Microbiology Flashcards
This Gram-negative organism grows well on
McConkey’s agar, is often green in color and
is oxidase positive.
What is Pseudomonas aeruginosa?
This common Gram-negative organism, many cases of which can be prevented by immunization, requires hemin and NAD for growth.
What is Haemophilus influenzae ?
Immunization against this organism has greatly reduced the incidence of meningitis in children.
Haemophilus species fail to grow on blood agar unless it is supplemented with X factor (hemin) and V factor (NAD).
Errors in Gram stain
- Gram-negative called gram-positive
- Gram-positive called gram-negative
- Gram-negative called gram-positive
Acinetobacter
- Gram-positive called gram-negative
Bacillus and Clostridium species typically are – 95% to 100% gram-positive early in their growth phase in broth cultures but become
– 40% to 50% gram-negative in the late growth phase and
– 90% to 95% gram-negative in the stationary phase.
Loss of Gram stain retention associated with aging of the culture most likely accounts for the frequency of misreading for these species.
Culture Media – Selective and Differential
Hectoin Enteric agar
- Selective for enteric bacteria
- Differential for Salmonella & Shigella
16s rRNA Gene Sequencing for Bacteria ID
16s rRNA Gene Sequencing
- highly conserved
- about 1,550 bp long variable and conserved regions.
- conserved regions at the beginning of the gene and at either the 540-bp region or at the end of the whole sequence (about the 1,550-bp region)
- variable region used for comparative taxonomy
MALDI-TOF for Bacteria ID
MALDI-TOF for Bacteria ID
- Matrix-Assisted Laser-Desorption/Ionization Time-of-Flight Mass Spectroscopy.
- Analyzes high-copy proteins in the bacterial cell; mostly ribosomal proteins.
- Sample is spotted onto plate, mixed with matrix, then fed into the instrument.
- Low reagent cost (pennies), extremely fast ID (minutes).
What is Mass Spectrometry and TOF?
Mass Spectrometry
- Charged molecules are accelerated by an electric field in vacuum.
- Requires generating gas-phase ionized material.
- The acceleration is proportional to the mass/charge ratio: m/z
- The charges may be deflected or flung about in various ways in different instruments.
- They’re detected by the charge passing through another electric field.
- Detect 10-15 to 10-18 molar concentrations.
- Time-of-flight (TOF) instrument
- The target is accelerated a defined amount, and flies linearly, without further acceleration, until it is detected.
This Gram-negative organism is best isolated on a selective agar containing cefsoludin, irgasan, and novobiocin?
Yersinia enterocolitica
- CIN agar (Yer-CIN-ia) is used to isolate this organism from the stool
CSF Major pathogens
- How do you detect pathogens?
- Young adults?
- Old adults?
- Babies?
- Fungal?
- Parasite?
CSF Major pathogens:
- Bacteria: cytospin and gram stain
- Young adults:
- N. meningitidis
- Listeria in pregnant women
- Old adults:
- S. pneumoniae
- Antigen in urine
- HSV
- S. pneumoniae
- Babies:
- Listeria
- group B Streptococci
- E. coli
- Fungal:
- Coccidiomycosis
- Cx
- Histoplasma
- Antigen in CSF, BAL, urine
- Cryptococcus (HIV+)
- Antigen in CSF and serum, follow titers
- Candida albicans
- Coccidiomycosis
- Parasite:
- Nagleria
- Look for motility (STAT)
- Nagleria
H. influenzae
in newborns
and newborns
Encephalitis and Brain abscesses
Encephalitis
- Viral usually; rarely rickettsial, syphilis, Lyme.
Brain abscesses
- Contiguous: from sinusitis or otitis media
- Upper respiratory flora
- Often polymicrobial
- Hematogenous, from bacterial endocarditis
- Usually monomicrobial, BE pathogens
Pleural and Peritoneal pathogens
Pleural fluid:
Pneumonia bugs
S. pneumoniae
S. aureus
S. pyogenes
Peritoneal fluid:
GI bugs
E. coli and other gram-negatives
mixed GI flora in surgical patients
GI and skin flora in spontaneous bacterial peritonitis in ascites/liver failure
Joint pathogens
Joints
- Staphylococcus aureus
- Neisseria gonorrhoeae
- Borrelia burgdorferi (Lyme disease)
- Culture / Gram stain for conventional bacteria
- Serology for Lyme
Blood pathogens
Major Blood pathogens:
- S. aureus
- S. coag neg.
