Lower and Upper Respiratory Tract Bacteriology

Sputum culture has limited value in mild community-acquired pneumonia, so it is ordered for selected patients. In hospitalized adults, a pretreatment lower respiratory Gram stain and culture is recommended for severe pneumonia, for patients being treated empirically for methicillin-resistant Staphylococcus aureus (MRSA) or Pseudomonas aeruginosa, for patients with prior respiratory isolation of either organism, and for patients hospitalized and given parenteral antimicrobials within the previous 90 days. When the patient is intubated, an endotracheal aspirate collected promptly after intubation gives better yield than expectorated sputum. Clinicians use negative results from these cultures when they review broad empiric coverage.^{11 Metlay JP, et al. Diagnosis and treatment of adults with community-acquired… 2019.}

Lower respiratory specimens include expectorated or induced sputum, tracheal aspirate, bronchoalveolar lavage (BAL) fluid, protected-brush specimens, and bronchial washings.^{2,32 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.3 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Expectorated sputum is best collected early in the morning before eating, after the patient rinses the mouth with water and produces a deep-cough specimen of 5 to 10 mL. A nonproductive cough can be induced with aerosolized saline. An induced specimen is thin and watery and can be mistaken for saliva on appearance alone, so the requisition identifies it as induced and the laboratory screens it accordingly.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Sputum collected for mycobacterial culture follows different rules. At least three specimens are collected, 8 to 24 hours apart, with at least one taken early in the morning.^{44 Lewinsohn DM, et al. Official American Thoracic Society/Infectious Diseases Society… 2017.} The patient must not rinse the mouth with tap water first, because a water rinse can introduce environmental mycobacteria such as Mycobacterium gordonae or the M. avium-intracellulare complex and seed the specimen with organisms that can be mistaken for a true pathogen.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Sputum passes through the oropharynx, so a screening Gram stain assesses whether it contains lower respiratory secretions or mostly saliva. Apply your laboratory's adopted screening criteria. The routine bacterial culture policy used here rejects specimens with more than 25 squamous epithelial cells per low-power field because they indicate salivary contamination. A specimen with 25 or fewer passes this cellular screen if the other specimen checks are acceptable. This policy does not require a minimum neutrophil count. Record a rejection and its reason, and request a fresh specimen.^{1717 National Jewish Health, et al. Respiratory culture.}

Recognizing indigenous oral and upper-airway flora prevents colonizers from being worked up as pathogens. These organisms account for most growth on throat cultures.

Mixed flora of this kind is expected in specimens that pass through the mouth and does not alone make the specimen unacceptable. Squamous epithelial cells are the best marker of oral contamination. Numerous squamous cells with mixed flora and few or no neutrophils indicate saliva.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

BAL fluid and protected-brush specimens are used for acute bacterial pneumonia, for ventilator-associated pneumonia in patients not yet treated, for opportunistic pathogens in immunocompromised patients, and for mycobacterial infection when a patient cannot produce sputum.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

A protected-brush specimen is collected through a double-cannula catheter holding only 0.001 to 0.01 mL of secretions. A displaceable plug seals the outer cannula until it reaches the sampling site. The brush and inner cannula retract into it before withdrawal to prevent upper-airway flora from contaminating the brush during passage. Place the brush in 1 mL of sterile saline or broth for transport.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

