Exam 1 Material Bacteriology II

Three essential characteristics of laboratory results

Accurate, Significant, Clinically relevant

Key considerations for proper specimen management

Select the correct anatomic site. Collect using proper technique and supplies. Package to promote survival of the organism and prevent leakage. Transport to the laboratory as soon as possible.

Common types of specimens

Urine, Tissue, Blood, Genital, Feces, Eyes, Fluids, Ears, Nasal, Sputum, Throat, Skin, Wound, Etc.

Specimen of choice for ear infections (otitis media)

The specimen of choice is fluid obtained by tympanocentesis.

Sputum specimens and bacterial pneumonia complications

All sputum specimens are contaminated to some degree with oropharyngeal flora.

Importance of rapid transport of specimens

Microorganisms multiply and die rapidly; if they do so during collection, transport, or storage, the specimen is no longer representative of the patient's disease process.

Reasons why a specimen might be rejected by the laboratory

Improper or no label, Improper container (nonsterile), Leaking container, Quantity not sufficient, Prolonged transport, Duplicate specimens submitted at same time.

Key steps in the laboratory workflow after specimen receipt

Specimen setup, Culture incubation (18-24 hrs), Read-out, Set-up ID and sensitivity tests, Result (computer, manually), Reports to physician.

Common media types used for culturing specimens

Sheep blood agar, Chocolate agar, MacConkey agar, Thayer-Martin agar, CDC anaerobic agars, Broth culture (Thioglycollate, BHI, etc.).

Typical incubation conditions for cultures

CO2​, Ambient air, Anaerobic atmosphere, 35-37°C, 18-24 hours incubation.

A "Big NO NO" in a microbiology lab

Sniffing a culture plate. While certain organisms have distinctive odors (e.g., Haemophilus - musty/mousy, Eikenella - bleach, Pseudomonas aeruginosa - sweet/fruity), it used to be a common practice but is no longer acceptable.

Identification (ID) and sensitivity tests setup

Manual identification (API, tubed media), Instrument identification (Vitek, Microscan), Manual sensitivity testing (Kirby Bauer, E-test), Instrument sensitivity testing (Vitek, Microscan).

Handling urine specimens in the lab

Transport to lab within 30 minutes or refrigerate. Use 1/1000 calibrated loop to streak plates (Sheep blood agar, MacConkey agar).

Process for handling blood cultures

10ml inoculated into each of 2 bottles (one aerobic, one anaerobic). Generally 2-3 sets in a 24-hour period. Bottles incubated for 5 days before being reported as negative. Bottles stained as soon as flagged positive. Organisms seen immediately reported to physician. Specimen subcultured and organisms identified.

"Panic" results requiring immediate reporting

Positive blood cultures, Positive CSF gram stains or India Inks, Positive CSF cultures, Positive Acid Fast Bacillus smears, Positive Acid Fast Bacillus cultures, Vancomycin resistant Enterococcus, Vancomycin resistant Staphylococcus.

Non-culture tests performed in microbiology

Rapid strep screens, Clostridium difficile toxin assays, Parasitology, Occult Bloods, Serology, Cryptococcal antigen testing, Rapid influenza testing, Other rapid viral tests.

Quantitation for "MANY" organisms in a wound/aspirate specimen

> 25 organisms per oil immersion field.

FEW organisms in a sterile body fluid

0-2 organisms per oil immersion field.

Importance of Quality Control (QC) in clinical microbiology

Accurate results are critical for proper diagnoses and treatment of infectious disease.

Agencies mandating operational guidelines for QC

The Joint Commission for the Accreditation of Healthcare organizations (JCAHO) and others.

Key requirements for QC guidelines and record keeping

All rules and procedures must be written and available in a QC manual. The manual must be reviewed and signed annually. All QC activities must be recorded, including tolerance limits and corrective actions.

Areas a QC program should include procedures for controlling

Temperatures, Equipment, Media, Reagents, Susceptibility testing, Personnel.

Daily QC requirements for temperature-dependent equipment

Daily temperature checks. Thermometers must be checked against a NIST-referenced thermometer before use. Certificates of calibration must be kept for the life of the thermometer. Non-mercury thermometers are recommended.

Monitoring culture media

Records must be maintained for 2 years. They should be checked for sterility and performance using stock cultures.

Commercial media types that must be retested by the laboratory

Chocolate, selective agar for pathogenic Neisseria, and Campylobacter.

Checks on 'in-house' prepared culture media before use

Each medium must be tested with organisms expected to grow and/or produce a positive reaction, as well as organisms that should not grow or produce a negative reaction.

Visual inspections on all media before use

Moisture (free of moisture but not dry), Sterility (free of contaminants), Breakage (petri dishes not cracked/broken), Appearance (blood-based plates no hemolysis, appropriate color).

Reactivity of chemical/biological reagents or stains

Generally, when a new lot is received, with positive and negative control organisms. Many reagents must be tested each day of use with both positive and negative controls.

Organization providing guidelines for antimicrobial susceptibility testing

The CLSI (Clinical and Laboratory Standards Institute).

