Enterococcus faecalis- An Overview

The GI tract is home to a particular kind of bacteria called Enterococci. These bacteria come in at least 18 different species. One of the most prevalent species is Enterococcus faecalis (E. faecalis). A member of the gut’s commensal gram-positive flora called Enterococcus faecalis is a diverse range of organisms’ microbiota. Since the introduction of antibiotic therapy, it has emerged as a hospital-acquired, multidrug-resistant pathogen.

Enterococcus faecalis Overview
Enterococcus faecalis Overview

What is Enterococcus faecalis?

Enterococcus faecalis is facultative anaerobes that are low-GC, gram-positive, non-sporulating, and currently rank among the most common multidrug-resistant hospital pathogens worldwide.

  • It is capable of causing several infections, including endocarditis, sepsis, surgical wound infections, and urinary tract infections. It is the third most frequently isolated healthcare pathogen.
  • Enterococci were included in Lancefield’s serological classification of group D streptococci in the 1930s.
  • In the 1980s, enterococci were separated from the genus Streptococcus and given their genus, Enterococcus, based on genetic differences. The genus name Enterococcus was used in place of Streptococcus to precede the previously used species designations, such as faecalis, faecium, durans, etc.
  • It can withstand extremely harsh conditions, such as alkaline pH (9.6) and high salt concentrations. It can withstand desiccation, ethanol, heavy metals, detergents, bile salts, and detergents.
  • Carbohydrates, lactate, malate, citrate, and many keto acids can all be catabolized to produce energy. 

Classification of Enterococcus faecalis

  • In the 1930s, Lancefield used serology to classify enterococcus as part of the group D Sreptococcus system.  Streptococcus species were divided into four subgroups by Sherman in 1937: faecal streptococci (enterococci), dairy streptococci, viridans group, and pyogenous streptococci.
  • The term “enterococcus” should only be used for Streptococci that can grow at both 10 and 45 degree celsius, at pH 9.6 and in 6.5 percent NaCl, survive at 60 degree celsius for 30 min., and have the ability to split esculin, according to Sherman’s 1937 classification scheme.
  • It was determined in 1984 that the species Streptococcus faecium and Streptococcus faecalis were sufficiently distinct from the other streptococci to be designated another genus: Enterococcus, through DNA hybridization and 16S rRNA sequencing. This indicates that both enterococci and streptococci contain the D group antigen. There are now 28 species in the Enterococcus genus after nine species were transferred from the Streptococcus groups. 
DomainBacteria
PhylumFirmicutes
ClassBacilli
OrderLactobacillales
FamilyEnterococcaceae
GenusEnterococcus
SpeciesE. faecalis

Enterococcus faecalis Habitat

Although they can be isolated from the oropharynx, female genital tract, and skin, the gastrointestinal tract of humans and other mammals is the typical habitat for these microorganisms. Because of animal hosts’ shedding, these bacteria are easily found in the environment and thrive in the nutrient-rich, oxygen-depleted environment of the intestinal tract.

  • E. faecalis typically exists in our intestines without causing harm. However, it can result in a more serious infection if it spreads to other areas of your body.
  • During surgery, the bacteria may enter the blood, urine, or wound. It can then spread to other locations, resulting in more severe infections like sepsis, endocarditis, and meningitis. In healthy individuals, E. faecalis bacteria typically do not result in issues.   
  • E. faecalis can become pathogenic in people who are immunocompromised (have a weakened immune system) or have an underlying disease (disease-causing). As a result, E. faecalis is regarded as an opportunistic pathogen, one that preys on the body’s weakened immune system.
  • E. faecalis can enter the body through surgery, improper hand hygiene, poorly cleaned medical equipment, and infected food and liquids.
Enterococcus faecalis gram stain
Figure: Enterococcus faecalis gram stain. Image Source: Dr. Sahay.

