Diagnostic Testing in the Microbiology Laboratory

Diagnostic Testing in the Microbiology Laboratory
Presentation by: Jane Wong
Public Health Microbiologist

Topics

* Some basic principles of microbiology testing
* A crash course in microbiology
* Follow a specimen through the lab
* Laboratory staffing issues

Media and Culture

* Media: Nutrients (agar, pH indicators, proteins and carbohydrates) used to grow organisms outside of their natural habitats
* Culture: The propagation of microorganisms using various media

Direct and Indirect Testing

* Direct: Demonstration of the presence of an infectious agent
o Culture
o Microscopy
o Molecular methods such as PCR
* Indirect: Demonstration of presence of antibodies to a particular infectious agent
o Serology

Sterile versus Non-sterile Body Sites

* Sterile body sites:
o These sites normally do not contain any bacteria, so any bacteria found there are significant
+ Blood
+ Spinal fluid
* Non-sterile body sites:
o These sites are open to the external environment and normally contain bacteria
+ Throat
+ Feces

Specimens from Sterile Sites
Specimens from Non-Sterile Sites
Sensitivity
Specificity
Documentation
Specimen

* Appropriateness
* Collection
* Transport to lab
* Inoculation of media
* Culture and isolation
* Confirmation
* Report
Appropriate Specimen
Collection
Transport to Laboratory
Inoculation of Media
Culture media

* Used to grow bacteria
* Can be used to:
o Enrich the numbers of bacteria
o Select for certain bacteria and suppress others
o Differentiate among different kinds of bacteria

Microbiological Culture Media
Isolation of Individual Bacteria
Growth of Colonies
Mixed Culture of Soil Organisms Containing Bacillus anthracis
Colony “Picking”
Colonies of Bacteria in Pure Culture
Pure Culture of Francisella tularensis
Colonies After 72 hours Growth
Pure Culture of Yersinia pestis
Colonies on Blood Agar After 48 hours of Growth
Yersinia pestis Colonial Morphology Viewed With Transmitted Light

Confirmation

* Now we have a pure culture of bacteria
* Testing is now done to confirm the identification of the bacteria culture
o Stains
o Biochemical tests
o Serological tests (using known antibodies)
o Molecular tests (nucleic acid probes)

Gram Stain of Streptococcus sp.
Yersinia pestis
Gram stain
Gram stain of Brucella sp.
B. anthracis Gram stain showing spores
Gram stain of B. anthracis from broth culture

Examples of Biochemical Tests
Left: API 50 Test
Above: Antimicrobial Sensitivity Test
Yersinia pestis E-Test (Antimicrobial Sensitivity Test)
Nitrate and Urea Reactions
Reactions on MacConkey Agar
Triple Sugar Iron (TSI) Test
Blood Bottles Incubated
18 Hours of Incubation
Microbiologist Suspects Bacillus anthracis
India Ink Preparation
Growth on a Blood Agar Plate (Petri Dish) After 18-24 Hours
Gram stain of B. anthracis from broth culture
Motility
Laboratory Cannot Rule Out Bacillus anthracis
* Refers the culture to a reference laboratory that is part of the Laboratory Response Network (LRN)
Report
Molecular Tests
The Flip Side!
Other Factors in Personnel Shortage
Licensing Applications/Year For Clinical Laboratory Scientist Certification

Diagnostic Testing in the Microbiology Laboratory

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The Microbiology of Wounds

The Microbiology of Wounds
Presentation by:Neal R. Chamberlain, Ph.D.,
Department of Microbiology/Immunology
KCOM

Microbes and Chronic Wounds

* All chronic wounds are contaminated by bacteria.
* Wound healing occurs in the presence of bacteria.
* Certain bacteria appear to aid wound healing.
* It is not the presence of organisms but their interaction with the patient that determines their influence on wound healing.

Definitions

* Wound contamination: the presence of non-replicating organisms in the wound.
* All chronic wounds are contaminated.
* These contaminants come from the indigenous microflora and/or the environment.
* Most contaminating organisms are not able to multiply in a wound. (Ex. Most organisms in the soil won’t grow in a wound).
* Wound colonization: the presence of replicating microorganisms adherent to the wound in the absence of injury to the host.
* This is also very common.
* Most of these organisms are normal skin flora.
* Staphylococcus epidermidis, other coagulase negative Staph., Corynebacterium sp., Brevibacterium sp., Proprionibacterium acnes, Pityrosporum sp..
* Wound Infection: the presence of replicating microorganisms within a wound that cause host injury.
* Primarily pathogens are of concern here.
* Examples include; Staphylococcus aureus, Beta-hemolytic Streptococcus (S. pyogenes, S. agalactiae), E. coli, Proteus, Klebsiella, anaerobes, Pseudomonas, Acinetobacter, Stenotrophomonas (Xanthomonas).

