MLAB 2434: Microbiology Keri Brophy-Martinez

1
MLAB 2434 MicrobiologyKeri Brophy-Martinez

• The Gram-Negative Cocci

2
Families

• Neisseriaceae
• Neisseria
• Kingella
• Eikenella
• Simonsiella
• Alysiella

• Moraxellaceae
• Moraxella
• Acinetobacter

3
General CharacteristicsNeisseria Species

• Aerobic, gram-negative diplococci
• Nonmotile
• Oxidase positive
• Catalase positive
• Fastidious, capnophilic

4
Neisseria Species and Moraxella catarrhalis

• Habitat
• Upper respiratory tract
• Genitourinary tract
• Alimentary(Digestive) tract
• Primary pathogens
• N. gonorrhoeae
• N. meningitidis

5
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

6
Clinical Conditions Neisseria gonorrhoeae

• 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

7
Clinical Infections Neisseria gonorrhoeae

• 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

8
Clinical Conditions Neisseria gonorrhoeae

• 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

9
Clinical Conditions Neisseria gonorrhoeae

• 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

10
Laboratory DiagnosisNeisseria gonorrhoeae

• Clinical specimens
• Genital sites
• Female endocervix
• Male urethra
• Anal
• Oral/pharyngeal
• Eye
• Blood/joint fluids

11
Specimen Collection

• Dacron/ Rayon swabs preferred
• Swabs transported in Amies medium with charcoal
• Inoculate media within 6 hours of collection,
  avoid drying

12
Transport Media

• Transgrow or JEMBEC
• JEMBEC James E Martin Biological Environmental
  Chamber

JEMBEC
13
Laboratory Diagnosis Neisseria gonorrhoeae

• Morphology
• Gram-negative, kidney-beanshaped diplococci

14
Laboratory Diagnosis Neisseria gonorrhoeae
15
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

16
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

17
Laboratory DiagnosisNeisseria gonorrhoeae

• 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

18
Laboratory Diagnosis Neisseria gonorrhoeae

• Oxidase Test
• Test on filter paper or directly on plate
• Oxidase reagent Dimethyl or tetramethyl oxidase
  reagent
• Violet-purple color indicates a positive result

19
Laboratory Diagnosis Neisseria gonorrhoeae

• 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

20
Laboratory Diagnosis Neisseria gonorrhoeae

• 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

21
Antimicrobial Resistance Neisseria gonorrhoeae

• 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

22
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

23
Virulence FactorsNeisseria meningitidis

• Pili
• Polysaccharide capsule
• Cellular membrane proteins
• Lipooligosaccharide/endotoxin

24
Clinical Infections Neisseria meningitidis

• 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

25
Clinical Infections Neisseria meningitidis
Hemorrhage in the adrenal glands in
Waterhouse-Fridericksen syndrome
26
Laboratory DiagnosisNeisseria meningitidis

• 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
27
Laboratory Diagnosis Neisseria meningitidis

• 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)
28
Laboratory Diagnosis Neisseria meningitidis

• Oxidase-test positive
• Conventional CTA carbohydrates for biochemical
  identification (glucose and maltose)
• Immunologic methods

29
Antibiotic Therapy Neisseria meningitidis

• Penicillin
• Other options rifampin or sulfonamide
• Vaccine
• For use with people aged 11-55
• Does not protect against all serotypes

30
Nonpathogenic Neisseria species

• Normal flora of upper respiratory tract
• Some members
• Neisseria cinera
• Neisseria lactamica
• Neisseria mucosa
• Neisseria sicca
• Neisseria subflava

31
Moraxella catarrhalis

• 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)

32
Virulence factorsMoraxella catarrhalis

• Endotoxin
• Pili
• Beta-lactamase

33
Laboratory DiagnosisMoraxella catarrhalis
Direct smear from an otitis media sample showing
intracellular gram-negative diplococci
34
Laboratory DiagnosisMoraxella catarrhalis

• 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
35
Laboratory Diagnosis Moraxella catarrhalis

• Oxidase positive
• Catarrhalis Disc
• Positive blue-gren
• All CTA sugars negative
• Produce beta- lactamase

36
Identification of Selected Neisseria Species
Moraxella
37
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-col
  lection-swab-amies-medium-w-charcoal-wwcsam-3450
• https//new.fishersci.com/ecomm/servlet/fsproductd
  etail_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.