Enterococcus faecalis Morphology

  • Enterococcus faecalis is gram-positive cocci or ellipsoid microorganisms arranged in pairs or short chains in liquid media.
  • A gram-positive, non-capsulated, fermentative, facultatively anaerobic, non-spore-forming coccus is called Enterococcus faecalis, whose cells are ovoid-shaped and measure 0.5 to 1 μm in diameter.
  • The DNA contains between 37 and 40 moles of G+C. 
  • They have a cell wall that contains the group D glycerol teichoic acid, a group-specific antigen.
  • 20–38% of the dry cell weight of E. faecalis is made up of the cell wall (in the exponential and stationary phase cells). Since E. faecalis is a Gram-positive bacterium, its cell wall primarily comprises peptidoglycan, teichoic acid, and polysaccharide. 
  • Peptidoglycan makes up 40% of the cell wall, with the remaining 60% consisting of a “ribitol-containing teichoic acid and a rhamnose-containing polysaccharide.” As in most Gram-positive cells, the peptidoglycan prevents bursting by high cytoplasmic osmotic pressure.
Enterococcus faecalis Morphology 
Figure: Scanning electron microscopic (SEM) image of Enterococcus faecalis. Image Source: CDC.

Enterococcus faecalis Cultural Characteristics

Incubating Enterococcus faecalis in routine non-selective media is simple, and it grows under conditions designed for streptococci. It grows in the 10°C to 45°C temperature range and reproduces in broth with a pH of 9.6, as well as in media with 6.5 percent NaCl (unlike streptococci) or 40 percent bile. Also, it can be cultured in different agar like chromogenic agar, blood agar, McConkey agar, etc., to identify different characteristics.

1) MacConkey agar

  • Minute, round and red colonies appeared.

2) Nutrient agar 

  • Colorless whitish colonies appeared.
  • It grows as smooth, small, protruding, opaque colonies with a dry consistency (similar to staphylococcal colonies.
Figure: Enterococcus faecalis on Blood Agar. Image Source: University of Copenhagen.

3) Blood agar 

  • Circular or smooth, convex colonies with an entire margin appeared. 
  • Non-hemolytic or exhibits a –hemolysis.

4) Chromogenic agar

  • Small turquoise blue colonies appeared.
Figure: Enterococcus faecalis on ChromID CPS chromogenic agar. Image Source: Nathan Reading.

Biochemical Characteristics of Enterococcus faecalis

The biochemical characteristics of Enterococcus faecalis can be tabulated as follow:

S.N.Biochemical characteristicsE. faecalis
1.Gram stainingGram-positive
2.ShapeCocci
3.MotileNon-motile
4.Spore formationNon-sporing
5.CatalaseNegative(-)
6.H2SNegative(-)
7.Bile esculin testPositive(+)
8.Gelatin hydrolysisVariable
9.HemolysisVariable
10.IndoleNegative(-)
11.CitrateNegative(-)
12.UreaseNegative(-)
13.Nitrate reductionPositive(+)
14.VPPositive(+)

Fermentation of 

S.N.SubstrateE. faecalis
1.D-GlucosePositive(+)
2.MannitolPositive(+)
3.ArabinoseNegative(-)
4.LactosePositive(+)
5.SucrosePositive(+)
6.PyruvatePositive(+)
7.D-FructosePositive(+)
8.MaltosePositive(+)
9.GlycerolPositive(+)
10.RibosePositive(+)
11.SorbitolPositive(+)

Enzymatic reactions

S.N.EnzymesE. faecalis
1.Pyrazin amidase(PYR)Positive (+)
2.Leucine aminopeptidase(LAP)Positive (+)
3.Alkaline phosphataseVariable

Virulence factors of Enterococcus faecalis

It has been discovered that several virulence factors increase a particular E. faecalis strain’s ability to either cause disease or make disease symptoms worse. Their high levels of antibiotic resistance and ability to adhere to host surfaces to form biofilms determine their virulence. For instance, numerous factors affect an Enterococcus species’ virulence.

Virulence factors of Enterococcus faecalis
Figure: Virulence factors of Enterococcus faecalis. Image Source: K. Waar.