BAL is performed by wedging the bronchoscope tip into an airway and instilling sterile saline in three or four aliquots, commonly more than 140 mL in total, which samples an estimated one million alveoli. Between 10 and 100 mL usually returns. The fluid should reach the laboratory within 30 minutes, because carryover from the procedure inhibits some bacteria. When a delay is unavoidable, refrigeration limits that loss only partly.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Vortex BAL fluid for culture inoculation and cytocentrifuge preparations. Keep small tissue fragments moist in sterile saline and process them separately. Treat viscous fluid suspected of harboring fungi, including Pneumocystis jirovecii, with a mucolytic agent and centrifuge it before staining. A Gram-stained cytospin showing one or more bacteria per oil-immersion field and no squamous epithelial cells strongly suggests acute bacterial pneumonia. A Papanicolaou-stained cytospin can show the cytopathic inclusions of cytomegalovirus pneumonia. Acid-fast and organism-specific antibody stains on cytospins extend testing to mycobacteria and targets such as Legionella species.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Quantitative culture was introduced to raise specificity, but published trials in intubated patients have not shown better outcomes than qualitative or semiquantitative culture. Current guidance recommends noninvasive sampling with semiquantitative culture over invasive sampling with quantitative culture, so the method in use is set by local practice.^{66 Kalil AC, et al. Management of adults with hospital-acquired and… 2016.} Where quantitative culture is performed, inoculate plates by serial dilution or calibrated loop and compare the colony count, in colony-forming units (CFU) per milliliter, with a threshold.

Report the quantitative result with its qualified interpretive comment. A count below the applicable threshold reduces the microbiologic support for infection. It does not establish colonization or contamination, even when a recognized respiratory pathogen is recovered, because the guideline threshold is a conditional clinical recommendation and counts can be misleadingly low.^{6,76 Kalil AC, et al. Management of adults with hospital-acquired and… 2016.7 Centers for Disease Control and Prevention. Pneumonia (ventilator-associated and non-ventilator-associated)…} Antimicrobial therapy before collection can falsely lower the count. Contamination during passage through the endotracheal tube or bronchoscope can falsely raise it. Interpret the count with the Gram stain and clinical findings.^{66 Kalil AC, et al. Management of adults with hospital-acquired and… 2016.}

Routine bacterial workup follows the general Gram stain and culture pathway. Mycobacterial workup adds decontamination and digestion to suppress faster-growing respiratory flora. All handling occurs in a biological safety cabinet, ideally in a negative-pressure room under biosafety level 3 practices.^{4,34 Lewinsohn DM, et al. Official American Thoracic Society/Infectious Diseases Society… 2017.3 Carroll KC, et al. Manual of Clinical Microbiology. 2023.} Fungal workup uses the same containment. Media may include blood enrichment and antimicrobial supplementation according to cost and the species expected in the local patient population.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

For P. jirovecii direct fluorescent antibody staining, first liquefy a viscous specimen with a mucolytic agent such as N-acetyl-L-cysteine or dithiothreitol, vortex it vigorously, and centrifuge it. Smear the sediment, fix it according to the stain manufacturer's instructions, and examine it microscopically.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Streptococcus pneumoniae and Haemophilus influenzae are identified from respiratory specimens by the same methods used for blood isolates. Their characteristics, X and V factor differentiation, and related species are covered in Blood Cultures and Gram-Positive Blood Isolates and Gram-Negative, Fastidious, and Vector-Borne Agents of Bloodstream Infection.

Molecular targets such as lytA, plyA, penicillin-binding protein genes, pneumococcal surface protein A, and the DNA polymerase gene identify S. pneumoniae at the species level. In a respiratory specimen, however, a positive result cannot separate infection from asymptomatic colonization, which is especially common in children. A pneumococcal urinary antigen assay is used in some settings as a nonculture adjunct in adults with severe pneumococcal pneumonia.^{2,32 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.3 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Moraxella catarrhalis is a gram-negative diplococcus carried in the oropharynx of healthy children and adults. It adheres through outer-membrane pili and causes bronchitis, otitis media, sinusitis, and pneumonia, particularly in patients with chronic lung disease. Bacteremia, endocarditis, meningitis, and ophthalmia neonatorum are rare. On Gram stain, it is indistinguishable from Neisseria. It grows on plain blood agar, unlike N. gonorrhoeae, and does not oxidatively utilize carbohydrates. All sugar reactions are negative, in contrast to the glucose-positive Neisseria species, and the DNase reaction is positive. Nearly all isolates produce beta-lactamase, detectable with nitrocefin, and are presumed penicillin resistant.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Legionella species

