Microbiology of enterococci

INTRODUCTION — A number of the characteristic features of enterococci (formerly called group D streptococci) have been recognized for at least 100 years, including their presence in feces and sewage, their ability to cause endocarditis and urinary tract infections (UTIs), and their ability to survive harsh environmental conditions including drying, high temperatures, and exposure to some antiseptics [1,2].

The common name "enterococcus," derived from the French "entérocoque" used in an 1899 publication, points out that these bacteria are cocci of intestinal (enteric) origin, and the 1906 use of Streptococcus faecalis and the 1919 use of Streptococcus faecium also emphasized their presence in feces.

Subsequent studies confirmed these early observations. Most humans and animals have enterococci in their intestinal tract; the counts in healthy humans often range from 10⁵ to 10⁷ bacteria per gram of stool.

●Enterococci, particularly Enterococcus faecalis, are a relatively common cause of endocarditis (third, after staphylococci and streptococci), causing 5 to 15 percent of community-acquired endocarditis and up to 30 percent of nosocomially acquired endocarditis), and have been recognized as the leading cause of infective endocarditis in patients who undergo transcatheter aortic valve implantation (TAVI) [3,4]. In addition, enterococci can be a common cause of nosocomial urinary tract infections (being recovered from up to 15 to 20 percent of UTIs in the hospital setting) [5,6].

MICROBIOLOGY CLASSIFICATION — Enterococci are gram-positive, facultatively anaerobic cocci that form short- to medium-length chains. In the 1930s, Lancefield devised a serologic typing system that classified enterococci as group D streptococci, along with some organisms (eg, Streptococcus gallolyticus, formerly S. bovis) that differed from enterococci in a number of other features. In 1937, Sherman divided streptococci into the pyogenic, viridans, lactic, and enterococcal groups. Enterococci were characterized by the following features:

●Their reaction with group D antiserum

●Their ability to grow in high salt, at 10ºC and 40ºC, and at high pH

●Their ability to survive exposure to 60ºC for 30 minutes

●Their ability to hydrolyze esculin in the presence of bile

In the ensuing 45 to 50 years, enterococci continued to be taxonomically classified in the genus Streptococcus. In the mid-1980s, with advances in taxonomic techniques based upon nucleic acid analyses, it was proposed that S. faecalis and S. faecium be removed from the genus Streptococcus, transferred to a new genus, Enterococcus, and reclassified as E. faecalis and E. faecium, respectively [7]. Subsequently, other enterococcal species were also reclassified from the genus Streptococcus to the genus Enterococcus; dozens of new species have now been recognized.

Problems with classification — The characteristic features of enterococci as being bile esculin positive, reactive with group D antiserum, and able to grow in 6.5 percent sodium chloride were used by clinical laboratories for decades to distinguish enterococci from other catalase-negative gram-positive cocci. However, some problems arise with the use of these criteria. As an example, the presence of group D antigen can be difficult to detect in some enterococcal isolates. In addition, other organisms such as lactococci also can grow in high salt and/or be bile esculin positive; however, lactococci do not grow (or grow very slowly) at 45ºC.

Two other genera that share a number of biochemical features with enterococci, including their ability to produce the group D antigen, are Pediococcus and Leuconostoc, which can also grow at 45ºC. These organisms are intrinsically vancomycin resistant, which was a reliable distinguishing trait prior to the emergence of vancomycin-resistant enterococci (VRE). These genera are now best distinguished from VRE biochemically by the addition of tests for production of pyrrolidonyl arylamidase (PYR) and leucine aminopeptidase (LAP). Expected results are: +/+ with enterococci; -/+ for Pediococcus; and -/- for Leuconostoc; Leuconostoc also produce gas from glucose [8].

Identification at the species level — Prior to the emergence of vancomycin resistance, the majority of clinical enterococcal isolates were E. faecalis (85 to 90 percent), but the incidence of E. faecium in the United States has increased (to about 32 percent of health care-associated enterococcal isolates), coincident with emergence of ampicillin and vancomycin resistance [9]. Although commercial kits for E. faecalis are generally reliable, commercial strips and kits for non–E. faecalis have been less reliable.

With the increase in non-faecalis (primarily E. faecium) and the increased attention paid to vancomycin-resistant organisms, including E. gallinarum and E. casseliflavus, there is renewed interest in identifying these organisms. One reason for this interest is related to species-specific antimicrobial resistance differences that may not be apparent from routine antibiograms.