Control organisms for susceptibility testing frequency

Usually daily until precision is demonstrated with 20 or 30 consecutive days. After this, QC organisms may be tested weekly instead of daily.

Proficiency testing

Carefully designed samples are given to techs as unknowns (can be purchased or prepared internally). All tests performed on patients must be subjected to proficiency testing twice per year.

Clinical Laboratory Improvement Act (CLIA) of 1988 mandate

That the competency of each employee be determined and verified upon employment, with re-verification annually. Proof of competency must be maintained in the employee's personnel file.

Maintaining stock cultures for QC

Grown in a large enough volume to be divided into small freezer vials to last a year. A new frozen vial can be removed weekly. An organism should be subcultured twice after thawing to return it to a healthy state.

Performance Improvement (PI)

A process to decrease sources of error in the clinical laboratory (formerly called Quality Control). Emphasized by JCAHO, College of American Pathologists (CAP), and mandated by CLIA.

Key challenge in clinical microbiology

Distinguishing between a patient's normal flora and the causative agent of an infection, as some microbes are normal flora at certain anatomical locations but considered pathogens when isolated from other sites.

Commensalism

Living in, on, or with another organism without causing damage.

Mutualism

The peaceful and profitable co-existence between two organisms.

Parasitism

An organism capable of causing harm or disease.

Indigenous flora

Normal flora, which are organisms commonly found on or in body sites of healthy people.

Resident flora

Organisms that colonize an area of the body for months or years.

Transient flora

Organisms that are temporarily present at a specific body site.

Carrier in microbiology

A person that has a pathogenic organism living on or in the body.

Acute vs Chronic carrier states

Acute is short-lived or transient carriage of a pathogenic organism, while chronic is carrying a pathogenic organism for months or years, which may be permanent.

Sterile body sites

Solid organs, Blood, CSF, Urine, Lower respiratory tract.

Bacteria contribution to human health

They are vital for the maintenance of human health. There are 10 times more bacterial cells than human cells in the human body, especially on the skin and in the digestive tract. They contribute to gut immunity and synthesize vitamins like folic acid, vitamin K, and biotin.

Beneficial bacteria like Lactobacillus

They convert milk protein to lactic acid in the gut. They also inhibit the growth of potentially pathogenic bacteria through competitive exclusion (competing for nutrients and space) and by producing toxins harmful to some pathogens.

Location of most normal flora

In the digestive tract (gut flora), and most are anaerobic.

Estimated bacterial species in human body

Over 200 different bacterial species.

Relationship between normal flora and host

They tend to be commensal or mutual with the host, but can be opportunistic pathogens in immunocompromised hosts.

What factors influence the composition of normal flora on the skin?

The type of sebaceous or sweat glands, with more organisms found in moist areas like the armpit, groin, and perineum.

Common microorganisms on the skin

Candida spp., Micrococcus spp., Staphylococcus spp., Diphtheroids, Propionibacterium, Clostridium spp.

Characteristic of mouth flora supporting anaerobes

Bacterial plaques contain a high number of streptococci (1011 per gram) and result in a low oxidation-reduction potential.

Common mouth flora

Staphylococcus epidermidis, Streptococcus spp., Peptostreptococcus spp., Veillonella spp., Lactobacillus spp., Actinomyces israelii, Bacteroides spp., Prevotella/Porphyromonas.

Distinguishing pathogenic Streptococcus pneumoniae

Both are alpha-hemolytic on blood agar, but S. pneumoniae is sensitive to the 'P' disk (optochin), while S. mitis is resistant.

Protection of the lower respiratory tract

Ciliary epithelial cells and the movement of mucus. These tissues are usually sterile.

Common organisms in the upper respiratory tract

Viridans streptococci (S. mitis, S. mutans, S. milleri, S. sanguis), Moraxella catarrhalis, Neisseria spp., Diphtheroids.

Common organisms in the nose and nasopharynx

Staphylococcus aureus, Staphylococcus epidermidis, Haemophilus parainfluenzae.

Why is the stomach usually sterile?

Due to gastric juices, acids, and enzymes.

Organisms in the GI tract

In the colon, with between 108 and 1011 bacteria per gram of solid material. 90% of these bacteria are anaerobes.

Common GI flora

Bacteroides spp., Clostridium spp., Enterobacteriaceae, Eubacterium spp., Fusobacterium spp., Peptostreptococcus spp., Peptococcus spp., Staphylococcus aureus, Enterococcus spp.

Normally sterile parts of the genitourinary tract

The kidneys, bladder, and fallopian tubes.

Composition change of vaginal flora

It is consistent with hormonal changes and age.

Common genitourinary flora

Lactobacillus spp., Bacteroides spp., Clostridium spp., Peptostreptococcus spp., Staphylococcus aureus, Enterococcus spp., Diphtheroids.

Nosocomial Infection

Hospital-acquired disease, often when commensal organisms from healthcare workers infect sick patients.

Endogenous infections

When a bacteria gains entry from its normal, benign location into a site where it can cause disease.

Components of the Upper Respiratory Tract

Nasal cavity, Pharynx, and Larynx.