Legionella species are obligately aerobic, faintly staining, small gram-negative bacilli first recognized in the 1976 Philadelphia pneumonia outbreak. More than 60 species are recognized, and L. pneumophila serogroup 1 causes most disease. The organisms persist in cooling towers, water heaters, warm-water spas, faucets, shower heads, and other water systems. They also survive inside environmental protozoa, including Acanthamoeba and Naegleria, which contributes to persistence between outbreaks. Transmission is usually from aerosolized environmental water. Person-to-person transmission is exceptionally rare, with a single probable case reported.^{1818 Correia AM, et al. Probable person-to-person transmission of Legionnaires’ disease. 2016.} Disease ranges from subclinical infection to severe fibrinopurulent pneumonia caused by intracellular infection of alveolar macrophages.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Isolation requires buffered charcoal yeast extract (BCYE) agar supplemented with L-cysteine, ferric salt, and alpha-ketoglutarate. Selective supplements such as cefamandole, polymyxin B, and anisomycin, or polymyxin B, anisomycin, and vancomycin, suppress contaminating flora in nonsterile specimens. Weak-acid or heat pretreatment of sputum reduces contamination, but overtreatment also lowers Legionella recovery. Incubate plates for 5 to 14 days in a humid, low-carbon-dioxide atmosphere. Colonies are iridescent and sticky. Failure to grow after subculture to BCYE without cysteine or to blood agar confirms the absolute cysteine requirement.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.} Routine respiratory media do not recover Legionella, so culture must be requested specifically.^{55 Centers for Disease Control and Prevention. Laboratory testing for Legionella.}

Current practice combines a urinary antigen test with culture or a molecular assay of a lower respiratory specimen. Collect both specimens at the same time and before antimicrobials when possible.

Nasopharyngeal specimens are used mainly for viral respiratory diagnosis by rapid molecular methods. The site is also sampled for B. pertussis, Chlamydia trachomatis, C. pneumoniae, and, rarely, Corynebacterium diphtheriae. Anterior-nares specimens are used to screen for MRSA carriage. Endoscopically or surgically obtained sinus specimens are submitted for chronic or complicated sinusitis, which typically involves S. aureus, gram-negative bacilli, streptococci, anaerobes, or fungi.^{2,32 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.3 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Aspirates and washings recover viruses better than swabs, but swabs are collected far more often for convenience. An aspirate is obtained through a small feeding-tube-style catheter attached to a 1-mL syringe or a mucus-trap suction catheter. A wash instills and withdraws 3 to 7 mL of sterile phosphate-buffered saline with a rubber suction bulb. A swab specimen is collected by clearing nasal mucus, passing a small flexible nasopharyngeal swab along the septum to the posterior pharynx, and rotating it against the mucosa. Swabs for molecular testing must have a plastic shaft and a polyester (Dacron), rayon, or nylon tip. Cotton and calcium alginate inhibit amplification, and cotton is toxic to B. pertussis.^{11,311 Centers for Disease Control and Prevention. Best practices for use of polymerase chain reaction for…3 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Place specimens collected for viral testing in the appropriate transport medium and transport them promptly, with brief refrigeration and packing in ice if a short delay cannot be avoided. When a molecular assay will be run, the manufacturer's collection instructions specify the swab, the medium, and the storage conditions.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Pertussis testing

The specimen must come from the posterior nasopharynx, since throat and anterior nares specimens miss the organism, and a flocked swab or an aspirate is preferred because it recovers more bacterial DNA. Polymerase chain reaction (PCR) is faster and more sensitive than culture. Culture is the more specific method and is the only one that yields an isolate for susceptibility testing and molecular epidemiology. Culture sensitivity is highest within the first 2 weeks after cough onset, and PCR remains useful through about 3 to 4 weeks. After roughly 4 weeks of cough, or about 5 days of therapy, a negative result has little value and does not exclude pertussis.^{12,1112 Centers for Disease Control and Prevention. Laboratory testing for pertussis.11 Centers for Disease Control and Prevention. Best practices for use of polymerase chain reaction for…}

