Sillanpaa et al. 2013). Deletion of the ebp locus resulted in a diminished capacity of E. faecalis OG1RF to colonize kidneys and bladders in a murine model of ascending UTI (Nallapareddy et al. 2011b) and in catheter-associated UTI (Nielsen et al. 2012).

9 Factors Contributing to Enterococcus Pathogenesis and Divergence from Commensals

Enterococci are part of normal GIT, and transition of commensals to pathogens is associated with change in ecology, acquisition of toxin genes, and mobile genetic elements carrying PAI. Comparative studies of the genomes of nosocomial and commensal strains show that nosocomial strains have acquired these virulent traits. Genome sequence analysis of V583, which is considered as representative strain of nosocomial lineage of E. faecalis, revealed that 26% of the genome comprised of mobile genetic elements, 38 insertion elements, remains of 3 integrative phages, 7 apparent phages, and 3 independently replicating plasmids (Paulsen 2003). Similar evolutionary sequences are found in case of nosocomial E. faecium strains which are found to possess large PAI carrying several IS and esp gene (Leavis et al. 2007). Further, these virulent genes synergize with each other to express the virulence phenotype; for example, in an endocarditis model, AS was found to synergize with cytolysin protein.

Further, the genome of V583 when compared with commensal strain OG1RF revealed the presence of CRISPR-cas in commensal strains which provides defense against plasmid acquisition and phage infection. The absence of CRISPR in V583 led to studies directed to find correlation between absence of CRISPR and accumulation of mobile genetic elements in nosocomial strains. Sixteen E. faecalis strains representing the deepest phylogenetic nodes were studied to identify polymorphism in location and content of CRISPR loci. Results suggested that CRISPR loci were highly conserved and influenced the movement of pheromone-responsive plasmids and phages. A complete absence of CRISPR-cas in MDR Enterococcus strains was also reported (Palmer and Gilmore 2010). Based on the whole genome data and the comparison of the 2113 core genes of the eight E. faecium strains, they were placed in two different clades. Clade A mainly comprised of nosocomial infection-causing strains, whereas clade B comprised of commensal strains. Clades A and B have similarity in the range 93.9–95.6% that shows the degree of divergence between the species. In case of E. faecalis, the commensal and nosocomial pathogens can also be similarly differentiated into different clonal complexes, but the division is overlapping in some cases (Gilmore et al. 2013).

10 Treatment

The treatment of infections caused by susceptible Enterococcus strains is mainly by using ß-lactam antibiotics. Monomicrobial Enterococcal infections that are susceptible are usually treated with ampicillin and penicillin alone. On the other hand, polymicrobial infections are treated with the combination of ampicillin and other antibiotics having broad spectrum of activity against aerobic, anaerobic, and Gram-negative bacteria. Combination drugs containing β-lactam along with β-lactamase inhibitor such as piperacillin-tazobactam or ampicillin-clavulanic acid can also be used. In case of penicillin allergies, a single agent like vancomycin or teicoplanin can be used to treat nonresistant Enterococcal infections. In case of penicillin-susceptible VRE infections like cystitis, oral application of nitrofurantoin, doxycycline, and fosfomycin can be considered (Heintz et al. 2010). Infections caused by VRE strains are mostly treated by antibiotic, linezolid, daptomycin, and quinupristin-dalfopristin (Q-D), but side effects are common in prolonged treatment. For E. faecium Q-D is not used as it is resistant. UTI and skin and soft tissue infections caused by VRE are treated by fluoroquinolones or doxycycline depending upon the susceptibility patterns (Landman and Quale 1997). Adjunct measures including drainage of abscesses and removal of infected foci can also be used wherever possible.

Treatment of IE caused by E. faecalis mainly includes combination of ß-lactams, gentamicin, and aminoglycosides. In case of IE caused by E. faecium, treatment is challenging due to higher resistance to ß-lactams and aminoglycosides. Daptomycin and ceftaroline have also been found to be effective for the treatment (Pericás et al. 2015).

11 Antibiotic Resistance

Increasing antibiotic resistance in enterococci is a matter of concern because the increase in the cases of Enterococcal infections in humans is partially due to its ability to escape the action of commonly used antibiotics. Enterococci are known to be intrinsically resistant to many antibiotics due to which they can exist in environments enriched for antibiotic resistance (Mundy et al. 2000; Murray 1998). They are known to be intrinsically resistant to semisynthetic penicillins and cephalosporins due to the presence of low-affinity penicillin-binding proteins; for example, E. faecalis expresses PBP4, and E. faecium expresses PBP5. The minimum inhibitory concentrations (MICs) for penicillins are typically 2–8 μg/ml for E. faecalis and 8–16 μg/ml for E. faecium (Sifaoui et al. 2001). They also show decreased susceptibility to penicillin, ampicillin, aminoglycosides, and clindamycin.