For example, revision of the Clinical and Laboratory Standards Institute (CLSI) daptomycin breakpoints for enterococci introduced species specific cutoffs based on different dosing regimens for E. faecium and all other enterococcal species (see "Treatment of enterococcal infections", section on 'Susceptibility breakpoints'). Pharmacokinetic-pharmacodynamic modeling and clinical data suggest higher daptomycin exposures are associated with increased probability of target attainment and better clinical outcomes in E. faecium infections, while data for increased dosing regimens in other enterococcal species is less robust [10]. Thus, the use of daptomycin for severe enterococcal infections such as endocarditis would require identification of the infecting organism to the species level. (See "Antimicrobial therapy of left-sided native valve endocarditis", section on 'Enterococci'.)

Another reason for species identification is that the appearance of a non-faecalis, non-faecium species in different patients may be a clue to an outbreak secondary to nosocomial spread. Repeated isolation of an unusual species from a single patient may also serve as a clue to relapse from an underlying focus (eg, renal abscess, endocarditis) rather than reinfection by a new organism.

Enterococcal species were traditionally distinguished based upon results from a complex set of biochemical reactions, including fermentation of carbohydrates, hydrolysis of arginine, tolerance to tellurite, and motility and pigmentation:

●Tellurite tolerance with production of black colonies and utilization of pyruvate are characteristic of E. faecalis.

●The primary motile species are E. casseliflavus (E. flavescens) and E. gallinarum; yellow pigment is produced by E. casseliflavus and E. mundtii.

Motility and pigment are particularly useful for distinguishing E. casseliflavus and E. gallinarum from E. faecium, which can be misidentified by kits that do not include these tests. However, occasional isolates display atypical reactions.

There are a number of difficulties with identification of enterococci to the species level. These include the necessity to perform multiple reactions, the analysis of which is best performed by computer application, and the difficulty in identifying enterococci with atypical phenotypic characteristics [11]. In many locations, genetic methods have largely replaced these biochemical tests for species identification. These include gene probes, polymerase chain reaction (PCR) for specific genes, 16S rRNA sequencing, proprietary multiplexed nucleic acid amplification, and matrix-assisted laser desorption/ionization–time of flight (MALDI-TOF) [12-17].

On a practical level, almost all E. faecalis isolates are susceptible to ampicillin but resistant to quinupristin-dalfopristin, whereas most clinical E. faecium isolates are resistant to ampicillin yet susceptible to quinupristin-dalfopristin.

ANTIBIOTIC RESISTANCE — Enterococci come well equipped with a variety of intrinsic (ie, naturally occurring species characteristics) antibiotic resistances. They are also capable of acquiring new resistance genes and/or mutations, particularly after exposure to antibiotics. (See "Mechanisms of antibiotic resistance in enterococci".)

In Europe, E. faecium strains resistant to vancomycin have been identified frequently in food animals, although vancomycin-resistant enterococci belonging to clones common in food animal feces rarely have been found to infect humans. E. faecium isolates resistant to vancomycin have only rarely been reported in animals in the United States; isolates resistant to other antibiotics, including quinupristin-dalfopristin, have been recovered from commercial poultry and meat products in the United States [18,19].

It is unclear whether the ingestion of these organisms leads to persistent intestinal carriage in humans. This issue was addressed in a study in which 12 healthy volunteers were fed glycopeptide and streptogramin-resistant E. faecium strains recovered from chickens or pork [20]. The strains were detectable in the stool of 8 of the 12 volunteers at one week, 1 of 12 at two weeks, and none at five weeks. This observation illustrates the potential for spread of these organisms as well as their resistance genes, which have the potential to transfer to the existing human enterococcal flora.

Nosocomial infection — Enterococci are the second to third most common organism recovered from hospital-acquired (nosocomial) infections, particularly in more severely ill patients who have been hospitalized for long periods of time and/or have received multiple antibiotics [6,9]. (See "Vancomycin-resistant enterococci: Epidemiology, prevention, and control".)

Until the appearance of vancomycin resistance, E. faecalis accounted for 85 to 90 percent of enterococcal isolates and E. faecium for ≤10 percent. However, E. faecium has been implicated in up to 40 percent of bloodstream isolates in high-risk populations such as liver or stem cell transplant recipients and up to 35 percent of enterococcal isolates from nosocomial infections [21,22].

The rise of E. faecium in the hospital setting has been attributed to its many antimicrobial resistances, which allow these organisms to survive and proliferate when antibiotic-susceptible organisms are eliminated [23].