Transport handling differs by method. For culture, plate the specimen directly as soon as possible, or place it in half-strength Regan-Lowe transport medium, or in Amies charcoal gel when the receiving laboratory accepts it. Plate within 24 hours, and keep and ship specimens at 2 to 8 °C. For PCR alone, use a dry sterile tube or the performing laboratory's approved medium. Routine liquid transport media are discouraged, because DNA released from a contaminated swab shaft into the liquid produces false-positive results.^{12,1112 Centers for Disease Control and Prevention. Laboratory testing for pertussis.11 Centers for Disease Control and Prevention. Best practices for use of polymerase chain reaction for…}

Plate cultures onto Regan-Lowe or freshly prepared Bordet-Gengou agar, often with an antibiotic such as cephalexin to suppress contaminants, and incubate them at least 7 days at 35 to 37 °C in humid ambient air. B. pertussis colonies are small, smooth, and glistening, described as resembling mercury drops, and do not grow on blood agar. A positive catalase and oxidase reaction with a negative urease presumptively identifies B. pertussis. B. parapertussis grows faster, shows variable growth on Columbia agar, and is oxidase negative with catalase and urease both positive. Direct fluorescent antibody staining of a nasopharyngeal smear gives a rapid result but has appreciable false-positive and false-negative rates. PCR has replaced it.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

IS481 is a multicopy insertion sequence with high analytical sensitivity but is not specific for B. pertussis because B. holmesii and B. bronchiseptica also carry it. IS1001 targets B. parapertussis, and the pertussis toxin promoter improves species discrimination. Multitarget assays are therefore preferred. Limit testing to symptomatic patients. Do not test asymptomatic contacts to guide prophylaxis. A high cycle-threshold value indicates little target DNA and is more likely to reflect contamination, although it may represent true infection. Interpret it with cough duration, therapy, vaccination history, and exposure history.^{1111 Centers for Disease Control and Prevention. Best practices for use of polymerase chain reaction for…}

Other nasopharyngeal targets

A polyester-tipped nasopharyngeal swab can be used for either culture or a direct fluorescent antibody smear for C. trachomatis. For C. pneumoniae, place the swab in transport medium and transport it promptly, with brief refrigeration if needed. Nucleic acid amplification is the recommended detection method.^{9,39 Kumar S, et al. Acute respiratory infection due to Chlamydia pneumoniae. 2007.3 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

For suspected diphtheria, swabbing multiple nasopharyngeal sites, including beneath any visible membrane, raises sensitivity. Send specimens to the laboratory immediately, or place them in semisolid Amies transport medium if a delay is unavoidable. Tinsdale medium, a tellurite-containing differential medium, gives the best yield but is expensive, has a short shelf life, and is not always available. Laboratories without experience in recovering and identifying C. diphtheriae refer these specimens.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

MRSA carriage screening uses a polyester-tipped swab of the anterior nares in a tube transport system, delivered promptly. Detection is by a commercial molecular assay, which returns a result in a few hours, or by culture on MRSA-selective chromogenic agar in 24 to 48 hours. When a molecular assay is used, the swab specified by the manufacturer is required.^{2,32 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.3 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Throat swabbing is contraindicated when epiglottitis is suspected, because of the risk of aspiration and airway obstruction.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Group A streptococcus causes fewer than one-third of pharyngitis cases but is sought to prevent acute rheumatic fever and suppurative complications and to interrupt transmission. Other bacterial targets with a clear treatment benefit are C. diphtheriae and Neisseria gonorrhoeae. Groups C and G streptococci also cause pharyngitis, although the benefit of identifying them specifically is less established. Herpes simplex virus, cytomegalovirus, enteroviruses, and other viruses shed in oral secretions may be recovered without causing the pharyngitis. Testing is not indicated when cough, rhinorrhea, hoarseness, oral ulcers, conjunctivitis, or other findings suggest a viral cause.^{13,1413 Shulman ST, et al. Clinical practice guideline for the diagnosis and management of… 2012.14 Centers for Disease Control and Prevention. Clinical guidance for group A streptococcal pharyngitis.}

