Enteric Bacteriology: Stool Workup and Enterobacterales

Transport and acceptability limits depend on the requested test and collection system, so follow your laboratory's validated, test-specific instructions. The limits below are examples for stool. Each assay's instructions set its own limits.

The preferred specimen is diarrheal stool that takes the shape of its container, collected in a clean, tightly lidded cup and free of urine and barium. Unpreserved stool should reach the laboratory within 2 hours, because pH falls as stool cools and inhibits Shigella in particular. When transport will take longer, place the specimen in Cary-Blair or the transport medium named by the assay manufacturer. It should arrive within 24 hours.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

Reject formed stool for routine enteric culture and for Clostridioides difficile testing, with one documented exception: a formed specimen from a patient whose physician records ileus or possible toxic megacolon. Laboratories also reject stool mixed with urine, a dry swab or a swab carrying no visible feces, a leaking container, frozen raw stool, a specimen in expired or parasitology fixative, and unpreserved stool that arrives more than 2 hours after collection.^{2,42 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.4 McDonald LC, et al. Clinical practice guidelines for Clostridium difficile infection… 2018.}

Serial specimens add little for bacterial pathogens. In adult studies of repeat submissions, the first sample detected 87% to 94% of bacterial pathogens and a second raised detection to about 98%. In children the first specimen detects about 98%. A second specimen is therefore reserved for selected adults with continued clinical suspicion after a negative first test.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

Diagnostic yield also depends on where the diarrhea began. Routine testing for community enteric pathogens other than C. difficile is commonly restricted once a patient has been hospitalized more than 3 days, because organisms acquired in the community are rarely responsible by then. The restriction is counted from the day of admission, so an illness that began before admission remains eligible however long the diarrhea has lasted. Laboratory policy defines the exceptions, which typically cover outbreak investigation and selected high-risk patients such as those with neutropenia or advanced HIV infection.^{2,52 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.5 Bauer TM, et al. Derivation and validation of guidelines for stool cultures for… 2001.}

A rectal swab is less sensitive than stool for adult bacterial culture and is not the preferred diarrheal specimen. In a symptomatic child, a swab with visible feces may substitute when stool cannot be obtained. No enteric NAAT is cleared by the Food and Drug Administration (FDA) for rectal swabs, so a laboratory that accepts that source must validate it. Rectal swabs collected for Neisseria gonorrhoeae and Chlamydia trachomatis are used for proctitis testing, and rectal or perirectal swabs are used for vancomycin-resistant Enterococcus (VRE) surveillance. Each of those uses requires the transport device specified for its assay. NAAT is more sensitive than culture for gonococcal and chlamydial infection. Its specificity depends on the assay, organism, and specimen site, since some gonococcal NAATs cross-react with commensal Neisseria in oropharyngeal specimens, and assays lacking extragenital clearance require in-house validation.^{1-31 Tille PM. Bailey & Scott's Diagnostic Microbiology. 2025.3 Shane AL, et al. 2017 Infectious Diseases Society of America clinical practice… 2017.}

Toxin-mediated food poisoning is handled outside the culture-based workup. Short-incubation illness from preformed Staphylococcus aureus or Bacillus cereus enterotoxin, and the longer-incubation illness caused by Clostridium perfringens, is referred to public health laboratories. Suspected botulism is referred as well, because most clinical laboratories are not equipped for toxin or organism detection in feces and Clostridium botulinum is a select agent.^{1,21 Tille PM. Bailey & Scott's Diagnostic Microbiology. 2025.2 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

Selective and differential media

Selective agents suppress competing flora. A differential indicator converts a metabolic difference into a visible colony color.

