Path 3100 Medical Microbiology PDF

PATH 3100. Medical Microbiology Neisseria species and Moraxella catarrhalis Dr. Elena Y Enioutina, M.D., Ph.D.  Objectives Compare two common strains of Neisseria: Neisseria gonorrhoeae and Neisseria meningitidis. Determine the appropriate test methods fo...

PATH 3100. Medical Microbiology Neisseria species and Moraxella catarrhalis Dr. Elena Y Enioutina, M.D., Ph.D.  Objectives Compare two common strains of Neisseria: Neisseria gonorrhoeae and Neisseria meningitidis. Determine the appropriate test methods for recovering, isolating, and identifying Neisseria and Moraxella. Describe the transmission and disease states for: N. gonorrhoeae, N. meningitidis, and Moraxella catarrhalis.  Neisseria: Biology - Many species isolated from humans, but not all are pathogens - N. gonorrhoeae and N. meningitidis are pathogens - N. gonorrhoeae is always a pathogen - N. meningitidis can be commensal Shared Characteristics of N. gonorrhoeae and N. meningitidis - Gram-negative diplococci - Have pili for attachment to the host cells https://en.wikipedia.org/wiki/Neisseria_gonorrhoeae - Aerobic - Prefer or require increased CO2 for growth - Optimal growth at 35-37°C in moist environment - FASTIDIOUS (sensitive to drying and temperature) - Non-motile - Affinity for mucous membranes  General Differences Between N. gonorrhoeae and N. meningitidis N. gonorrhoeae N. meningitidis 1. Isolated from urethra, 1. Isolated from nasopharynx, cervix, rectum, pharynx blood, CSF 2. Sometimes isolated from 2. Colonizes nasopharynx CSF, joints, skin 3. Transmitted primarily by 3. Transmitted by direct contact sexual contact with respiratory secretions 4. Causes localized infection 4. Causes systemic infection 5. “Gonococcus” or “GC” 5. “Meningococcus”  General Differences Between N. gonorrhoeae and N. meningitidis N. gonorrhoeae N. meningitidis 6. Rarely lethal 6. Life threatening 7. Killed rapidly in blood 7. Multiples rapidly in blood 8. Very fastidious 8. Not as fastidious – grows on sheep blood agar 9. Not encapsulated 9. Capsules present 10. Humans are the only 10. Nasopharynx of humans is known reservoir the most common reservoir  Neisseria Identification Gram-negative cocci (diplo, kidney-bean shape) Colonies are usually oxidase positive. Neisseria Identification Oxidase positive  “Lancet-shaped” (spear point) Gram-positive diplococci: Think Streptococcus pneumoniae May appear in chains. May see a capsule. Appear as two beans: flat sides together Not in chains. Often clustered. Often intracellular. Gram-negative diplococci: Think Neisseria species  Rapid tests for Neisseria ID 1. Direct Fluorescent Antibody 2. DNA probes 3. MALDI-TOF 4. DNA Sequencing 5. Antigenic tests Activity! – 5 minutes Neisseria gonorrhoeae  Gonorrhea Gonorrhea is acute pyogenic infection of mucosa – vaginal/urethral, anal, pharyngeal N. gonorrhoeae can cause pharyngitis, suppurative arthritis, and ophthalmitis in newborns Can cause or be a co-infector in pelvic inflammatory disease Male urethral exudate Important! N. gonorrhoeae has pili. Pili help bacteria to attach and infect male urethra  Case study A 19-year-old female was seen in clinic. On physical exam, she had a swollen right knee and decreased range of motion of her right shoulder. She also had a thick vaginal discharge. Her temperature was 38.4ºC, and she had a WBC of 15,7000mm3. She gave a history of having two recent sexual partners. Blood, vaginal, and joint fluid cultures were performed. Both the vaginal and joint fluid cultures grew the same organism. N. gonorrhoeae: Laboratory Testing Specimen: – Exudate from infected area (genital, throat, anal) immediately placed into transport media or plate if culturing – Urine placed into transport media (NAAT) – Swab from infected area (as per exudate) Methods: – Nucleic Acid Amplification Tests (NAAT) for Detection – preferred test for N. gonorrhoeae detection – Culture – Gram Stain  Nucleic Acid Amplification Tests (NAAT) for Detection Urine or urethral swab, limited other sources Amplifies and detects N. gonorrhoeae DNA (if present) Results typically available within ≤ 24 hours  N. gonorrhoeae: Specimen