1) Bio-film formation

  • Endodontic and urinary tract infections, as well as endocarditis, are all primarily brought on by enterococci’s capacity to produce biofilms. 
  • Biofilm formation requires the development of pili by enterococci, and the ebp gene cluster is associated with this (endocarditis-and biofilm-associated pili). The genes srtC (encoding sortase C), ebpA, ebpB, and ebpC make up the ebp operon E. faecalis non-piliated mutant was unable to form a biofilm. 
  • Two minor pilins can be found in the pilus shaft of enterococcal pili, which are heterotrimeric. Gram-positive pili are distinguished by the dedicated sortase used in their assembly.
  • The sortases covalently anchor proteins with a C-terminal pilin-associated motif to the peptidoglycan crosslink several classes of precursor proteins to form the pili. 
  • E. faecalis has two types of sortases in it: Sortase C is known as Bps (biofilm and pilus-associated sortase) and links the pilin subunits, whereas Sortase A links the majority of proteins with a C-terminal sortase motif to cell wall peptidoglycan.
Figure: Enterococcus faecalis biofilm formation. Image Source: Jun-Hong Ch’ng et al. 2018.

2) Proteins

  • A cell-wall-associated protein called extracellular surface protein (ESP) was first identified in Enterococcus species. 
  • The esp gene has a length of 5622 bp and is prevalent in isolates derived from infections. 
  • It is believed to encourage colonization, immune system evasion, adhesion, and antibiotic resistance. Esp gene also aids in forming enterococcal biofilms, which may make them more resilient to environmental stresses and allow them to adhere to eukaryotic cells like those in the urinary tract. 
  • Studies have demonstrated that E. faecalis can form biofilms less when the esp gene is disrupted.

Pathogenesis of Enterococcus faecalis

Although it is present in most healthy people, E. faecalis can infect humans and cause endocarditis, sepsis, urinary tract infections (UTIs), meningitis, and other infections. E. faecalis infections are thought to be influenced by several virulence factors. 

  • To colonize the gastrointestinal tract, which is their natural habitat;
  • To adhere to a variety of extracellular matrix proteins, such as thrombospondin, lactoferrin, and vitronectin; and 
  • To colonize human embryonic kidney cells, oral epithelia, and urinary tract epithelia.

The steps are;

1) Adherence to host tissues, especially urinary tract infections, is the first important step in the pathogenesis of E. faecalis. Aggregation substance (Agg), a surface protein expressed in response to pheromone induction that regulates E. faecalis adhesion to renal epithelial cells, is one of the virulence factors connected to adherence. The adhesin to collagen of E. faecalis is another surface protein that helps bacteria adhere to collagen (Ace). In the adherence to and colonization of host tissues, Agg and Ace both play important roles.

2) Additionally, E. faecalis can permanently cling to biotic and abiotic surfaces, forming biofilms. It has been demonstrated that these biofilms give the bacteria more antibiotic resistance than planktonic cells, which are more exposed to and susceptible to antibiotic therapy. In addition to adhesion, E. faecalis also secrete virulence factors that increase the severity of infection. Blood hemolysis brought on by the secreted toxin cytolysin, which E. faecalis produces in response to pheromones, adds to the organism’s pathogenicity.

3) The cytolysin, a plasmid-encoded hemolysin, is crucial for pathogenesis in animal models of infection and is linked to a five-fold increase in the risk of death in patients with bacteremia in humans when combined with high levels of gentamicin resistance. 

4) Gelatin and casein are hydrolyzed by the enzymes serine protease (SprE) and gelatinase (GelE), respectively. Enterococci’s ability to spread throughout their host is crucial to their ability to do so. Gelatinase is crucial for developing biofilms. Gelatinase has been shown to facilitate the aggregation of cells into microcolonies, which is the first step in forming biofilms.

Causes of Enterococcus faecalis Infection

  • Infections with E. faecalis can spread from one person to another due to improper hygiene. 
  • People who don’t wash their hands after using the restroom risk spreading the infection because these bacteria are found in feces. 
  • The bacteria can contaminate food or land on objects like telephones, computer keyboards, and doorknobs. They can then be transferred to others from there. 
  • Hospitals frequently experience E. faecalis outbreaks. If healthcare workers don’t wash their hands, the bacteria may spread. 
  • Dialysis ports, catheters, and other medical equipment that hasn’t been properly cleaned can also harbor E. faecalis.
  •  People who undergo organ transplantation, kidney dialysis, or cancer treatment are, therefore, more likely to contract infections due to compromised immune systems or contamination from their catheters.

Clinical manifestations of Enterococcus faecalis Infection

E. faecalis bring on several different types of infections in people. Some of them are:

1. Bacteremia

The risk of death from enterococcal bloodstream infections (BSI) is high. It is believed that enterococci translocate from the gut into the bloodstream in most cases of enterococcal BSI. Intravenous lines, endocarditis, urinary tract infections, and other abscesses are examples of additional infection routes.

