• samples processed > 2 hrs from time of collection

  • can interefere with the viability of potential pathogens and allow for overgrowth of bacterial contaminants from the upper respiratory tract

• Gram positive cocci in pairs or chains

• Hemolysis- alpha (greenish zone- partial hemolysis)

• Catalse- negative

• Optochin disc- sensitive

• Bile solubility- positive (organism's growth is inhibited by bile salts)

Staphylococcus aureus

• Gram positive cocci in clusters (grape like)

• Growth on BAP and MAC (golden colony)

• Hemolysis- beta (clear zone)

• Catalase- positive

• Coagulase- positive

• Mannitol- ferments mannitol on mannitol salt agar (yellow colony)

• Cefoxitin disc- determine if MRSA

Pseudomonas aeruginosa

• Gram negative rods (grape smell)

• Growth on BAP and MAC

• Hemolysis- gamma

• Oxidase- positive

• Motility- motile

• Pigment production- blue-green pigment (pyocyanin and pyoverdine)

Legionella pneumophilia

• Gram negative bacilli, thin, pleomorphic

• Buffered charcoal yeast extract (BYCE) agar, requires L-cysteine for growth

• Catalase- positive

• Urine antigen test- rapid test

• Direct Fluorescent Antibody (DFA) staining

Haemophilus influenzae

• Gram negative coccobacilli

• Growth on CHOC

• Require V and X factor for growth (satellite phenomenon around S. aureus)

• Hemolysis- gamma

• Oxidase- positive

Klebsiella pneumoniae

• Gram negative rod bacteria (capsule)

• BAP and MAC (lactose fermentor) growth

• Oxidase- negative

• Indole- negative

• Citrate- positive

Moraxella catarrhalis

• Gram negative diplococci

• Growth on BAP and CHOC, not on MAC

• Hemolysis- gamma

• Oxidase- positive

• Catalase- positive

• Carb fermentation- assacharolytic

• DNase- positive

• Specimen type

• Overcrowding of contaminants

• Lack of culture sensitivty

• Patient empirically treated prior to culture

• Other agents of infection (viral, fungal, etc.)

• Improper collection

• Transport media

  • fastidious organism

• Color: can be yellow, green, red (blood)

• Consistency: thick

• Gram stain:

  • <10 epithelial

  • > 25 WBC/lpf

• Reject specimen if contaminated with saliva

• Expectorated sputum- Patient does not clean mouth and is asked to cough deeply to produce the sputum specimen

• Induced sputum- Patient rinses mouth with sterile water or sailine before collection

• Best for non intubated patients who are able to produce a good sputum sample

Endotracheal Tube (ET) Aspirate

• Patients who are intubated, sample is aspirated from the ET tube

Bronchial Washing/Bronchoalveolar Lavage (BAL)

• Collected during bronchoscopy procedure, saline is put into the airway and then aspirated back up for analysis

• Considered gold standard when they need to obtain a sample from deeper parts of the lungs

• Required for patients on ventilators

Lung aspirates

• Removes fluid or air from the pleural space

Lung biopsy

Types of Susceptibility Testing

• Disk Diffusion

  • Looking at the zone of inhibition are measured to determine the effectiveness of the drug

  • Simple and cost effective

  • More limited

• Broth Microdilution method

  • Determining the MIC

  • More precise and allows us to test more antibiotics

• E test

  • Antibiotic strip has a gradient of antibiotic concentrations is placed on the agar and determines the MIC

  • More expensive

• Automated systems

  • VITEK - more rapid results and testing a variety antibiotics at once

Interpretation

• Sensitive (S)- pathogen is inhibited by antibiotic

• Intermediate (I)

• Resistant (R)- pathogen is not inhibited by the antibiotic

• Consider the quantity of organism

  • Is there a predominance of one of these organisms?

  • Are they present in large amounts?

    • growth in quadrants II, III, IV?

Honorable mentions:

• Nocardia

• Histoplasma capsulatum

• Coccidioides immitis

• Cryptococcus sp.

• Aspergillus sp.

• Bordetella pertussis

• C. diptheriae

• Mycobacterium tuberculosis

• Group B Strep (pediatric patients)

• Influenza A, B

• Parainfluenza

• Adenovirus

vs.

Ventilator-Associated Pneumonia (VAP)

• likely to have more resistance to commonly used antibiotics

• Common pathogens:

  • E. coli

  • Pseudomonas aeruginosa

  • Acinetobacter baumannii

  • MRSA

Cystic Fibrosis Patients

• Decontamination methods for culture (NTM)

• 16S rRNA gene sequencing

• Common pathogens:

  • Nontuberculosis mycobacterium

  • Burkholderia cepacia complex

  • MRSA