Collection for Culture Use Dacron or rayon swab (no cotton or calcium alginate) Organisms are sensitive to atmosphere and temperature. Send specimens to lab at no less than room temperature (22-25°C) immediately Direct inoculation of selective media for N. gonorrhoeae (e.g. Modified Thayer-Martin) Culture for Neisseria gonorrhoeae Chocolate agar Modified Thayer Martin - Lysed sheep red blood agar cells - Lysed sheep blood - Vancomycin - Colistin - Nystatin - Trimethoprim Incubate in 3-5% CO2  Thayer Martin Agar Enzymatic Digest of Casein 7.5 g Enzymatic Digest of Animal Tissues 7.5 g Corn Starch 1.0 g Dipotassium Phosphate 4.0 g Monopotassium Phosphate 1.0 g Sodium Chloride 5.0 g Sodium Chloride 5.0 g Agar 17.0 g Horse Blood, defibrinated 50.0 ml Vitalex Growth Supplement 10.0 ml Vancomycin 1.0 mg. Colistin Sulfate 3.75 mg Amphotericin B 0.5 mg Trimethoprim 1.5 mg Final pH 7.2 ± 0.2 at 25°C N. gonorrhoeae: Direct inoculation of plate at bedside GC Culture Plates (Jembec plate)  Direct Gram Stain Male urethral discharge – used as a diagnostic test and may still be done in some STD clinics Female endocervix – low sensitivity; must follow with culture or molecular testing The laboratory diagnosis of Neisseria gonorrhoeae Can J Infect Dis Med Microbiol. 2005 Jan-Feb; 16(1): 15–25  Gonorrhea Epidemiology 468,514 identified cases in U.S. https://www.cdc.gov/std/stats16/Gonorrhea.htm  Rates of Reported Cases by Sex, United States, 2010–2019 “CDC estimates that approximately 1.6 million new gonococcal infections occurred in the United States in 2018, and more than half occur among young people aged 15-24”. https://www.cdc.gov/std/statistics/2019/overview.htm#Gonorrhea  N. gonorrhoeae Treatment 1. Ceftriaxone with azithromycin is the treatment of choice 2. Strains are now resistant to penicillin 3. Fluoroquinolones (ciprofloxacin) not recommended because of emerging resistance 4. Pan-resistant gonorrhea? – 7/7/2017 WHO announced that “at least three people worldwide are infected with totally untreatable ‘superbug’ strains of gonorrhea which they are likely to be spreading to others through sex.” Neisseria meningitidis  Neisseria meningitidis: Transmission Nasopharynx of humans is the only reservoir Spread by respiratory droplets 5-15% of teenagers and young adults are carriers Outbreaks in closed populations such as frat houses, military barracks, dorms  N. meningitidis Disease Meningitis – **medical emergency** Fever, headache, neck stiffness, photophobia, irritability Meningococcemia – disseminated disease Petechial rash Shock and disseminated intravascular coagulation (DIC) Usually in kids and young adults, but can strike all age groups  Case Study A 27 year old mother of four comes to the medical clinic complaining of a scratchy throat. The physician takes a throat culture, tells her to take some aspirin and to call the next day. She went home and felt worse with an increased headache. She came back to the ER and now says she has the worst headache she has ever had, and has vomited since talking to you. A quick physical exam shows a low blood pressure and tiny red petechiae (pinpoint hemorrhages) in the skin. Her neck is slightly stiff. Petechial rash seen in N. meningitidis meningitis  N. meningitidis: Laboratory Testing Specimen: – CSF transported to laboratory immediately at no less than room temperature – Blood (culture) Methods: – Culture – Molecular detection (such as PCR)  Carbohydrate utilization test Carbohydrate utilization test is used to validate the identification of N. meningitidis. Different carbohydrates: – glucose, maltose, lactose, and sucrose are added to tubes containing a cystine trypticase agar https://www.cdc.gov/meningitis/lab-manual/chpt07-id-characterization-nm.html  Acid production Organism Glucose Maltose Lactose Sucrose Neisseria + + – – meningitidis Neisseria + + + – lactamica Neisseria + – – – gonorrhoeae Neisseria + + – + sicca Moraxella – – – – catarrhalis https://www.cdc.gov/meningitis/lab-manual/chpt07-id-characterization-nm.html  Neisseria meningitidis: Epidemiology Most frequent cause of bacterial meningitis in infants, children, adolescents, and young adults in U.S.