- Enterobacteriaciae
- enterococci
- Pseudomonas
- S. pneumoniae
- viridans streptococci
- yeasts
Blood culture
Typical Media:
- blood culture bottles (aerobic ± anaerobic)
- Incubated in automatic reader which detects CO2 production (BactAlert, Bactek) or pressure change (Trek)
- Usually incubate 5 days, occasionally fewer, more for special pathogens ( Brucella , Legionella )
Blood Culture Contamination
Blood Culture Contamination
- Skin flora
- Coagulase-negative staph
- Corynebacterium spp.
- Rarely viridans streps
- A cost issue - Several thousand dollars
- QA guidelines suggest getting rate to 2-3%
- Chlorhexidine or iodine skin preps
- Dedicated IV / blood culture teams
Bloodstream Infections – Bacterial Endocarditis
Bloodstream Infections – Bacterial Endocarditis
Native Valve Endocarditis
- Streptococci, 60-80%
- Viridans streps
- Beta-streps
- Enterococci
- Staphylococci, 20-35%
- S. aureus
- Gram-negatives, 1-5%
- Fungi, others
Prosthetic valve endocarditis
- Coag negative staph
Risk factors – existing valve disease, IVD
Bloodstream Infections – Lines
Bloodstream Infections – Lines
- Coag-negative staph
- Enterococcus
- S. aureus
- Enterobacteriaciae
- Pseudomonas
- Acinetobacter
Maki roll-plate method
- Catheter tip rolled on BAP
- >15 colonies of a single isolate considered significant
- Should be paired with peripheral culture
UTI in Community
- List in order of frequency
- Pathogens and susceptibilities
- Gram + chains?
- Gram + clusters?
UTI in Community
- 95% E.coli
- No resistance
- Klebsiella
- Resistant to ampicillin
- Give ciprofloxacin or nitrofurantoin
- Proteus
- Resistant to nitrofurantoin and tetracyclin
- Give fosfomycin (one dose)
- Group B strep
- Gram + in chains
- Give PCN
- If PCN allery give clyndamycin and erythromycin
- Staph saprophyticus
- Gram + in clusters
- Give cefalosporins
Interpretation of Urine Cultures
- Quantity
- Purity
Interpretation of Urine Cultures
- Quantity important;
- >10^5 significant, >10^4 possibly significant
- 10^2/ml in sterile collection technique
- 10^2/ml in pregnancy
- Purity is important:
- 1 isolate, likely
- 2 isolates, perhaps
- ≥3 isolates, contaminated
- Special populations: spinal cord injury, neurogenic bladder
Pneumonia in Community
- Ambulatory
- Older patients
Pneumonia in Community
Ambulatory
- Mycoplasma
- Dx serologically (IgM) or by molecular
- Give erythromycin or tetracyclin
- Legionella
- Urine antigen only detects legionella pneumophila type 1
- If negative Cx or DFA bronchoscopy specimen
- Haemophilus influenzae
- Give rocephin or ceftriaxone
Older patients
- Strep pneumoniae
- Urine antigen
- Cx resp specimen
- MRSA
Pneumonia in Hospital
Pneumonia in Hospital
- Moraxella
- aspiration
- pure Cx
- Give non-Betalactams
- Klebsiella and Pseudomonas
- Gram -
- Water
Bronchitis
Bronchitis
- Culture results don’t correlate with symptoms
- H. influenzae
- S. pneumoniae
- Moraxella catarrhalis
- Viruses may be important in exacerbations
- influenza
- parainfluenza
- RSV
- human metapneumovirus
Lung abscess
Lung abscess
- Destruction of lung parenchyma to produce cavities with air-fluid levels
- Usually polymicrobic with anaerobes and upper respiratory tract flora:
- Peptostreptococcus
- Prevotella
- Bacteroides (nonfragilis )
- Fusobacterium
- occasionally
- Clostridium, Actinomyces, alpha-hemolytic Streptococcus, Enterobacteriaceae.
Grading Sputum
Grading Sputum
- Squamous Screen
- Acceptable: <10 SEC / 10x field
- Unacceptable: >10 SEC / 10x field
- Murray and Washington
- Culture only group 5 ( >25 WBC and <10 SEC)
- And culture if >10 WBC and <10 SEC
This oxidase-positive Gram-negative rod associated with bite wounds and endocarditis does not grow on MacConkey agar, and its name reflects the fact that it often eats its way into the agar.
