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MLAB 2434: Microbiology Keri Brophy-Martinez. The Gram-Negative Cocci. Families. Neisseriaceae Neisseria Kingella Eikenella Simonsiella Alysiella. Moraxellaceae Moraxella Acinetobacter. General Characteristics: Neisseria Species. Aerobic, gram-negative diplococci Nonmotile

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families
Families
  • Neisseriaceae
    • Neisseria
    • Kingella
    • Eikenella
    • Simonsiella
    • Alysiella
  • Moraxellaceae
    • Moraxella
    • Acinetobacter
general characteristics neisseria species
General Characteristics:NeisseriaSpecies
  • Aerobic, gram-negative diplococci
  • Nonmotile
  • Oxidase positive
  • Catalase positive
  • Fastidious, capnophilic
neisseria species and moraxella catarrhalis
Neisseria Species and Moraxellacatarrhalis
  • Habitat
    • Upper respiratory tract
    • Genitourinary tract
    • Alimentary(Digestive) tract
  • Primary pathogens:
    • N. gonorrhoeae
    • N. meningitidis
virulence factors
Virulence Factors
  • Fimbrae (common pili)-
    • enhance the ability of bacterial cells to adhere to host cells and to each other
  • Lipooligosaccharide:
    • endotoxin involved in damage to host tissue
  • Capsule
  • Cell membrane proteins
  • IgA protease-
    • cleaves IgA on mucosal surfaces
clinical conditions neisseria gonorrhoeae
Clinical Conditions:Neisseriagonorrhoeae
  • Pyogenic (pus-producing) infection of columnar and transitional epithelial cells
    • urethral, endocervix, anal canal, pharynx, and conjunctiva
    • Incubation period: 2 to 7 days
    • Transmitted by sexual contact
clinical infections neisseria gonorrhoeae
Clinical Infections:Neisseriagonorrhoeae
  • Disease in the male
    • 95% show symptoms of acute infection
    • Symptoms include dysuria, urethral discharge
    • Complications include epididymitis and urethral stricture, and prostatitis
  • Disease in the female
    • 20% to 80% are asymptomatic
    • Symptoms include:
      • Burning or frequency of urination, vaginal discharge, fever and abdominal pain
    • Complications include pelvic inflammatory disease (PID), sterility and ectopic pregnancy
clinical conditions neisseria gonorrhoeae1
Clinical Conditions:Neisseriagonorrhoeae:
  • Disseminated gonococcal disease
    • Acute form has the following symptoms: fever, chills, malaise, intermittent bacteremia, and skin lesions
    • If untreated will progress to septic joint form of the disease (inflamed joints, swollen, hot, full of pus and fluid)
    • Gonococcal arthritis occurs as a result of disseminated gonococcal bacteremia
clinical conditions neisseria gonorrhoeae2
Clinical Conditions:Neisseriagonorrhoeae:
  • Disease in children
    • In infancy, an eye infection (ophthalmia neonatorum) may occur during vaginal delivery
    • May cause blindness if not treated
    • Infection is preventable with the application of antibiotic eye drops at birth
  • Extragenital infections
    • Pharyngitis
    • Anorectal infections
laboratory diagnosis neisseria gonorrhoeae
Laboratory Diagnosis:Neisseriagonorrhoeae
  • Clinical specimens
    • Genital sites
      • Female: endocervix
      • Male: urethra
    • Anal
    • Oral/pharyngeal
    • Eye
    • Blood/joint fluids
specimen collection
Specimen Collection
  • Dacron/ Rayon swabs preferred
  • Swabs transported in Amies medium with charcoal
  • Inoculate media within 6 hours of collection, avoid drying
transport media
Transport Media

Transgrow or JEMBEC

JEMBEC= James E Martin Biological Environmental Chamber

JEMBEC

laboratory diagnosis neisseria gonorrhoeae1
Laboratory Diagnosis: Neisseriagonorrhoeae
  • Morphology
    • Gram-negative, kidney-bean–shapeddiplococci
media selection
Media Selection
  • Chocolate agar
    • Subject to overgrowth of normal flora
  • Thayer-Martin agar is chocolate agar with vancomycin, colistin, and nystatin
  • MTM contains the above plus trimethoprin
  • Specimen MUST be plated on warmed media ASAP
incubation
Incubation
  • Inoculated culture media must be incubated at 350 C in 3% to 5% CO2 or candle jar
  • Candle jar must use white wax candles
laboratory diagnosis neisseria gonorrhoeae3
Laboratory Diagnosis:Neisseriagonorrhoeae
  • Colony morphology on modified Thayer-Martin (MTM) agar
    • Small, beige- gray
    • Translucent, smooth
  • Fresh growth must be used for testing, because N. gonorrhoeae produces autolytic enzymes
laboratory diagnosis neisseria gonorrhoeae4
Laboratory Diagnosis: Neisseriagonorrhoeae
  • Oxidase Test
    • Test on filter paper or directly on plate
    • Oxidase reagent =Dimethyl or tetramethyl oxidase reagent
    • Violet-purple color indicates a positive result
laboratory diagnosis neisseria gonorrhoeae5
Laboratory Diagnosis: Neisseriagonorrhoeae
  • Carbohydrate utilization
  • Cystine trypticase agar (CTA)
    • Contain 1% of a single carbohydrate
      • Glucose, maltose, lactose, sucrose
    • Phenol red is pH indicator
      • Read in 24-72 hours
laboratory diagnosis neisseria gonorrhoeae6
Laboratory Diagnosis: Neisseriagonorrhoeae
  • Immunologic methods
    • Use colonies from primary plate
    • Organisms do not need to be viable
      • Fluorescent antibody technique
      • Coagglutination
  • Non-culture methods
    • Use direct patient specimen
    • ELISA, nucleic acid probe, and PCR testing
    • Expensive; usually used in high-risk populations with large volume of testing
    • Unable to perform on all sources
antimicrobial resistance neisseria gonorrhoeae
Antimicrobial Resistance: Neisseriagonorrhoeae
  • PPNG = Penicillinase Producing Neisseria gonorrhoeae
    • First seen in 1976
    • Plasmid-mediated
    • Beta-lactamase testing should always be done on N. gonorrheoae
  • Treatment = Penicillin
  • Tetracycline if beta-lactamase positive strain. Can also use cephalosporins and flouroquinolones
neisseria meningitidis
Neisseria meningitidis