Components of the Lower Respiratory Tract

Trachea, Bronchi, and Bronchioles.

Non-specific host protection factors of the respiratory tract

Nasal hairs, convoluted passages, mucous membranes, secretory IgA and lysozyme secretion, cilia, and reflexes (coughing, sneezing, swallowing).

Pathogens found in the Naso/Oropharynx

Acinetobacter, Pseudomonas spp., Streptococci spp., Filamentous fungi, Staphylococcus aureus, Eikenella corrodens, Neisseria meningitidis, Mycoplasma spp., Bacteroides spp., Peptostreptococcus, Actinomyces spp., Haemophilus spp., Moraxella catarhallis, Candida albicans, HSV, Enterobacteriaceae, and Mycobacterium spp.

Organisms rarely considered pathogens in the Naso/Oropharynx

Nonhemolytic strep, Staphylococci, Micrococci, Corynebacterium spp., CNS (Coagulase-negative Staphylococci), other Neisseria spp., Lactobacillus spp., Campylobacter spp., and Veillonella spp.

General mechanisms of bacterial pathogenesis

Adherence, Toxins, Growth, and Evasion of host immune responses (e.g., capsule production).

Bacteria that produce capsules to evade host immune responses

S. pneumoniae, N. meningitidis, H. influenzae, Klebsiella pneumoniae, and some strains of P. aeruginosa.

Bacteria that multiply within host cells

Chlamydia, Legionella, and Mycobacterium tuberculosis.

Definite respiratory pathogens

Corynebacterium diphtheriae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Chlamydia trachomatis and pneumoniae, Bordatella pertussis, Legionella spp., Nocardia spp., RSV, HSV, adenoviruses, rhinoviruses, and Cryptococcus neoformans.

Rare respiratory pathogens

Francisella tularensis, Bacillus anthracis, Yersinia pestis, Coxiella burnetti, Brucella spp., Pasteurella multocida, and Chlamydia psittaci.

Diseases of the Upper Respiratory Tract

Laryngitis, Laryngotracheobronchitis, Epiglottitis, Pharyngitis, Tonsillitis, Rhinitis, and Stomatitis.

Main symptom of Laryngitis

Hoarseness.

Usual causes of acute laryngitis

It is usually viral, caused by influenza virus, rhinovirus, or adenovirus.

Causes of laryngitis with exudate or membrane

Streptococcus, mononucleosis, or diphtheria.

Another name for Laryngotracheobronchitis

Croup.

Common symptoms of Croup

Variable fever, difficulty moving air through the larynx, hoarseness, and a barking non-productive cough. Severe respiratory distress and fever are common in children.

Primary viral causes of Croup

Parainfluenza virus, influenza virus, RSV, and adenovirus.

Age group common for Epiglottitis

Children 2-6 years of age.

Symptoms of Epiglottitis

Fever, difficulty swallowing, drooling, and respiratory obstruction.

Usual bacterial causes of Epiglottitis

H. influenzae type B, Streptococci, and Staphylococci.

Culture contraindication for suspected Epiglottitis

Swabbing may lead to respiratory obstruction.

Symptoms of Pharyngitis and Tonsillitis

Pharyngeal pain with inflammatory exudates, vesicles, mucosal ulceration, or swollen lymph nodes.

Primary bacterial cause of pharyngitis

S. pyogenes (treatable with penicillin).

Common complication of tonsillitis

Peritonsillar abscess.

Common etiologic agents of peritonsillar abscess

Non-spore forming anaerobes (Fusobacterium, Bacteroides, and anaerobic cocci), Streptococcus pyogenes, and viridans Streptococci.

Symptoms of Rhinitis

Variable fever, increased mucous secretions, edema of nasal mucosa, sneezing, and watery eyes.

Common viral causes of Rhinitis

Rhinoviruses, Coronaviruses, Adenoviruses, Parainfluenza and influenza virus, and Respiratory syncytial virus (RSV).

Type of swabs for respiratory tract specimen collection

Cotton or Dacron swabs.

Time swabs should remain moist and be cultured after collection

Within 4 hours. If more than 4 hours is expected, the specimen must be placed in transport medium.

Characteristic of specimens for Group A Streptococci regarding desiccation

They are highly resistant to desiccation.

Handling of specimens for B. pertussis

They should be aspirated nasopharyngeal secretions, inoculated directly at the patient bedside, and transported no more than 2 hours in special media like 1% casamino acid medium, charcoal horseblood transport medium, or 5% serum inositol in fetal bovine serum.

Usefulness of Gram-stain of the upper respiratory tract for direct examination

No.

Primary cause of bacterial pharyngitis and routine screening method

S. pyogenes. It is routinely screened using 5% sheep blood agar for Beta hemolysis and a 0.04 unit bacitracin filter paper disk (showing susceptibility). Direct antigen tests (agglutination reactions) and the PYR test are also used.

Special media for culturing C. diphtheriae

Loeffler's agar and Cystine-tellurite agar.

Special media needed to culture B. pertussis

Bordet-Gengou agar, Regan-Lowe agar, and Charcoal horse blood agar.