Eikenella corrodens
- pits the agar when it grows
- oxidase-positive
- Gram-negative rod
- bite wounds and endocarditis
- does not grow on MacConkey agar
Animal bites (including human)
Animal bites (including human)
- Oral flora; mixed aerobic/anaerobic flora
- Pasturella multocida – cats and dogs
- Eikenella corrodens
- Capnocytophaga canimorsus – dogs
Diabetic foot ulcers
Cellulitis
Toxic Shock Syndromes
Diabetic foot ulcers
- Mixed flora, Gram negatives, anaerobes, streps and staphs
Cellulitis
- Staph and Strep
Toxic Shock Syndromes
- toxigenic strains of S. aureus and S. pyogenes
- Skin and soft tissue infections
- vaginal/tampon use for S. aureus
- Desquamating skin rash, shock out of proportion to the infected area
Throat pathogens
Throat pathogens
- Group A strep
- other beta-hemolytic strep
- N. gonorrhoeae
- C. diphtheriae
Typical Media: blood agar, selective media for specific pathogens by request
- Tinsdale agar for C. diphtheriae
This specimen is used to isolate Streptococcous agalactiae (group B strep) during prenatal screening. It is inoculated into broth enrichment followed by subculture. Be specific about the specimen source.
Streptococcous agalactiae (group B strep)
- The most sensitive specimen is rectovaginal; various culture schemes are most commonly used, but amplification methods are also employed.
Stool pathogens
Stool
- Major pathogens:
- Salmonella
- Shigella
- Campylobacter
- E. coli O157 & others
- Yersinia enterocolitica
- Typical Media: MacConkey’s, Hectoin enteric or XLD, Campy, CIN, sorbitol-MacConkey’s
- Antigen tests for Shiga-like toxins or for O157 antigen
- Campylobacter by antigen
- Special media and procedures for Vibrios
Hemolytic-Uremic Syndrome
Hemolytic-Uremic Syndrome
- Primarily in children (peak 5-10 y/o)
- 5-10d after diarrhea
- Renal failure + microangiopathic hemolysis
- Other end-organs affected include CNS
- Associated with shiga-like-toxin producing enteric bacteria
- E. coli strains: O:157 H:7 and others
- Shigella species
- No effective therapy, antibiotics NOT recommended
Clostridium difficile – disease and diagnosis
Clostridium difficile – disease and diagnosis
- Toxin-mediated illness
- Culture for epidemiologic purposes only
- Toxins A and B
- Antigen tests: rapid or conventional EIA
- Kits that detect both A and B are more sensitive
- GDH antigen is more sensitive than toxin, but is unspecific
- PCR for toxin gene
- Cytopathic effect in cell culture
- Labor-intensive, takes a day or two, more sensitive than toxin EIA.
- Toxigenic culture is ‘gold standard’
- Culture in selective broth, assay for toxin in supernatant.
This test is used to distinguish a catalase positive gram-positive coccus, whose colonies are often golden-yellow in color, from other members of the same genus.
Coagulase
- Catalase distinguishes between staph (catalase-positive) and strep (catalasenegative)
- Used to separate S. aureus from coagulase negative staphylococci.
Bacterial Vaginosis
Bacterial Vaginosis
- Major Etiology
- Overgrowth of mixed anaerobes (e.g. Mobiluncus, Gardnerella )
- Loss of Lactobacillus morphotypes
- Laboratory Diagnosis
- Wet mount for clue cells
- Gram stain for clue cells
- Elevated pH >=4.5
Important Bacterial Diseases Not diagnosed by conventional culture
Important Bacterial Diseases Not diagnosed by conventional culture
- Spirochetal diseases
- Syphilis
- Lyme
- Diseases caused by rickettsia and rickettsia-like organisms
- Rocky Mountain Spotted Fever
- Ehrlichiosis
Syphilis
Syphilis
- Primary lesion
- mucosal surface
- Secondary lesions
- mucocutaneous lesions
- Late manifestations
- neurologic, cardiac
Diagnosis
- Non-treponemal tests
- RPR; VDRL
- For screening and following therapy
- Decline after effective treatment
- Specific treponemal tests
- FTA-abs; MHA; EIA
- Confirmatory; positive for life
- Reverse algorithms using automated treponemal test as primary, non-treponemal test as confirmation.
Lyme Disease
Lyme Disease
- tick-transmitted disease caused by B. burgdorferi
- Peak ages bimodal: children 5-9 and adults 20-54
- Peak months: May-Oct
- Europe: Ixodes ricinus vector
- Different Borrelia strains
- Different presentation; more neurologic disease, chronic skin lesions
- Early disease: cutaneous, musculoskeletal, menigoencephalitis, cranial nerve palsies, heart block
- Late disease: chronic arthritis, subtle CNS effects
Lyme Disease Diagnosis
Lyme Disease Diagnosis
- Early disease: clinical diagnosis by rash; requires up to 4-6 weeks for IgM response to appear
- Lyme serology and Western blot
- DNA probes and PCR investigational
- CSF titers in late CNS disease
This large aerobic spore-forming Gram-positive rod is non-motile and nonhemolytic.
