Microbiology: Infection and Specimen Collection

Skin and Soft Tissue Infections

• Skin is the outermost layer, consisting of epidermis (outermost), dermis (contains hair follicles, sebaceous glands, sweat glands), and subcutaneous layer (fat).
• Fascia (fibrous tissue) and muscles are also associated with skin.
• Wound infections are caused by trauma (minor or severe), obstruction of oil/sweat glands, or inflammation of hair follicles.
• Infecting organisms can be endogenous (normal flora) or exogenous (outside the body).
• Common skin flora includes Staphylococci (S. epidermidis & S. aureus), diptheroids (Corynebacterium), micrococci, streptococci (non-hemolytic), Propionibacterium acnes, anaerobes, and yeast.
• Dermatitis is inflammation of the skin, which can be caused by Candida spp., S. aureus, coliforms, Corynebacterium spp., and molds (dermatophytes)
• Pyoderma is inflammation of skin with pus. Examples include: Impetigo (blister-like superficial skin infection caused by Group A streptococci & S. aureus); Erysipelas (superficial, painful skin infection caused by Group A streptococci & S. aureus); Anthrax (rare, caused by Bacillus anthracis).
• Erysipeloid is a superficial soft skin infection, associated with animal/meat/hides, caused by Erysipelothrix rhusiopathiae.
• Cellulitis, a diffuse infection of deep epidermis and subcutaneous tissue, is caused by Group A streptococci and S. aureus.
• S. aureus and MRSA can cause folliculitis (infected hair follicle, sometimes due to contaminated hot water tubs), furuncles (boils), and carbuncles (involve multiple hair follicles).
• Abscesses are collections of pus in skin and subcutaneous tissue.
• Soft tissue (wound) infections can be caused by many organisms, including S. aureus, streptococci, and anaerobes, common causes in surgical wounds and burns, and animal/human bites (e.g., Pasteurella multocida).
• Myonecrosis is a serious muscle infection causing gas gangrene, commonly caused by C. perfringens.
• Necrotizing fasciitis is a severe fascia infection, typically caused by Group A strep and S. aureus.
• Decubitus ulcers (bed sores or pressure sores) are caused by bacteria near the rectum, commonly Enterobacteriaceae, Pseudomonas, and Enterococci.
• Diabetic foot ulcers are slow-healing injuries caused by S. aureus, streptococci, Enterobacteriaceae, Pseudomonas aeruginosa, and anaerobes.
• Nodular lymphangitis is characterized by Sporothrix schenckii, Nocardia spp., Actinomyces spp., and Mycobacteria.
• Dermatologic manifestations of systemic infections can result from various pathogens including Borrelia burgdorferi, T. pallidum, Rickettsiae, Leptospira, and Mycobacterium leprae, resulting in erythema migrans, rashes.
• Viral infections leading to dermatologic manifestations include measles (Rubeola and Rubella), Chickenpox/Shingles (Varicella-Zoster Virus), Herpes Simplex Virus, Warts (HPV).
• Toxin-mediated skin diseases include staphylococcal scalded-skin syndrome, toxin shock syndrome (S. aureus), and scarlet fever (S. pyogenes).
• Specimen collection and transport protocols include avoiding surface contamination, decontamination of skin/mucous membranes, preference for tissue/pus aspirates, and maintaining moist tissue.
• Swabs are less desirable in specimen collection.
• For anaerobic infections, the use of anaerobic transport media is required.
• Microscopic examination includes gram stains for detection of clinically significant organisms (evaluation, rejection of specimens with high epithelial quality).
• KOH and calcofluor white mounts, and acid-fast stains may be used for microscopic examination.
• Cultures are conducted using media such as BAP, CHOC, MAC, PEA, with variations based on the suspected organism and the site of infection.
• Anaerobic cultures are particularly important for closed wounds or abscesses.
• Other cultures may involve Lowenstein-Jensen, Middlebrook media, viral culture in shell vials, and Sabouraud's agar for fungi.

Respiratory Tract Infections

• URT infections involve the oral cavity and neck including the nose, mouth, throat, epiglottis, larynx, middle ear and paranasal sinuses.
• LRT infections include the trachea, bronchi, bronchioles, and lung alveoli.
• The area below the larynx is usually sterile.
• Normal flora of the URT includes Staphylococci (including CONS and S. aureus), Streptococci (including viridans and pneumococci), micrococci, diphtheroids, Neisseria spp., Haemophilus, anaerobes, spirochetes and Candida spp.
• URT infections include thrush, laryngitis, epiglottitis, pharyngitis, and tonsillitis. Pathogens include respiratory viruses (influenza, parainfluenza, RSV, adenovirus, rhinovirus, coronavirus, coxsackie A, EBV, CMV), S. pyogenes, B-hemolytic strep groups C, F, and G, and Arcanobacterium haemolyticum, H. influenzae; parainfluenzae; N. gonorrhoeae; Corynebacterium diptheriae; and Bordetella pertussis/parapertussis, yeast.
• URT specimen collection may include swabs, syringes/needles, biopsies. Diphtheria specimens are cultured on BAP, Loeffler, and tellurite media; pertussis specimens are cultured on Bordet-Gengou blood agar.
• Throat cultures often include antigen testing for group A strep followed by confirmation cultures.
• Microscopic examination via gram stain is often not an appropriate diagnostic method due to high numbers of normal flora.
• Cultures for group A strep are common but other potential pathogens may be sought when indicated (e.g., GNR, Arcanobacterium haemolyticum, Corynebacterium diphtheriae).
• Common laboratory procedures for cultures include BAP, CHOC, and MAC, often cultured at 35 °C in CO2. Specific media for cystic fibrosis patients may also be required.
• Methods for sputum collection include expectorated, induced, tracheal aspirates, bronchial washings & brushings, and biopsies. Invasive procedures like bronchoscopy may be reserved for unusual cases
• Processing specimens for gram stains, bacteria, and cultures is generally required, often within 2 hours.
• Evaluation of sputum via gram stain may reveal etiologic agents and sputum suitability for culture, and the presence of contaminants based on number of epithelial cells.
• A good quality sputum sample will show less than 10 epithelial cells per low power field, and greater than 25 PMN per low power field.
• Routine cultures for sputum, tracheal aspirates, bronchial washings, and biopsies (including BAP, CHOC, and MAC) and special media for cystic fibrosis (В. серасіа, MRSA, Pseudomonas) is commonly conducted
• Anaerobic cultures are recommended for particular situations (lung aspirates/ open lung biopsies)
• Bioterrorism agents (including anthrax, plague, Q fever, and tularemia are commonly evaluated from samples.

