neisseria meningitidis

background information

Neisseria meningitidis is a pathogenic bacterium. The variety of illnesses it causes is referred to as Meningococcal Disease. This includes bacterial meningitis (infection of meninges in brain and spinal cord), meningococcemia (blood infection), pneumonia, septic arthritis, pericarditis (inflammation of pericardium), and urethritis (inflammation of urethra).

Other symptoms may include sudden onset fever, headache, nausea, vomiting, headache, severe myalgias, nonspecific rash, sore throat, other upper respiratory symptoms, neck stiffness, photophobia, petechiae or hemorrhagic rash, altered mental status, shock, abnormal skin color, purpura fulminans, or even disseminated intravascular coagulation.

Meningococcal disease caused by Neisseria meningitidis is important to be studied because there is a 50% fatality rate once an individual has contracted the disease. This is also an infectious disease, meaning it spreads easily from human to human contact. Meningococcal disease is also a huge public health burden to the Meningitis Belt of Africa, which several outside parties, including WHO, are committed to fixing.

Meningitis belt

Meningococcal diseases are most commonly found in the meningitis belt of Africa (sub-Saharan) with about 30,000 cases reported each year, but is present all throughout the world.

Growing neisseria meningitidis: BSL-2

Preparing the Agar and Broth:

Agar: 814 GC Agar/Broth medium

1. Agar Medium
  1. Solution A: 36.0 g GC agar base with 500 mL DI water
  2. Solution B: 10.0 g dried bovine hemoglobin with 500 mL DI water
  3. Autoclave Solutions A and B separately at 121ºC. Cool to 50ºC. Aseptically add Solution B to Solution A. Mix very well. Rehydrate 10.0 mL IsoVitalex according to package instructions and add aseptically to medium
2. Broth Medium
  1. Solution A: 15.0 g proteose peptone, 1.0 g corn starch, 4.0 g dipotassium phosphate, 1.0 g monopotassium phosphate, 5.0 g sodium chloride, 500 mL DI water
  2. Solution B: 10.0 g dried bovine hemoglobin with 500 mL DI water
  3. Autoclave Solutions A and B separately at 121ºC. Cool to 50ºC. Aseptically add Solution B to Solution A. Mix very well. Rehydrate 10.0 mL IsoVitalex according to package instructions and add aseptically to medium.
3. Mix well and dispense into appropriate vessel

Growth Conditions: 5% Carbon Dioxide at 37°C

Procedure:

Open vial according to enclosed instructions
Using a single tube of #814 broth (5 to 6 mL), withdraw approximately 0.5 to 1.0 mL with a Pasteur or 1.0 mL pipette. Rehydrate pellet.
Aseptically transfer this aliquot back into the broth tube. Mix well.
Use several drops of the suspension to inoculate a #814 agar slant and/or plate.
Incubate the tubes and plate under an atmosphere of 5% CO2 at 37°C for 24-48 hours

Special Instructions: store at -80°C or lower if sample frozen, store between 2-8°C if sample freeze dried

vizualize neisseria meningitidis

differentiating neisseria meningitidis

Blood Agar

Observation: Alpha Hemolysis- Greenish color will appear around the colonies, but will not contain colorless areas.

Meaning: Incomplete hemolysis of the red blood cells. Greenish color caused by hydrogen peroxide production of the bacteria.

macconkey agar

Observations: Colorless areas on the MacConkey agar.

Meaning: N. meningitidis is non-lactose fermenting, and therefore it will appear colorless on the plate.

mannitol

Observation: There will be no growth on the plate, and the MSA agar will remain red.

Meaning: MSA is selective for ability to grow in high salt concentrations, which N. meningitidis cannot. N. meningitidis also cannot ferment Mannitol, therefore, the agar will not turn yellow.

dnase

Observation: No colorless areas around the streak. Agar should remain original color.

Meaning: This test is differential to bacteria that produce deoxyribonuclease, which breaks down the DNA in the plate. N. meningitidis does not produce this molecule, which is why the agar remains unchanged.

gram stain

Observation: Bacterial cells appear pink/red under the microscope, indicating a gram-negative bacteria.

Meaning: A gram-negative test result indicates that the bacteria has an outer membrane, a thin peptidoglycan layer, and a large periplasmic space. It is also a good indicator that the bacteria is pathogenic.

phenol red broth

Observation: The medium will turn from red to yellow, and a gas bubble will be present in the inverted tube.

Meaning: The color of the phenol red broth is a pH indicator, changing from red to yellow when acid is produced when the organism ferments the glucose. The gas bubble is also a product of glucose fermentation, and indicates an aerobic species.

sim medium deep agar

Observation: The H₂S test will remain unchanged (doesn’t turn black), motility test will develop a stab line, and indole test will appear yellow.

Meaning: SIM medium deep agar test for sulfide production, motility of the cells, and indole formation. N. meningitidis has negative results for all three of these tests, meaning that it doesn’t produce hydrogen sulfide, is not cellularly motile, and does not possess the enzyme tryptophanase to produce indole.

catalase

Observation: Many bubbles will appear.

