Enterococcus faecalis

Zuhair Syed

Enterococcus faecalis is a pathogen. It causes several different kinds of infections such as endocarditis, prostatitis, intra-abdominal infection, cellulitis, concurrent bacteremia, and is especially known for causing urinary tract infections. In serious cases, the bacteria can also cause meningitis or Periodontitis. E. faecalis does not provide any benefits to the host and is normally a commensal bacteria primarily in the human gut and also in the mouth and vagina, and only causes infections if it spreads to other parts of the body like the blood, urinary tract, or surgical wounds.

Background

Where is E. faecalis traditionally found?

The bacteria are naturally found in the human gut and in human feces. It is also found in smaller numbers in a person’s vagina or mouth. Also, it is present in animal gastrointestinal tracts. Furthermore, it is commonly found on medical equipment that interacts with the human gastrointestinal system such as dialysis machines and catheters.

Why care about E. faecalis?

It is important because, as previously mentioned, it causes a lot of infections and spread very easily from essentially any person without good hygiene (particularly bathroom hygiene practices) and poses a great risk to immunosuppressed patients like cancer treatment or surgery patients in the hospital or people with wound openings. Due to its tendency to cause nosocomial infections, hospitals pay close attention to it and take care to sterilize surfaces and equipment in the hospital and also staff and visitors should avoid touching things they don’t have to in order to prevent spread or contamination.

Commercial or ecological applications of E. faecalis

Despite its pathogenic nature, E. faecalis does have commercial or ecological applications. Due to its presence in both human and animal feces, it is in a position to be used as a “surrogate" for waterborne pathogens. So, E. faecalis and other Enterococci are used as fecal indicator bacteria in research labs and water quality testing worldwide such as being used to assess health risks from recreational bodies of water suspected of being polluted or assess the safety of sources of drinking water.

Treatments

How can infection or contraction of disease from E. faecalis be prevented?

Infection by E. faecalis can be prevented by following good hygiene practices. E. faecalis infections spread from one person to another due to lack of proper hygiene and since it is found in human feces, not washing hands after using the bathroom allows the bacteria to be spread onto surfaces that the person touches such as food, doorknobs, and tables. Therefore, washing hands after using the restroom is an effective way to prevent E. faecalis infections. Also, infections can be prevented by cleaning and sanitizing equipment in hospitals since E. faecalis is often spread in hospitals especially through medical equipment like dirty catheters and dialysis ports. Furthermore, infections can be prevented by carefully sterilizing the rooms that immunosuppressed patients, such as those who have undergone an organ transplant or dialysis, are kept in and the surfaces and equipment that they may come into contact with.

Using antibiotics and other treatment methods against E. faecalis

Antibiotics are not very effective for fighting the bacteria. It has resistance to multiple antibiotics and gains resistance to new antibiotics quickly. It is even resistant to Vancomycin which is a last resort drug for dealing with many gram-positive infections. However, the preferred medicine for dealing with E. faecalis is Ampicillin and other options include daptomycin, gentamicin, linezolid, nitrofurantoin, streptomycin, tigecycline, and vancomycin (some strains are not resistant to vancomycin). Also, other drugs such as Arbekacin and Dibekacin, which inhibit ribosomal binding of formylmethionyl-tRNA and Abacavir, a drug that attacks the inhibits the reverse transcriptase abilities of the bacteria, are used in treatments to the bacteria. There aren’t any other treatments for it, but for severe infections like endocarditis or meningitis, combined mixtures of multiple classes of drugs are prescribed by doctors.

Identification

Tests to identify Enterococcus faecalis:

The best test to use to identify Enterococcus faecalis from other related bacteria is to use MSA. E. faecalis and many other Enterococcus have the same results for all the other tests that we discussed in this class. If we include tests not discussed in class, then tests for glycerol, sucrose, D-Sorbitol, and Arginine dihydrolase would be helpful to distinguish E. faecalis from other Enterococcus bacteria.

To identify Enterococcus faecalis from other bacteria in general, the tests to use are gram staining, blood agar, macconkey agar, catalyse, oxidase, phenol broth, and SIM because E. faecalis shows very strong results on each of these tests that can be used to distinguish it from other microbes.

Identification Errors

Enterococcus faecalis can be easily confused for other species of Enterococci bacteria. Enterococci bacteria are typically easy to distinguish from other bacteria and Bile esculin (bile salt) agar tests are actually used to very easily identify Enterococci. However, because of their close properties, similar appearance, and ability to grow in the same conditions, they have very similar results on many tests. Enterococcus faecium in particular has very similar results on tests and can be easily identified in place of Enterococcus faecalis.

Blood Agar

Enterococcus faecalis normally shows gamma hemolysis (however some strains show alpha-hemolytic or even beta-hemolytic behavior). This means that E. faecalis does not produce the exotoxin hemolysin and does not destroy red blood cells and hemoglobin.