Microbiology of Wounds

* The microbial flora in wounds appear to change over time.
* Early acute wound; Normal skin flora predominate.
* S. aureus, and Beta-hemolytic Streptococcus soon follow. (Group B Streptococcus and S. aureus are common organisms found in diabetic foot ulcers)
* After about 4 weeks
o Facultative anaerobic gram negative rods will colonize the wound.
o Most common ones= Proteus, E. coli, and Klebsiella.
* As the wound deteriorates deeper structures are affected. Anaerobes become more common. Oftentimes infections are polymicrobial (4-5).
* Long-term chronic wounds oftentimes contain more anaerobes than aerobes.
* Aerobic gram-negative rods also infect wounds late in the course of chronic wound degeneration. Usually acquired from exogenous sources; bath and foot water
* Ex. Pseudomonas, Acinetobacter, Stenotrophomonas (Xanthomonas).
* Organisms like Pseudomonas are not very invasive unless the patient is highly compromised (ex. Ecthyma gangrenosum in neutropenic patients).
* These organisms are associated with marked wound deterioration due to endotoxin, enzymes, and exotoxins.

* As the wounds go deeper and become more complex they can infect the underlying muscles and bone causing osteomyelitis.
* Coliforms and anaerobes are associated with osteomyelitis in these patients. You also see Staphylococcus aureus.
* Enterococcus and Candida are often isolated from wounds.
* Treating a patient for these organisms is only indicated if there are no other pathogens present and the organisms are present in high concentrations (106 CFU’s per gram of tissue)

From Colonization to Infection?
Dose of Bacteria
Bacterial Problems to Consider
Virulence
Host Resistance
Wound Depth can Result in Different Diseases
How do you know when a wound is infected?

* This can be very difficult.
* A continuum exists between when pathogens colonize the wound and then start to cause damage.
* There is no absolutely foolproof laboratory test that will aid in this diagnosis.
* One feature is common to all infected chronic wounds;
* The failure of the wound to heal and progressive deterioration of the wound.
* Unfortunately, wound infections are not the only reasons for poor wound healing.
* The typical features of wound infections:
* increased exudate
* increased swelling
* increased erythema
* increased pain
* increased local temperature
* Periwound cellulitis, ascending infection, change in appearance of granulation tissue (discoloration, prone to bleed, highly friable).

Specimen Collection and Culture Techniques.

* There is a good deal of controversy concerning specimen collection.
* The gold standard collection method is to do a tissue biopsy or needle aspirate of the leading edge of the wound after debridement.
* >105 CFU/gm of tissue= greater likelihood of sepsis developing.
* Indicate the specific anatomic site the biopsy is collected from.
* Indicate whether this is a surface or deep wound. Ask for a smear and gram stain of the tissue.
* Surface wounds are NOT cultured for anaerobes.
* Deep wounds are cultured for anaerobes.
* If a tissue biopsy is not possible;
* cleanse the wound with sterile saline
* vigorously swab the base of the lesion
* Surface wounds place the swab in a sterile container for transport.
* Deep wounds place the swab in a sterile anaerobic container for transport.

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Microbiology, Infections, and Antibiotic Therapy

Microbiology, Infections, and Antibiotic Therapy
Presentation by: Elizabeth J. Rosen, MD
Francis B. Quinn, MD


Basic Bacteriology
Shape
Arrangement
Gram Staining
Cell Wall Characteristics

* Gram Positive
* Gram Negative

Bacterial Growth

* Binary Fission = Exponential Growth
* Four Phases of Growth

Normal Bacterial Flora
Host Defense Mechanisms

* Nonspecific Immunity
o barriers
o inflammatory response
* Specific Immunity
o Passive
o Active
+ humoral
+ cell-mediated

Clinical Microbiology

* Gram Positive Cocci
* Gram Positive Bacilli
* Gram Negative Cocci
* Gram Negative Bacilli
* Anaerobes
* Spirochetes
* Mycobacteria

Gram Positive Cocci

* Staphylococcus
* Streptococcus

Staphylococcus

* S. aureus, S. epidermidis, S. saprophyticus
* S. aureus

Streptococcus

* S. viridans
o oral flora
o infective endocarditis
S. pyogenes

* Group A, beta hemolytic strep
* pharyngitis, cellulitis
* rheumatic fever
+ fever
+ migrating polyarthritis
+ carditis
+ immunologic cross reactivity
* acute glomerulonephritis
+ edema, hypertension, hematuria
+ antigen-antibody complex deposition
S. pneumoniae