Meaning: Catalase test is used to determine if a species is aerobic, meaning they respire using oxygen. It also indicates that the species possesses catalase, which protects them from the toxic byproducts of oxygen respiration.

oxidase

Observation: The test will turn blue/purple within 30 seconds.

Meaning: The oxidase test detects the presence of a cytochrome oxidase in the organism’s respiratory chain. This positive result is indicative of aerobic organisms, especially ones that use oxygen as their final electron acceptor during respiration.

molecular diagnostics

16S primers for Neisseria meningitidis PCR:

8F (5′-AGTTGATCCTGGCTCAG-3′) and (3’-TCAACTGGACCAGTC-5’)

Melting Temperature: 47.1° C

GC Content: 53%

1492R (5′-ACCTTGTTACGACTT-3′) and (3’-TGGAACAATGCTGAA-5’)

Melting Temperature: 36.5° C

GC Content: 40%

16S rRNA Sequence determined from primers:

>2720789915

TTGAACATAAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCATGCT

TTACACATGCAAGTCGGACGGCAGCACAGAGAAGCTTGCTTCTTGGGTGG

CGAGTGGCGAACGGGTGAGTAACATATCGGAACGTACCGAGTAGTGGGGGATAACTGATCGAAAGATCAGCTAATACCGCATACGTCTTGAGAGAGAAAGCAGGGGACCTTTGGGCCTTGCGCTATTCGAGCGGCCGATATCTGATTAGCTGGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCAGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGTCTGAAGAAGGCCTTCGGGTTGTAAAGGACTTTTGTCAGGGAAGAAAAGGCTGTTGCTAATACCAGCGGCCGATGACGGTACCTGAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGGGCGCAGACGGTTACTTAAGCAAGATGTGAAATCCCCGGGCTCAACCCGGGAACTGCGTTCTGAACTGGGTGACTAGAGTGTGTCAGAGGGAGGTAGAATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCTCCTGGGACAACACTGACGTTCATGCCCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAATTAGCTGTTGGGCAACTTGATTGCTTAGTAGCGTAGCTAACGCGTGAAATTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACTCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATGTACGGAATCCTCCAGAGACGGAGGAGTGCCTTCGGGAGCCGTAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCCATCATTCAGTTGGGCACTCTAATGAGACTGCCGGTGACAAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGACCAGGGCTTCACACGTCATACAATGGTCGGTACAGAGGGTAGCCAAGCCGCGAGGCGGAGCCAATCTCACAAAACCGATCGTAGTCCGGATTGCACTCTGCAACTCGAGTGCATGAAGTCGGAATCGCTAGTAATCGCAGGTCAGCATACTGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGGGATACCAGAAGTAGGTAGGGTAACCGCAAGGAGTCCGCTTACCACGGTATGCTTCATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGGAACCTGCGGCTGGATCACCTCCTTTCT

treatments

Individuals can prevent contraction by use of meningococcal vaccination, which exist in two types: polysaccharide and conjugated polysaccharide.

Meningococcal disease is usually treated with penicillin, ampicillin, or a combination of penicillin and chloramphenicol. However, several serogroups are becoming increasing resilient to penicillin.

references

Arreaza L, Fuente L, Vazquez J. 2000. Antibiotic Susceptibility Patterns of Neisseria meningitidis Isolates from Patients and Asymptomatic Carriers. Antimicrob Agents Chemother 44(6): 1705-1707. 10.1128/aac.44.6.1705-1707.2000

Charles-Orszag A, Tsai FC, Bonazzi D, Manriquez V, Sachse M, Mallet A, Salles A, Melican K, Staneva R, Bertin A, Millien C, Goussard S, Lafaye P, Shorte S, Piel M, Krijnse-Locker J, Brochard-Wyart F, Bassereau P, Dumenil G. 2018. Adhesion to nanofibers drives cell membrane remodeling through one-dimensional wetting. Nat Commun 9:4450. 10.1038/s41467-018-06948-x.

Daraghma R, Sapra A. Meningococcal Vaccine. 2020. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan-. https://www.ncbi.nlm.nih.gov/books/NBK553102/

Nguyen N, Ashong D. Neisseria Meningitidis. 2020. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan-. https://www.ncbi.nlm.nih.gov/books/NBK549849/

Rouphael NG, Stephens DS. 2012. Neisseria meningitidis: Biology, Microbiology, and Epidemiology. Methods Mol Biol 799:1-20. 10.1007/978-1-61779-346-2_1.

Sacchi C, Whitney A, Reeves M, Mayer L, Popovic T. 2002. Sequence Diversity of Neisseria meningitidis 16S rRNA Genes and Use of 16S rRNA Gene Sequencing as a Molecular Subtyping Tool. J Clin Microbiol 40(12): 4520–4527. 10.1128/JCM.40.12.4520-4527.2002

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