mlab 2434: microbiology keri brophy-martinez
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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 - PowerPoint PPT PresentationTRANSCRIPT
MLAB 2434: MLAB 2434: MicrobiologyMicrobiologyKeri Brophy-MartinezKeri Brophy-Martinez
The Gram-Negative Cocci
FamiliesFamiliesNeisseriaceae
◦ Neisseria◦ Kingella◦ Eikenella◦ Simonsiella◦ Alysiella
Moraxellaceae◦ Moraxella◦ Acinetobacter
General Characteristics:General Characteristics:NeisseriaNeisseria Species Species
Aerobic, gram-negative diplococci
NonmotileOxidase positiveCatalase positiveFastidious, capnophilic
NeisseriaNeisseria Species and Species and Moraxella Moraxella catarrhaliscatarrhalis
Habitat◦Upper respiratory tract◦Genitourinary tract◦Alimentary(Digestive) tract
Primary pathogens:◦N. gonorrhoeae◦N. meningitidis
Virulence FactorsVirulence 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:Clinical Conditions: Neisseria gonorrhoeae 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
Clinical Infections:Clinical Infections: Neisseria gonorrhoeae 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
Clinical ConditionsClinical Conditions:: Neisseria gonorrhoeae 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
Clinical ConditionsClinical Conditions:: Neisseria gonorrhoeae 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
Laboratory Diagnosis:Laboratory Diagnosis:Neisseria gonorrhoeaeNeisseria gonorrhoeae
Clinical specimens◦Genital sites Female: endocervix Male: urethra
◦Anal◦Oral/pharyngeal◦Eye◦Blood/joint fluids
Specimen CollectionSpecimen Collection
◦Dacron/ Rayon swabs preferred◦Swabs transported in Amies medium with
charcoal◦Inoculate media within 6 hours of collection,
avoid drying
Transport MediaTransport Media
Transgrow or JEMBECJEMBEC= James E Martin Biological Environmental Chamber
JEMBEC
Laboratory Diagnosis: Laboratory Diagnosis: Neisseria gonorrhoeaeNeisseria gonorrhoeae
Morphology
◦Gram-negative, kidney-bean–shaped diplococci
Laboratory Diagnosis: Laboratory Diagnosis: Neisseria gonorrhoeaeNeisseria gonorrhoeae
Media SelectionMedia 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
IncubationIncubation
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:Laboratory Diagnosis:Neisseria gonorrhoeaeNeisseria 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
Laboratory Diagnosis: Laboratory Diagnosis: Neisseria gonorrhoeaeNeisseria 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
Laboratory Diagnosis: Laboratory Diagnosis: Neisseria gonorrhoeaeNeisseria 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
Laboratory Diagnosis: Laboratory Diagnosis: Neisseria gonorrhoeaeNeisseria 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
Antimicrobial Resistance: Antimicrobial Resistance: Neisseria gonorrhoeaeNeisseria 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
Neisseria meningitidisCommensal of carriers in the nasopharynxCross 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
MeningitisHighly fatal (25% even if treated)
◦ Encapsulated strains A, B, C, Y, W-135
Virulence Factors:Virulence Factors:Neisseria meningitidisNeisseria meningitidis
PiliPolysaccharide capsuleCellular membrane proteinsLipooligosaccharide/endotoxin
Clinical InfectionsClinical Infections:: Neisseria meningitidis 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
Clinical Infections:Clinical Infections: Neisseria meningitidis Neisseria meningitidis
Hemorrhage in the adrenal glands in Waterhouse-Fridericksen syndrome
Laboratory Diagnosis:Laboratory Diagnosis:Neisseria meningitidisNeisseria 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
Neisseria meningitidis growing on sheep blood agar (right) and chocolate agar (left)
Laboratory Diagnosis: Laboratory Diagnosis: Neisseria meningitidisNeisseria meningitidis
Examine cultures on blood agar & chocolate agar after incubation in increased CO2
Colony Morphology
◦Small
◦Tan-grey color
◦Smooth
Laboratory Diagnosis: Laboratory Diagnosis: Neisseria meningitidisNeisseria meningitidis
◦ Oxidase-test positive
◦ Conventional CTA carbohydrates for biochemical identification (glucose+ and maltose+)
◦ Immunologic methods
Antibiotic Therapy:Antibiotic Therapy: Neisseria meningitidisNeisseria meningitidis
PenicillinOther options: rifampin or
sulfonamide
Vaccine◦For use with people aged 11-55◦Does not protect against all serotypes
Nonpathogenic Nonpathogenic Neisseria speciesNeisseria species
Normal flora of upper respiratory tract
Some members◦Neisseria cinera◦Neisseria lactamica◦Neisseria mucosa◦Neisseria sicca◦Neisseria subflava
Moraxella catarrhalisMoraxella catarrhalis
Previously known as Branhamella catarrhalis
Normal commensal of the respiratory tractHas 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:Virulence factors:Moraxella catarrhalisMoraxella catarrhalis
EndotoxinPiliBeta-lactamase
Laboratory Diagnosis:Laboratory Diagnosis:Moraxella catarrhalisMoraxella catarrhalis
Direct smear from an otitis media sample showing intracellular gram-negative diplococci
Laboratory Diagnosis:Laboratory Diagnosis:Moraxella catarrhalisMoraxella 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
Laboratory Diagnosis :Laboratory Diagnosis :Moraxella catarrhalisMoraxella catarrhalis
Oxidase positiveCatarrhalis Disc
◦ Positive= blue-grenAll CTA sugars negative
Produce beta- lactamase
Identification of Identification of Selected Selected NeisseriaNeisseria Species & Species & MoraxellaMoraxella
Species GrowthBAP R.T T/M
Acid productionGluc Mal Lac Suc
N. gonorrhoeae
N. meningitidis
N. lactamica
N. sicca
M. catarrhalis
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ReferencesReferences
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.