MacConkey

E. faecalis will not grow or will hardly grow on MacConkey agar, showing colorless colonies This indicates that the bacteria is gram positive and that it can not ferment lactose.

Mannitol Salt Agar

The bacteria colonies turn the medium yellow. This means that the bacteria can digest mannitol and grow in 6.5 %/ 7.5% NaCl environments meaning it is salt tolerant. Bacteria that have this result are typically pathogenic, correctly indicating that E. faecalis is pathogenic.

DNAse

The bacteria shows negative DNAase indicated by absence of colored zones around the bacteria. This means that the bacteria does not carry out dexoyribonuclease activity. It is not able to depolymerize DNA.

Anaerobic Chamber

E. faecalis grows in an anaerobic chamber. This means the bacteria can survive without oxygen and either is anaerobic or has the capability to switch to being anaerobic. In fact, E. faecalis is a facultative anaerobe meaning it can survive with or without oxygen.

Gram Stain

E. faecalis stains purple so it is gram positive. This means the bacteria has a thick peptidoglycan layer and also does not have an outer lipid membrane.

Phenol Red Broth

The bacteria show a yellow broth without bubbles. This means that it is a glucose fermenter but is not a glucose oxidizer. It is a homofermentative bacteria.

SIM medium deep agar

E. faecalis is not motile. This means that it does not move and it has either weak or nonexistent flagella. The nonmotile E. faecalis bacteria stays in a human’s or animal’s gi tract. This has important health implications because the bacteria would cause infections if it did leave the GI tract. Normally SIM indole tests provide readings on hydrogen sulfide or indole, but this data is not readily available for E. faecalis.

Catalase Test

The bacteria reads a negative response for catalyse indicating it does not the catalsyze enzyme. It is not able to detoxify hydrogen peroxide by breaking it into water and oxygen. In fact, it actually does have catalyse enzymes but can only catalyze in the presence of heme, making E. faecalis one of the only species of lactic acid bacteria that have the capability to use catalyse activity.

Oxidase Test

It has negative oxidase results. This means that E. faecalis does not have oxidase ( cytochrome c oxidases, to be exact) and is not able to catalyze the transfer of electrons by reducing cytochrome C to O2.

Visualizing Enterococcus faecalis

Morphology

The morphology of E. Faecalis is cocci. This is visible in the gram stains shown. It can appear as cocci with pairs and short chains or as clusters. Furthermore, E. Faecalis can be diplococcus or single cocci bacteria depending on strain.

Gram Stains

The color of the gram stain is purple revealing that Enterococcus Faecalis is a gram positive bacteria. The bacteria in the image above are shown on blood agar.

On the right is another gram stain. The image labelled A is a single colony variant and the image labelled B is wild type. The images were taken after 24 hours of incubation in CASO blood agar.

SEM - Scanning Electron Microscope

These are several SEM images of E. Faecalis. The images on the top marked A are from single colony variant and the images on the bottom marked B are wild type. No stain was used, and the images were taken at 20,000 times magnification.

TEM - Transmission Electron Microscope

These are several TEM images of E. Faecalis. The images marked A to D show single colony variant and the images marked E and F are wild type. No stain was used, and the images were taken at varying magnifications shown in the bottom right of each image.

Confocal Microscopy

These images show confocal microscopy images of E. Faecalis biofilm in various media over time. The images labeled as A are Vancomycin susceptible strain FA2-2 and the images labeled B are Vancomycin resistant strain V583. Orange areas show live bacteria and green areas show dead bacteria. A florescent dye was used and then the images were digitally enhanced for color.

Growing Enterococcus Faecalis

1. BSL Level

The BSL for Enterococcus Faecalis is Level 2, however, genomic DNA is classified as BSL 1

2. Agar Needed

The bacteria can be cultured in ATCC Medium: 44 Brain Heart Infusion Agar/Broth or ATCC Medium: 260 Tryptic Soy Agar/Broth with 5% Sheep Blood

3. Growth conditions

It needs Aerobic conditions to live.

4. Procedure for growing upon arrival

Rehydrate the entire pellet with 0.5mL of ATCC® Medium 44: Brain Heart Infusion Agar/Broth and aseptically transfer the entire solution to a 56 mL tube of the broth. To inoculate the bacteria into more test tubes, take 0.5 mL from this 56 mL test tube and place into each additional test tube. Use several drops of the bacteria-broth solution in the test tube to inoculate a plate or a slant of ATCC® Medium 260: Trypticase soy agar/broth with defibrinated sheep blood. Incubate the bacteria at 37°C for 24 hours.

