MLAB 2434: Microbiology Keri Brophy-Martinez
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Transcript of MLAB 2434: Microbiology Keri Brophy-Martinez
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MLAB 2434: MLAB 2434: MicrobiologyMicrobiologyKeri Brophy-MartinezKeri Brophy-Martinez
The Gram-Negative Cocci
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FamiliesFamiliesNeisseriaceae
◦ Neisseria◦ Kingella◦ Eikenella◦ Simonsiella◦ Alysiella
Moraxellaceae◦ Moraxella◦ Acinetobacter
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General Characteristics:General Characteristics:NeisseriaNeisseria Species Species
Aerobic, gram-negative diplococci
NonmotileOxidase positiveCatalase positiveFastidious, capnophilic
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NeisseriaNeisseria Species and Species and Moraxella Moraxella catarrhaliscatarrhalis
Habitat◦Upper respiratory tract◦Genitourinary tract◦Alimentary(Digestive) tract
Primary pathogens:◦N. gonorrhoeae◦N. meningitidis
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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
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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
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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
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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
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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
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Laboratory Diagnosis:Laboratory Diagnosis:Neisseria gonorrhoeaeNeisseria gonorrhoeae
Clinical specimens◦Genital sites Female: endocervix Male: urethra
◦Anal◦Oral/pharyngeal◦Eye◦Blood/joint fluids
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Specimen CollectionSpecimen Collection
◦Dacron/ Rayon swabs preferred◦Swabs transported in Amies medium with
charcoal◦Inoculate media within 6 hours of collection,
avoid drying
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Transport MediaTransport Media
Transgrow or JEMBECJEMBEC= James E Martin Biological Environmental Chamber
JEMBEC
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Laboratory Diagnosis: Laboratory Diagnosis: Neisseria gonorrhoeaeNeisseria gonorrhoeae
Morphology
◦Gram-negative, kidney-bean–shaped diplococci
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Laboratory Diagnosis: Laboratory Diagnosis: Neisseria gonorrhoeaeNeisseria gonorrhoeae
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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
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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
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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
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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
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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
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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
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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
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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
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Virulence Factors:Virulence Factors:Neisseria meningitidisNeisseria meningitidis
PiliPolysaccharide capsuleCellular membrane proteinsLipooligosaccharide/endotoxin
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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
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Clinical Infections:Clinical Infections: Neisseria meningitidis Neisseria meningitidis
Hemorrhage in the adrenal glands in Waterhouse-Fridericksen syndrome
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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
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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
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Laboratory Diagnosis: Laboratory Diagnosis: Neisseria meningitidisNeisseria meningitidis
◦ Oxidase-test positive
◦ Conventional CTA carbohydrates for biochemical identification (glucose+ and maltose+)
◦ Immunologic methods
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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
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Nonpathogenic Nonpathogenic Neisseria speciesNeisseria species
Normal flora of upper respiratory tract
Some members◦Neisseria cinera◦Neisseria lactamica◦Neisseria mucosa◦Neisseria sicca◦Neisseria subflava
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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)
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Virulence factors:Virulence factors:Moraxella catarrhalisMoraxella catarrhalis
EndotoxinPiliBeta-lactamase
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Laboratory Diagnosis:Laboratory Diagnosis:Moraxella catarrhalisMoraxella catarrhalis
Direct smear from an otitis media sample showing intracellular gram-negative diplococci
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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
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Laboratory Diagnosis :Laboratory Diagnosis :Moraxella catarrhalisMoraxella catarrhalis
Oxidase positiveCatarrhalis Disc
◦ Positive= blue-grenAll CTA sugars negative
Produce beta- lactamase
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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.