Gram Negative Cocci

* Neisseria
o meningitidis
o gonorrhea
* Moraxella catarrhalis

Gram Positive Bacilli

* Clostridium
* Bacillus
* Corynebacterium
* Listeria
* Actinomyces
* Nocardia

C. tetani
C. botulinum

* Descending weakness-->paralysis
* diplopia, dysphagia-->respiratory failure

C. perfringens
C. diphtheriae

* Fever, pharyngitis, cervical LAD
* thick, gray, adherent membrane
* sequelae-->airway obstruction, myocarditis
* colony morphology
L. monocytogenes
Actinomyces

* Part of normal oral cavity flora
* 50% of infections occur in face & neck
* forms abscesses with sulfur granules
* draining sinus tracts

Nocardia
Gram Negative Bacilli

* Facultative Anaerobes
o Respiratory
# Haemophilus
# Bordetella
# Legionella
o Zoonotic
# Yersinia
# Francisella
# Pastuerella
o Enteric
# Klebsiella
# Serratia
# Proteus
# Enterobacter

* Strict Aerobes
o Pseudomonas
* Anaerobes
o Bacteroides

Enterobacteriaceae
K. rhinoscleromatis
* Catarrhal
o purulent rhinorrhea
* Granulomatous
o mucosal nodules
* Cicatricial
o fibrosis
o stenosis

H. influenzae
Legionella

* Community and Nosocomial pneumonia
* contaminated water sources
B. pertussis
Zoonotic Gram Negative Rods

* Yersinia
o plague
* Franciscella
o tularemia
* Pasturella
o dog/cat bites

Pseudomonas
Anaerobic Bacteria

* Bacteroides
* Fusobacterium
* Peptostreptococcus
* Actinomyces
* Prevotella

Spirochetes

* Treponema
* Borrelia

Manifestations of Syphilis
Lyme Disease

* Cutaneous lesions
o erythema chronicum migrans
* Nonspecific symptoms
o malaise, fatigue, headache, fevers, chills, myalgias, arthralgias, lymphadenopathy
* Late manifestations
o neurologic
o cardiac

M. tuberculosis

* Pulmonary disease (82%)
* Extrapulmonary disease (18%)

ENT Manifestations of TB

* Scrofula
o matted lymphadenopathy: posterior triangle
* Laryngeal TB
o edema, ulcers, polypoid changes: arytenoids
* Oral TB
o painless ulcers: tongue
* Aural TB
o thickened TM-->hyperemia-->multiple perfs
o thin, watery otorrhea-->thick, cheesy d/c
M. leprae

Antibiotic Therapy

* Identify infecting organism
* Evaluate drug sensitivity
* Target site of infection
* Drug safety/side effect profile
* Patient factors
* Cost

Classification of Antibiotics

* Bacteriostatic
* Bactericidal
* Chemical Structure
* Spectrum of Activity
* Mechanism of Action

Mechanism of Action
Inhibitors of Cell Wall Synthesis
Beta Lactam Antibiotics

* Penicillins
* Cephalosporins
* Carbapenems
* Monobactams
Penicllins

* Derived from the fungus Penicillium
* Therapeutic concentration in most tissues
* Poor CSF penetration
* Renal excretion
* Side effects: hypersensitivity, nephritis, neruotoxicity, platelet dysfunction

Natural Penicillins

* Penicillin G, Penicillin V

Antistaphylococcal Penicillins
Aminopenicillins
Antipseudomonal Penicillins
Cephalosporins
Generations of Cephalosporins
Monobactams
Carbapenems
Vancomycin
Protein Synthesis Inhibitors
Tetracyclines
Aminoglycosides
Macrolides
Erythromycin
Alternate Macrolides
Chloramphenicol
Clindamycin
Inhibitors of Metabolism
Sulfonamides
Trimethoprim
Co-Trimoxazole (TMP/SMX)
Inhibitors of Nucleic Acid Function/Synthesis
Fluoroquinolones
Antimycobacterial Therapy
First-Line Agents
Antimycobacterials for Leprosy
Antibiotic Prophylaxis
Classification of Wounds
Classification of Wounds
Prophylactic Antibiotics
Effective Prophylactic Regimens
Topical Antibiotic Prophylaxis
Indications for Antibiotic Prophylaxis in ENT Surgery

Microbiology, Infections, and Antibiotic Therapy.ppt

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