Bacillus anthracis
- large Gram-positive rod
- aerobic spore-forming group is genus Bacillus
- lack of hemolysis and motility used to screen for isolates suspicious for B. anthracis
Rocky Mountain Spotted Fever : Epidemiology
Rocky Mountain Spotted Fever : Epidemiology
- Dermacenter ticks
- US: SE, Midwest, northern Rockies
- Tick is vector and main reservoir
- Animal reservoirs include dogs and wild animals
Rocky Mountain Spotted Fever : Clinical
Rocky Mountain Spotted Fever : Clinical
- Incubation 2-14 days, median 7
- Fever, myalgia, headache
- GI symptoms
- Rash
- 90% of patients have the rash
- Can occur up to 5d after fever
- Extremities first, then spreads centrally (“centripetal”)
Rocky Mountain Spotted Fever : Diagnosis
Rocky Mountain Spotted Fever : Diagnosis
- Characteristic rash (only present in ~90%)
- Clinical presentation and risk factors
- Laboratory
- Serology only turns positive during recovery
- Immunohistology of skin biopsy
- PCR on skin biopsies possible
Ehrlichia & Anaplasma Types
Ehrlichia & Anaplasma Types
- Ehrlichia chaffeensis
- Human monocytic ehrlichiosis (HME)
- Anaplasma (formerly Ehrlichia) phagocytophila
- Human granulocytic ehrlichiosis (HGE)
- Ehrlichia ewingii and other species
- Emerging diseases
- Tick-borne illnesses, first recognized in humans in the 1980s
Ehrlichia chaffeensis :
Human monocytic ehrlichiosis (HME)
Ehrlichia chaffeensis :
Human monocytic ehrlichiosis (HME)
- Lone Star tick, Amblyomma americanum
- US: South central, SE states
- Diagnosis
- Laboratory
- leukopenia, thrombocytopenia, elevated LFT
- Morulae seen in <10%
- Serology
- PCR
- Laboratory
Anaplasma (formerly Ehrlichia) phagocytophila : Human granulocytic ehrlichiosis (HGE)
Anaplasma (formerly Ehrlichia) phagocytophila : Human granulocytic ehrlichiosis (HGE)
- Spread by Ixodes ticks (like Lyme)
- Northeast and upper Midwest (reported Europe)
- Peak incidence in July and November
- Most infections probably asymptomatic
- Cytopenias
- leukopenia, anemia, thrombocytopenia
- may be associated with clinically significant bleeding or immunosupression
- Morulae seen in 20-80%
- Serology
- PCR
Most members of this catalase-negative genus of Gram-positive cocci hydrolyze esculin and PYR.
Enterococcus
- catalase-negative
- Gram-positive cocci
- hydrolyze esculin and PYR
Bacteria With Bioweapons Potential
Bacteria With Bioweapons Potential
- Bacillus anthracis (anthrax)
- Francisella tularensis (tularemia)
- Yersinia pestis (plague)
- Others
- Brucella
- Coxiella
- What is it?
- Aerobic/Anaerobic?
- Sporeforming?
- Sites of disease?
- Case-fatality rate?
Bacillus anthracis
- Aerobic sporeforming gram-positive rod
- Causes cutaneous, GI, or inhalational disease
- Highly pathogenic, 60% case-fatality rate in inhalation disease in the 2001 outbreak
What is it?
Shape?
Gram positive/negative?
How is it spread?
Where is it found?
Francisella tularensis
- A fastidious, poorlystaining Gram negative rod.
- A zoonotic organism, spread to humans from small mammals by direct contact or by insects
- Endemic in US: West, Midwest, Southeast
What is it?
Where is it?
How is it spread?
Types of infection?
Yersinia pestis
- A gram-negative rod
- Member of the Enterobacteriaceae
- Zoonosis in the US West
- Oriental rat flea (Xenopsylla cheopis)
- Bubonic
Septicemic
Pneumonic
Anaerobic Gram-positive Rods
Anaerobic Gram-positive Rods
Large, spore-forming
- Clostridium
- C. perfringens: associated with severe soft-tissue infections and in vivo hemolysis; double-zone hemolysis
Nonsporeformers
- Actinomyces (filamentous, often branching, sulfur granules)
- Eubacterium
- Bifidobacterium
- Proprionibacterium
PNA: Host factor/Agents suggested
Syndrome/Causative Agent
Syndrome/Causative Agent
Syndrome/Causative Agent
Syndrome/Causative Agent
Syndrome/Causative Agent
Syndrome/Causative Agent
Syndrome/Causative Agent
Syndrome/Causative Agent
Syndrome/Causative Agent
Syndrome/Causative Agent
Organism/Vector
Organism/Vector
Organism/Vector
Organism/Vector
Organism/Vector
Human Herpes Virus - Latency, Clinical
Viral classification
Viral Cell Culture
Viral Cell Culture
EBV Serology
EBV Serology