Commensal of carriers in the nasopharynx

Cross the epithelium and enter the circulatory system

Primarily affects the immunocompromised, young children, trauma victims

Leads to septicemia and localization to the meninges causing inflammation of the brain

Meningitis

Highly fatal (25% even if treated)

Encapsulated strains A, B, C, Y, W-135

virulence factors neisseria meningitidis
Virulence Factors:Neisseriameningitidis
  • Pili
  • Polysaccharide capsule
  • Cellular membrane proteins
  • Lipooligosaccharide/endotoxin
clinical infections neisseria meningitidis
Clinical Infections:Neisseriameningitidis:
  • Bacterial meningitis
    • Transmission is by respiratory droplets and requires both close contact (ex: dormitories, military barracks, in institutions) and lack of specific antibody (susceptibility)
    • Symptoms include fever, headache, stiff neck, nausea, vomiting, and purulent meningitis with increased WBCs
    • Serotypes B and C most common in US
  • Other infections include meningococcemia, pneumonia, purulent arthritis, & endophthalmitis
  • May be seen in genital tract with oral-genital contact
clinical infections neisseria meningitidis1
Clinical Infections:Neisseriameningitidis

Hemorrhage in the adrenal glands in Waterhouse-Fridericksen syndrome

laboratory diagnosis neisseria meningitidis
Laboratory Diagnosis:Neisseriameningitidis
  • Identification
    • Examine direct smear from CSF for intra & extra cellular g- dc
    • Examine smear for halo
    • Other body sites include nasopharyngeal swabs, sputum, and urogenital specimens

Gram-stained smear of CSF showing the extra cellular and intracellular gram-negative diplococci

laboratory diagnosis neisseria meningitidis1
Laboratory Diagnosis: Neisseriameningitidis
  • Examine cultures on blood agar & chocolate agar after incubation in increased CO2
  • Colony Morphology
    • Small
    • Tan-grey color
    • Smooth

Neisseria meningitidis growing on sheep blood agar (right) and chocolate agar (left)

laboratory diagnosis neisseria meningitidis2
Laboratory Diagnosis: Neisseriameningitidis
  • Oxidase-test positive
  • Conventional CTA carbohydrates for biochemical identification (glucose+ and maltose+)
  • Immunologic methods
antibiotic therapy neisseria meningitidis
Antibiotic Therapy:Neisseriameningitidis
  • Penicillin
  • Other options: rifampin or sulfonamide
  • Vaccine
    • For use with people aged 11-55
    • Does not protect against all serotypes
nonpathogenic neisseria species
Nonpathogenic Neisseriaspecies
  • Normal flora of upper respiratory tract
  • Some members
    • Neisseria cinera
    • Neisseria lactamica
    • Neisseria mucosa
    • Neisseria sicca
    • Neisseria subflava
moraxella catarrhalis
Moraxellacatarrhalis
  • Previously known as Branhamella catarrhalis
  • Normal commensal of the respiratory tract
  • Has become an important opportunistic pathogen
    • Predisposing factors
      • Advanced age, Immunodeficiency, Neutropenia, Other debilitating diseases
  • Clinical infections
    • Pneumonia
    • Sinusitis
    • Otitis media (3rd most common cause)
virulence factors moraxella catarrhalis
Virulence factors:Moraxellacatarrhalis
  • Endotoxin
  • Pili
  • Beta-lactamase
laboratory diagnosis moraxella catarrhalis
Laboratory Diagnosis:Moraxellacatarrhalis

Direct smear from an otitis media sample showing intracellular gram-negative diplococci

laboratory diagnosis moraxella catarrhalis1
Laboratory Diagnosis:Moraxellacatarrhalis
  • Colonies appear smooth with a grayish- white color
  • When colonies pushed with loop, they “scoot” across media

Moraxella catarrhalis growing on chocolate agar after 24 hours of incubation

laboratory diagnosis moraxella catarrhalis2
Laboratory Diagnosis :Moraxellacatarrhalis
  • Oxidase positive
  • Catarrhalis Disc
    • Positive= blue-gren
  • All CTA sugars negative
  • Produce beta- lactamase
references
References
  • Engelkirk, P., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of Infectious Diseases: Essentials of Diagnostic Microbiology . Baltimore, MD: Lippincott Williams and Wilkins.
  • http://www.awinhospitalproducts.com/product/35-collection-swab-amies-medium-w-charcoal-wwcsam-3450
  • https://new.fishersci.com/ecomm/servlet/fsproductdetail_10652_606366_29104_-1_0
  • https://picasaweb.google.com/pia8628/0411microlab#5596104725803822690/
  • Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.
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