Initially, most clinical isolates of multiresistant E. faecium appeared to belong to a single clonal complex (CC17) of related multilocus sequence types. Subsequently, two distinct clades of E. faecium, an animal associated A (which also has most healthcare-associated strains) and human commensal clade B, were identified using whole genome sequencing techniques. The core genes of these two groups differ by an average of 3 to 5 percent and a molecular clock analysis suggests they diverged from each other thousands of years ago, leading some to propose that these differences warrant classification of clade B as a different species, E. lactis [10,24,25]. The timing and frequency of the emergence of a subclade designated "A1" and whether the animal-associated clade ("A2") is a truly distinct group is still debated, as the structure of this subclade is dependent on both differences in geographic sampling and recombination events [26,27]. Regardless, clade A, particularly the subgroup A1, contains almost all the clinical and multidrug-resistant E. faecium strains [28].

The population structure of E. faecalis is much more variable than that of E. faecium; however, several hospital-adapted lineages have been identified. In one study of 515 E. faecalis from the United Kingdom and United States, isolates belonging to one of three hospital-adapted lineages were enriched for multiple antibiotic resistance determinants, including vancomycin resistance [29]. Further studies are needed to determine if these E. faecalis lineages reflect a worldwide phenomenon.

In addition to having more antibiotic resistance genes conferring resistance to ampicillin and high levels of vancomycin, the hospital-associated clade of E. faecium also has genes that animal and community human fecal isolates do not have, including proven and putative virulence and colonization factors, and a much higher rate of mutation. In addition, many multidrug-resistant E. faecalis and E. faecium lack a functional CRISPR-cas9 system, which usually serves to protect bacteria from phages, but can also prevent the acquisition of resistance determinants on mobile genetic elements [30]. This suggests that the hospital-associated clade is not just more antibiotic resistant but also more capable of adapting to antibiotics, the environment, and possibly to the human immune response [28,31-34].

Enterococci can often persist in the face of antibiotics use and therefore have enhanced opportunity to cause opportunistic infection [35], particularly in patients with altered immune defenses and/or multiple potential portals of entry. Enterococci also have intrinsic factors such as a number of potential adhesin genes, which may explain the propensity of enterococci to cause endocarditis.

Once enterococci cause infection, their antibiotic resistances can create even greater dilemmas. Our worst therapeutic problems arise when a multidrug-resistant hospital-acquired E. faecium causes endocarditis. (See "Treatment of enterococcal infections".)

SUMMARY

●General – Enterococci (formerly called group D streptococci) are gram-positive cocci of intestinal origin that usually form short chains and are a common cause of endocarditis and nosocomial infections. (See 'Introduction' above.)

●Microbiology classification

•Microbiologic characteristics of enterococci include reactivity with group D antiserum in approximately 80 percent; ability to grow in high salt, at 10ºC and 40ºC, and at high pH; and survival at 60ºC for 30 minutes. They hydrolyze esculin in the presence of bile and produce pyrrolidonyl arylamidase (PYR). (See 'Microbiology classification' above.)

•Species differentiation, traditionally based on results from a complex set of biochemical reactions but more recently on DNA-based testing, is important, as species-specific antimicrobial resistance differences can affect therapeutic decisions. The identification of non-faecium, non-faecalis species can be helpful in detecting nosocomial outbreaks or relapsing infection. (See 'Identification at the species level' above.)

•Almost all E. faecalis isolates are susceptible to ampicillin but resistant to quinupristin-dalfopristin. Most clinical E. faecium isolates are resistant to ampicillin, susceptible to quinupristin-dalfopristin, and have moderately high-level resistance to tobramycin; in the United States, most clinical E. faecium are resistant to moderate to high levels of vancomycin. E. gallinarum and E. casseliflavus are intrinsically resistant to low levels of vancomycin. (See 'Identification at the species level' above.)

●Antibiotic resistance

•Enterococci resistant to antibiotics have been recovered from commercial poultry and meat products in the United States, but it is unclear whether the ingestion of these organisms leads to persistent intestinal carriage in humans. (See 'Antibiotic resistance' above and "Mechanisms of antibiotic resistance in enterococci".)

•Their many antimicrobial resistances, which allow them to survive, proliferate, and fill the void when antibiotic-susceptible organisms are eliminated, likely contribute to the frequency of enterococci in nosocomial infections. (See 'Nosocomial infection' above.)