2. Urinary tract infection (UTI)

E. faecalis cause over 50% of UTIs. A few investigations into how enterococci interact with uroepithelial tissue suggested that the plasmid-encoded aggregation substance may play a part in how enterococci adhere to renal epithelial cells. Infections of the urinary tract can affect the kidneys, urethra, and bladder.

3. Endocarditis

It is an infection of the endocardium, the inner lining of the heart. Infective endocarditis (IE) is one of the most therapeutically difficult infections by enterococci. Between 10 and 20 percent of all cases of IE are caused by enterococci, with E. faecalis being the most frequent cause. In IE, bacterial colonization of the endocardium results in vegetation within the tissue and a matrix that resembles a biofilm around the bacteria, making it challenging for the immune system or antibiotics to get through.

  • Inflammation of the membranes enclosing the brain and spinal cord is known as meningitis.
  • Periodontitis: This severe gum disease weakens the jawbones that support your teeth. It frequently affects people who have had a root canal.
  • If bacteria enter an open cut, such as during surgery, you could develop an infection. At all anatomical sites, including the eye, enterococci are frequent causes of surgical site infection.
  • Immune evasion

Enterococci must overcome specific and generalized host defense mechanisms to maintain an infection. It has been discovered that E. faecalis can actively cleave the complement protein C3 through the metalloprotease GelE, which lowers phagocytosis and increases bacterial survival rates.

Lab Diagnosis of Enterococcus faecalis

Bacterial culture and an antibiotic sensitivity test will be requested to detect E. faecalis. Depending on the infection site, a urine sample or blood drawn into designated vials and sent to the lab may be required. The samples will be tested in the lab to see if bacteria grow and to identify any that do.

1) Morphological and biochemical characteristics

The sample is streaked on the Enterococcal agar like chromogenic agar. Then, it will be incubated at 35 degrees C for 18-24 hrs. Identification will be done according to the gram staining and biochemical results. To determine which antibiotics will or won’t effectively treat the infection, they will undergo testing. The quality of the sample, as well as the collection, transportation, and handling of the sample to the laboratory, determines the reliability of the results.

2. Molecular Method

By using specific primers and PCR, the given gene of the reference strain E. faecalis can be amplified for species-level identification (positive control).

Treatment of Enterococcus faecalis Infection

  • Antibiotics are used to treat E. faecalis infections. The fact that these bacteria are now resistant to many different kinds of antibiotics presents a problem. As a result, some antibiotics are no longer effective in treating these bacteria. 
  • The antibiotic of choice for treating E. faecalis infections is ampicillin. Other antibiotic options include daptomycin, gentamicin, linezolid, nitrofurantoin, streptomycin, tigecycline, vancomycin, etc. 
  • A combination of antibiotics is used to treat more serious infections, like endocarditis or meningitis. Physicians frequently combine two distinct antibiotic classes. This may consist of ampicillin, vancomycin, gentamicin, and streptomycin in addition.

Prevention of Enterococcus faecalis Infection

Throughout the day, wash your hands with warm water and soap. Always wash your hands after using the restroom and before cooking or eating food. Use a hand sanitizer with an alcohol base if you do not have access to soap and water.

  • Never share personal items with anyone, particularly sick people. Towels, toothbrushes, forks, and spoons are examples of this.
  • Use an antibacterial disinfectant to clean communal items like phone handsets, doorknobs, and television remotes.
  • Make sure medical staff members wash their hands before caring for you while in the hospital, or have them wear clean gloves.
  • Request that all thermometers, blood pressure cuffs, IVs, catheters, and other equipment used in your treatment be cleaned and sterilized.

Application to Biotechnology

It has been investigated whether Enterococci could be used as a probiotic (a dietary supplement that contains living, non-virulent microbial cells that, when ingested, are thought to beneficially affect the composition of the intestinal microflora). It has been demonstrated that giving the E. faecalis strain reduces diarrhea. To reduce animal diarrhea, E. faecalis probiotics are added to the feed for chickens, pigs, and cattle. Due to E. faecalis high propensity for causing disease, more research has been done on how to reduce its virulence than on how to use it for good.

References

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