5. Special Instructions

There are no special instructions for the bacteria. However, there are instructions to make the ATCC Medium: 260 Tryptic Soy Agar with 5% Sheep Blood (defibrinated), which is the agar it grows in, if it is not already available. To make it, autoclave the ATCC Medium: 260 Tryptic Soy Agar at 121ºC and then cool the sterilized medium to about 47ºC, and after cooling, aseptically add 50 mL of defibrinated sheep blood at room temperature (https://www.atcc.org/products/all/29212.aspx#history). Gently mix the medium and then use as needed. The same process is followed if Tryptic Soy Broth is used with broth used as a substitute for whenever agar is used.

History: When was the first documentation of the bacteria and who discovered it?

The first documentation of Enterococcus faecalis was in 1899 in “A Case of Acute Endocarditis Caused By Micrococcus Zymogenes (Nov. Spec.), With A Description Of The Microorganism by William G. MacCallum and Thomas W. Hastings. MacCallum and Hastings discovered Enterococcus faecalis from a lethal case of endocarditis and provided the first detailed description of the pathogenic properties of the bacteria.

Molecular Diagnostics

Using 16s primers

2. In Silico PCR

Amplifying 16s sequence in the In Silico PCR program and the specific 16s sequence:

Partial 16s sequence in FASTA format from In Siloco PCR:

>NC_017312 Enterococcus faecalis 62 - nucleotides 375281-375602 (322)

CTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTTTG

TCCCCGAAGGGAAAGCTCTATCTCTAGAGTGGTCAAAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTG

CTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCG

GTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTTGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTT

AGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATC

Nucleotide BLAST: 16s rRNA sequence

NCBI Blast only showed partial sequences for Enterococcus faecalis :

>NR_159231.1 Enterococcus wangshanyuanii strain MN05 16S ribosomal RNA, partial sequence

AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCTTCTTTTC

TACCGAGTGCTTGCACTCATTTGAAAAGAGGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCA

TCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAGTCGACACCGCATGGTGTTGATTT

GAAAGACGCTTTCGGGTGTCACTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTC

ACCAAGGCGATGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGAC

TCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGT

GAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGTAGGAGAGTAACTGCTCTTACCT

TGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGC

GTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCT

CAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGC

GGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGA

GGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAA

GTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGACCG

CAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCA

ACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTTCCCTTCGGGGACA

AAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCG

CAACCCTTATTGTTAGTTGCCATCATTTAGTTGGGCACTCTAGCGAGACTGCCGGTGACAAACCGGAGGA

AGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAGTAC

AACGAGTCGCTAGGCCGCGAGGTCATGCAAATCTCTTAAAGCTTCTCTCAGTTCGGATTGTAGGCTGCAA

CTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCC

TTGTACACACCGCCCGTCACACCACGAAAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCC

AGCCGCCTAAGGTGGGATAGATGATTGGGGTGAAGTCGTAACAAGGTA

About The Author

Zuhair Syed is a second year microbiology major at the University of Florida on the pre-med track. He aims to become a physician and conduct innovative medical research at an academic medical center.

He is from Tallahassee, FL. In his free time he likes to play video games, watch tv shows, and hang out with friends. His favorite food is pizza and he is passionate about teaching other students. He is really interested in microbiology and biotechnology and their use for medical applications.

References

Byappanahalli MN, Nevers MB, Korajkic A, Staley ZR, Harwood VJ. 2012. Enterococci in the Environment. Microbiol Mol Biol Rev 76:685–706.

Kau AL, Martin SM, Lyon W, Hayes E, Caparon MG, Hultgren SJ. 2005. Enterococcus faecalis Tropism for the Kidneys in the Urinary Tract of C57BL/6J Mice. Infect Immun 73:2461–2468.

Kumar L, Cox CR, Sarkar SK. 2019. Matrix metalloprotease-1 inhibits and disrupts Enterococcus faecalis biofilms. Plos One 14.

Ledeboer NA, Das K, Eveland M, Roger-Dalbert C, Mailler S, Chatellier S. 2007. Evaluation of a novel chromogenic agar medium for isolation and differentiation of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis isolates. J Clin Microbiol.;45:1556–60. doi: 10.1128/JCM.02116-06.

Wanger A, Chavez V, Huang RS, Wahed A, Actor JK, Dasgupta A. 2017. Overview of Bacteria. Microbiology and Molecular Diagnosis in Pathology 75–117.

Wellinghausen, N., Chatterjee, I., Berger, A., Niederfuehr, A., Proctor, R., & Kahl, B. (2009, September 01). Characterization of Clinical Enterococcus faecalis Small-Colony Variants. JCM Aug 2009, 47 (9) 2802-281

William G. MacCallum and Thomas W. Hastings. 1899. A Case of Acute Endocarditis Caused by Micrococcus Zymogenes (Nov. Spec.), With a Description of The Microorganism. J Exp Med. 4(5-6):521-534

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