For throat collection, depress the tongue and pass the swab between the tonsillar pillars and behind the uvula without touching the buccal mucosa. Swab the posterior pharynx back and forth. Place bacterial culture swabs in modified Stuart transport medium and viral specimens in viral transport medium. N. gonorrhoeae requires a plastic-shafted Dacron, rayon, or nylon swab. Fatty acids in cotton and some calcium alginate lots kill gonococci, and calcium alginate also inhibits amplification.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Detecting group A streptococcus

Culture on sheep or horse blood agar remains a sensitive method and shows complete beta-hemolysis, but it requires overnight incubation. Rapid antigen detection tests give a same-visit result with high specificity, so a positive result needs no confirmatory culture, but their sensitivity is lower. Back up a negative rapid antigen test in a child 3 years or older with culture or a validated molecular test. Adults do not need routine backup culture, because their baseline risk of rheumatic fever is low.^{13,1413 Shulman ST, et al. Clinical practice guideline for the diagnosis and management of… 2012.14 Centers for Disease Control and Prevention. Clinical guidance for group A streptococcal pharyngitis.} A validated nucleic acid amplification test is the most sensitive rapid option, and some platforms are waived under the Clinical Laboratory Improvement Amendments (CLIA) for point-of-care use. When such an assay is the primary test, a negative result generally needs no reflex culture.^{2,142 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.14 Centers for Disease Control and Prevention. Clinical guidance for group A streptococcal pharyngitis.}

Other throat targets

Pharyngeal gonococcal infection is detected by nucleic acid amplification using an assay cleared or validated for that site, which is more sensitive than culture. Culture on a selective medium such as modified Thayer-Martin is still used where an isolate is needed for susceptibility testing. Inoculate the plate as promptly as possible to maximize yield.^{15,315 Workowski KA, et al. Sexually transmitted infections treatment guidelines, 2021. 2021.3 Carroll KC, et al. Manual of Clinical Microbiology. 2023.} For suspected diphtheria, both nasopharyngeal and throat swabs are collected and delivered immediately.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Vincent's angina, an acute necrotizing ulcerative tonsillitis caused by mixed oral fusobacteria and spirochetes, classically Fusobacterium nucleatum and Treponema vincentii, is diagnosed presumptively on a Gram-stained smear of the ulcerated lesion that shows fusiform gram-negative bacilli together with spirochetes. The dense mixed anaerobic flora normally present in the mouth makes culture unhelpful, so the smear is the main laboratory test. Blood cultures are reserved for systemic illness, and F. necrophorum sepsis is associated with Lemierre syndrome (septic thrombophlebitis of the internal jugular vein).^{16,316 Riordan T. Human infection with Fusobacterium necrophorum… 2007.3 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Because throat flora is dense and mixed, read the plate for specific organisms without identifying every colony. Examine it for beta-hemolytic streptococci and, when the request or the colony morphology suggests them, for N. gonorrhoeae, C. diphtheriae, or Arcanobacterium haemolyticum. Report growth of the expected mixed flora, with none of those organisms present in excess, as normal respiratory or oral flora. Identifying every colony type to genus and species adds cost and does not change patient care.^{3,133 Carroll KC, et al. Manual of Clinical Microbiology. 2023.13 Shulman ST, et al. Clinical practice guideline for the diagnosis and management of… 2012.}