What is the BSL level for your microbe?

The Biosafety classification for Neisseria gonorrhoeae according/based on U.S Public Health Service Guidelines is of level 2.

What type of agar do you need?

GC Agar/Broth Medium. Also, chocolate agar base (with the addition of supplements) promotes excellent growth; however, as mentioned before, GC agar gives slightly better results than chocolate agar.

Does it need to be in aerobic/anaerobic conditions?

Well, Neisseria gonorrhoeae generally is associated to be an “obligate aerobe”; however, it can grow in the absence of oxygen (anaerobic respiration) by using nitrite as a terminal electron acceptor.

What is the procedure for growing it once it arrives?

According to the ATCC and the CDC, the right procedures to follow for growing Neisseria gonorrhoeae once it arrives are:

• 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 mL pipette. Rehydrate the entire 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 at 37°C for 24 to 48 hours in an atmosphere of 5% CO2.

• Assure that screw caps are loose during incubation period.

Are there any other special instructions?

Appropriate safety procedures should always be used with this material. ATTC recommends the microbial cultures to be handled only by qualified professionals (microbiologists) by using appropriate PPE (personal protective equipment) and precautions.

When was the first documentation of it? Who discovered it? (Usually the first author)

The first documentation of Neisseria gonorrhoeae dates back to approximately 3,500 years ago; however, it was not until 1879 when Albert Neisser “determined the etiologic agent of the disease”.

Determine the tests used to identify your microbe your microbe and distinguish from related microbes.

According to Gideon, Neisseria gonorrhoeae can grow on Thayer Martin agar, which is an agar that contains 5% chocolate sheep blood and antibiotics. In addition, Neisseria gonorrhoeae presents no growth on nutrient agar.

Can it be confused for something else?

No other bacteria have similar combined test results to Neisseria gonorrhoeae. The only way in which Neisseria gonorrhoeae is mistaken is when its symptoms are mild or nonspecific, since they are mistaken for a “bladder or vaginal infection”.

Does anything grow in the conditions and appear the same?

No, the closest bacterium which may have a few similar test results is Neisseria meningitidis. They (Neisseria gonorrhoeae and Neisseria meningitidis) are both Gram-negative that can grow on Thayer Martin agar. They have the same positive results for glucose and the same negative results for sucrose, and lactose. Also, they have the same negative result in DNAse test. Even when they have a few similar results for some of the tests, they can be distinguished from one another by performing more tests. Some of the tests in which they differ (and make them unique) are: Growth on ordinary blood agar, maltose test, and nitrite to gas test.

The 16s primers

What are the primers you are using?

N. gonorrhoeae porA pseudogene (source: Journal of Molecular Diagnostics)

• Forward primer sequence: 5’- CAGCTCGTGTCGTGAGATGT- ‘3

(Left primer)

• Reverse primer sequence: 5’- CGGACTACGATCGGTTTTGT- ‘3

(Right primer)

What is the melting temperature?

N. gonorrhoeae porA pseudogene (source: Journal of Molecular Diagnostics)

• The melting temperature of N. gonorrhoeae forward primer is: 60.1*C

• The melting temperature of N. gonorrhoeae reverse primer is: 60.0*C

What is the GC content?

N. gonorrhoeae porA pseudogene (source: Journal of Molecular Diagnostics)

• The GC content of the forward primer: 55.0%

• The GC content of the reverse primer: 50.0%

In Silico PCR

Amplify 16s sequence in the program and include the specific 16s sequence (in FASTA format) in your submission.

In silico PCR amplification

N. gonorrhoeae porA pseudogene (source: Journal of Molecular Diagnostics)

>NR_026079.2 Neisseria gonorrhoeae strain NCTC 8375 16S ribosomal RNA, complete sequence

GAACATAAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCATGCTTTACACATGCAAGTCGGACGGC

AGCACAGGGAAGCTTGCTTCTCGGGTGGCGAGTGGCGAACGGGTGAGTAACATATCGGAACGTACCGGGT

AGCGGGGGATAACTGATCGAAAGATCAGCTAATACCGCATACGTCTTGAGAGGGAAAGCAGGGGACCTTC

GGGCCTTGCGCTATCCGAGCGGCCGATATCTGATTAGCTGGTTGGCGGGGTAAAGGCCCACCAAGGCGAC

GATCAGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAG

GCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGTCTGAAGAAGGCCT

TCGGGTTGTAAAGGACTTTTGTCAGGGAAGAAAAGGCTGTTGCCAATATCGGCGGCCGATGACGGTACCT

GAAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAA

TTACTGGGCGTAAAGCGGGCGCAGACGGTTACTTAAGCAGGATGTGAAATCCCCGGGCTCAACCCGGGAA

CTGCGTTCTGAACTGGGTGACTCGAGTGTGTCAGAGGGAGGTGGAATTCCACGTGTAGCAGTGAAATGCG

TAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCTCCTGGGATAACACTGACGTTCATGTCCGAAAGC

GTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAATTAGCTGTTGGGCAA

CTTGATTGCTTGGTAGCGTAGCTAACGCGTGAAATTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAAC

TCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAGAACC

TTACCTGGTTTTGACATGTGCGGAATCCTCCGGAGACGGAGGAGTGCCTTCGGGAGCCGTAACACAGGTG

CTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCA

TTAGTTGCCATCATTCGGTTGGGCACTCTAATGAGACTGCCGGTGACAAGCCGGAGGAAGGTGGGGATGA

CGTCAAGTCCTCATGGCCCTTATGACCAGGGCTTCACACGTCATACAATGGTCGGTACAGAGGGTAGCCA

AGCCGCGAGGCGGAGCCAATCTCACAAAACCGATCGTAGTCCGGATTGCACTCTGCAACTCGAGTGCATG

AAGTCGGAATCGCTAGTAATCGCAGGTCAGCATACTGCGGTGAATACGTTCCCGGGTCTTGTACACACCG

CCCGTCACACCATGGGAGTGGGGGATACCAGAAGTAGGTAGGGTAACCGCAAGGAGTCCGCTTACCACGG

TATGCTTCATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGGAACCTGCGGCTGGATCACCTCCTT

T

Is it a pathogen? What disease does it cause? Or does it have any benefits to the host?

Yes, Neisseria gonorrhoeae is categorized as an “obligate human pathogen”. N. gonorrhoeae causes mucosal surface infections on male and female reproductive tracts, pharynx, rectum, and conjunctiva (eyes).

How can infection or contraction of the disease be prevented

According to the Centers for Disease Control and Prevention (CDC) the surest way to avoid transmission of N. gonorrhoeae is to abstain from vaginal, anal, and oral sex. Also, another method of prevention is to be in a long-term mutually monogamous relationship (with an uninfected partner)

Where is it traditionally found?

Any sexually active individual may be infected with gonorrhoeae. Here in the United States, the highest “reported” rates of infection are among sexually active teenagers, young adults, and African Americans.

Why do people care about it?

People should care about N. gonorrhoeae because it is transmitted through sexual contact with the vagina, penis, mouth, or anus of an infected individual. Another reason why it matters, and people should care, is because it is so contagious that ejaculation does not have to occur in order to be acquired. In addition, N. gonorrhoeae can be spread perinatally from mother to newborn during childbirth, and those people who have had gonorrhoeae and received specific treatment may be reinfected if they have contact with an infected person.

Is there a commercial or ecological application, other oddities about your adopted microbe?

• Neisseria gonorrhoeae is the second most common bacterial sexually transmitted infection (STI) and results in substantial morbidity and economic costs worldwide

• This bacterial infection is also known as “the clap”

• Most women do not have symptoms. If they do, they often mistake them for something else

• If not treated, N. gonorrhoeae can cause long-lasting problems including infertility, infection in other parts of the body like the skin or joints, or even increased chance of getting HIV

• These bacteria cannot live long outside of the body; therefore, it cannot be contracted by touching objects such as toilet seats or clothes.

What antibiotics work against your bacteria? Are there any other treatments for it?

Neisseria gonorrhoeae has developed resistance to antibiotic drugs that are prescribed to treat it. After the spread of gonococcal fluoroquinolone (targeting RNA replication) resistance, the cephalosporin antibiotics (B-lactams-cell wall synthesis inhibitors) have been the recommended treatment for gonorrhoeae. If an individual is allergic to cephalosporin antibiotics, such as ceftriaxone, then gemifloxacin may be administered by mouth or gentamicin may be injected and azithromycin be administered by mouth. The emergence of a cephalosporin-resistant N. gonorrhoeae would complicate the ability to treat these bacteria effectively since there are only few antibiotic options left.

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