Among aminoglycosides, the MIC of streptomycin and kanamycin in E. faecalis is 250 μg/ml, whereas that of tobramycin and gentamycin is 8–64 μg/ml (Chow 2000). Aminoglycoside resistance is due to the inability of the aminoglycoside to enter the thick cell wall (Aslangul et al. 2006). Therefore in the presence of cell wall-inhibiting antibiotics such as β-lactams, the enterococci become susceptible to aminoglycosides. The presence of aminoglycoside-converting enzymes such as acetyltransferases, phosphotransferases, and nucleotidyl transferases in E. faecium and E. faecalis also contributes to aminoglycoside resistance (Chow 2000; Miller et al. 2014). In E. faecium chromosomally encoded genes, rRNA methyltransferase (efmM) (Galimand et al. 2011) and a 6′-N-aminoglycoside acetyltransferase (aac(6′)- Ii) (Costa et al. 1993) are associated with intrinsic resistance to tobramycin and kanamycin. Resistance to clindamycin in E. faecalis is probably due to the presence of protein product of lsa (lincosamide and streptogramin A resistance) gene, i.e., ATP-binding cassette (ABC)-efflux pumps, i.e., ABC-23 (Singh et al. 2002). Similarly, putative ABC transporter was identified in all E. faecium isolates that result in resistance to erythromycin, and chloramphenicol resistance has been linked to efflux of the antibiotic out of the cell (Aakra et al. 2010).

Enterococcus spp. are known to acquire resistance to various antibiotics such as vancomycin, chloramphenicol, erythromycin, and fluoroquinolones and high-level resistance to aminoglycosides and penicillin. Acquired antibiotic resistance in a pathogen is due to either of the two mechanisms, i.e., either by mutation of the gene or by horizontal transfer of antibiotic resistance genes. However, in Enterococcus antibiotic resistance problem mainly stems from horizontal gene transfer. Enterococci exist in complex microbial environments where they come in contact with large diversity of genetic material, and in the presence of antibiotic pressure, they tend to acquire resistance to antibiotics. For example, there is a direct relationship between exposure to parenteral antibiotics, especially cephalosporins, antibiotics for anaerobes, and high-level gastrointestinal colonization by ampicillin-resistant E. faecium (Rice et al. 2004).

Enterococci were recognized as important agents of MDR nosocomial infections after vancomycin resistance was observed. In the USA, 87% E. faecium and 14% E. faecalis isolated from nosocomial infections are vancomycin resistant (Edelsberg et al. 2014). Vancomycin acts by binding to D-alanine-D-alanine moiety of peptide chain of peptidoglycans, thus preventing cross-linking of peptidoglycans. Enterococcus strains become resistant to vancomycin by altering peptidoglycan precursors from d-alanine-d-alanine to d-Ala-d-lactate or to d-Ala-d-serine. Nine gene clusters involved in vancomycin resistance are vanA, vanB, vanD, and vanM (Xu et al. 2010) leading to the formation of d-Ala-d-Lac and vanC, vanE, vanG, vanL, and vanN catalyzing d-Ala-d-Ser formation (Boyd et al. 2008; Lebreton et al. 2011). VanA and vanB are the most relevant vancomycin-resistant determinants and are located on transposons (Courvalin 2006). In the 1970s Enterococcus acquired high level of resistance against ampicillin due to specific mutations in pbp genes, which encode for penicillin-binding proteins (Galloway-Peña et al. 2011).

12 Conclusion

The emerging problem of hospital-associated Enterococcal infections appears to be man-made due to injudicious use of antibiotics especially for non-therapeutic purposes such as avoparcin which was employed as growth promoter in the animal feed for the first time in 1975. The overuse of avoparcin led to avoparcin resistance in the gut flora of animals. The avoparcin-resistant gut flora showed cross-resistance toward vancomycin (Mudd 2000). Thus, the first VRE was isolated from the gut flora of farm animals in the 1990s. Subsequently, the use of avoparcin was banned (Casewell et al. 2003) It is hypothesized that the VRE from farm animals were acquired by humans. This is a classic case that highlights the serious repercussions of overuse of antibiotics both for therapeutic and non-therapeutic purposes. To treat Enterococcal infections, antibiotics should be selected wisely based on various factors such as the age, immunocompromised status, type and location of infection, and antibiotic sensitivity spectrum of the Enterococcal pathogen. Further, general measures to prevent the spread of Enterococcus in the hospital settings should be strictly followed such as sanitization of hospital environment and equipments.