Other Potential Infection Types

• Anthrax (Bacillus anthracis): three forms (cutaneous, gastrointestinal, inhalation).

  • Cutaneous anthrax is the most common form, occurring as a lesion developing into a black eschar.
  • Inhalation anthrax presents as flu-like symptoms followed (in ~1-2 days) by respiratory failure. Radiographic findings indicate mediastinal widening. Gastrointestinal anthrax involves ingestion of spores, resulting in bloody diarrhea and vomiting and 50% mortality rate if untreated.
  • Laboratory identification commonly includes gram staining (GPR spore-forming bacilli) and aerobic growth, nonhemolytic colonies, with a medusa-head morphology, and catalase-positive, nonmotile characteristics. Specimens for laboratory analysis include sputum, blood, gastrointestinal contents (blood/stool), environment samples, and evidentiary materials.

• Plague (Yersinia pestis): bubonic (infected lymph nodes), septicemic (blood-borne, necrotic changes—referred to as Black Death), and pneumonic (airborne transmission, highest mortality).

  • Bubonic plague demonstrates flu-like symptoms with painful buboes; the septicemic form shows no swelling and is similar to the bubonic form; pneumonic plague has the highest mortality rate and presents with flu-like symptoms followed by rapid transmission and hemoptysis (coughing up blood).
  • Laboratory identification often involves Gram-staining of specimens (GNR, slow-growth in 2 days, safety-pin appearance, nonmotile, occasionally oxidase, urea, indole negative), testing via SBA, and MAC. Specimen selection is important and specific to the form (bubonic: lymph node aspirate; septicemic: blood; pneumonic: sputum/bronchial wash/aspirate).

• Tularemia (Francisella tularensis): A zoonotic infection spread from animals, showing various clinical presentations and with flu-like symptoms (incubation 1-21 days, mostly 3-5 days; mortality can be low (with treatment to about 10% with no treatment), can last up to 2 weeks, persists in moist soil for months). The three most common presentations include pneumonic (flu-like symptoms over about 3-5 days), typhoidal (initial symptoms), and ulceroglandular (ulcer and adenopathy).

  • Laboratory identification may include slow growth of pleomorphic, small GNR that grow on SBA, CHOC, and MTM (buffered charcoal yeast-extract), nonmotile bacteria, catalase-positive and oxidase/urea negative characteristics. Specimens may include serum, blood, tissue samples, lesion/swab specimens, sputum, and environmental samples.

• Brucellosis (Brucella species): transmission occurs with unpasteurized dairy products, direct animal contact, or aerosols. The course of illness can last from weeks to months with fever, profuse sweating, malaise, and headache and muscle/back pain; mortality is typically under 5%.

  • Laboratory identification relies on GNCB slow-growth, aerobic growth, but some may grow on MAC, and nonmotile organisms that are catalase, oxidase, and urea positive. Blood or bone marrow, serum, tissue (spleen/liver), and environmental/evidentiary samples may be examined.

• Q Fever (Coxiella burnetii): an infection acquired via inhalation from infected animals. Spore-like bodies are produced. The incubation period is about 2-3 weeks showing flu-like symptoms, and in some cases, pneumonia and hepatitis. Mortality rate is ~2%.

  • Laboratory specimens include blood, serum, body fluids, visceral tissue, biopsies, and autopsy materials (stored at 4°C). Isolation testing and direct detection are conducted at reference labs, using BSL level 3 precautions.

• Smallpox (Variola major): a rare, highly contagious disease spread by infected droplets. The incubation period is about 8-16 days. Clinical presentations such as orinary (90% of cases), modified (mild, in previously vaccinated), flat (usually fatal), and hemorrhagic (usually fatal).

  • Laboratory identification usually relies on identifying the brick-shaped, DNA Orthopox-virus structure (200 nm). Collection of specimens for testing is often from tissues of the affected skin.

• Other Viral Hemorrhagic Fevers (Filoviruses, Arenaviruses, Bunyaviruses, Flaviviruses): are potentially severe, often life-threatening, viral infections (e.g., Ebola, Marburg, Lassa, Hantavirus, Tick-borne encephalitis, dengue, yellow fever) requiring BSL-3 precautions and testing at dedicated reference labs.

• Other cultures (Eye, Bone, Bone Marrow, External Ear): these infections can involve gram-positive/negative bacteria as normal flora and/or pathogens leading to infection. Special methods for collection via swabs, corneal scrapings, and/or enriched broth and/or media-based cultures, depending on the suspected organism, are needed.

• Botulism (Clostridium botulinum): a neuroparalytic syndrome with seven types of toxin; the most common are types A, B, E and F in humans, and C and D in animals. Presentations include foodborne (infant/non-infant), and wound-associated types. Common specimens for testing include serum, gastric contents, stool, and food/environmental samples.