The Enterobacterales are facultatively anaerobic, non-spore-forming gram-negative bacilli that ferment glucose with acid production, reduce nitrate to nitrite, and are typically oxidase-negative. Clinically important genera include Citrobacter, Cronobacter, Enterobacter, Escherichia, Hafnia, Klebsiella, Morganella, Proteus, Providencia, Salmonella, Serratia, Shigella, and Yersinia. Plesiomonas shigelloides belongs to the order and is its only oxidase-positive member of clinical importance. Nitrate-negative isolates are rare among the organisms a clinical laboratory reports.^{1,81 Tille PM. Bailey & Scott's Diagnostic Microbiology. 2025.8 Janda JM, et al. The changing face of the family Enterobacteriaceae (order. 2021.}

Endotoxin, the lipopolysaccharide of the outer membrane, drives much of the morbidity of gram-negative infection: fever, hypotension, granulocytosis followed by consumption, thrombocytopenia, disseminated intravascular coagulation, and complement activation that can end in septic shock. Individual organisms add their own virulence factors. The K1 capsule appears in E. coli strains causing neonatal meningitis, K. pneumoniae carries an antiphagocytic capsule, and the Vi capsular antigen of S. Typhi reduces complement deposition and neutrophil phagocytosis. Fimbrial adhesins anchor the organism to the mucosa.^{1,91 Tille PM. Bailey & Scott's Diagnostic Microbiology. 2025.9 Hiyoshi H, et al. The Vi capsular polysaccharide of Salmonella Typhi promotes… 2022.}

Diarrheagenic E. coli are grouped by pathotype: Shiga toxin-producing (STEC), enterotoxigenic (ETEC), enteroinvasive (EIEC), enteropathogenic (EPEC), enteroaggregative (EAEC), and possibly diffusely adherent (DAEC). Enterohemorrhagic E. coli names only the STEC strains that cause human illness, particularly bloody diarrhea and hemolytic uremic syndrome. Hybrid strains carrying virulence factors from more than one pathotype occur. In enterotoxigenic disease, heat-labile toxin activates adenylate cyclase and raises cyclic AMP. Heat-stable toxin activates guanylate cyclase. Both toxins produce secretory watery diarrhea.^{1010 Centers for Disease Control and Prevention. Technical information. 2024.}

Colony morphology narrows the possibilities but does not identify an isolate. Useful clues include swarming Proteus on blood agar, mucoid and lactose-positive Klebsiella, H₂S-producing Salmonella on HE or XLD, indole-positive E. coli, and the occasional red-pigmented S. marcescens. Prodigiosin is often absent at 35 to 37 °C, and most clinical isolates never show it. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry identifies most Enterobacterales within minutes, but commercial databases do not reliably separate Shigella species from E. coli, including enteroinvasive strains. Report such isolates as E. coli/Shigella until motility, lysine decarboxylase, acetate or mucate reactions and serology, or an equivalent public-health algorithm, finish the identification.^{1,111 Tille PM. Bailey & Scott's Diagnostic Microbiology. 2025.11 Association of Public Health Laboratories. Identification Guide. 2025.}

Biochemical differentiation of representative organisms, where + is at least 90% positive, − is at least 90% negative, V is variable, and H₂S is read on triple sugar iron (TSI) agar:

Anaerobes dominate the flora of the gastrointestinal tract and appear routinely in mixed wound and abscess cultures after mucosal or tissue disruption. For this reason, stool is not an acceptable specimen for general anaerobic culture. An anaerobe recovered from feces does not indicate infection. Two targeted exceptions exist. Toxigenic C. difficile culture is a reference procedure, and public health laboratories test feces for C. botulinum and for C. perfringens foodborne enterotoxin. Perform general anaerobic culture on tissue, aspirates, and other properly collected specimens.^{2,222 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.22 Nagy E, et al. How to isolate, identify and determine antimicrobial… 2018.}

For those specimens, obligate anaerobes are defined operationally: they form no colonies on solid media incubated in air or in 10% CO₂ in air. Oxygen tolerance varies within the category. Some strict species are inhibited near 0.5% O₂. Many clinically important moderate anaerobes, including the Bacteroides fragilis group, tolerate about 2% to 8%. Aerotolerant anaerobes may produce scant growth in room air with CO₂ but do not use oxygen for energy. Plate specimens to an enriched non-selective medium such as Brucella, CDC, or Columbia agar with sheep blood, vitamin K, and hemin, along with selective and differential agars including kanamycin-vancomycin laked blood, Bacteroides bile esculin, phenylethyl alcohol, and colistin-nalidixic acid, plus a broth. Incubate at 35 to 37 °C in an oxygen-free atmosphere that typically contains 5% to 10% H₂ and 5% to 10% CO₂ with the balance nitrogen, verified by a chemical or biologic indicator. Keep jars, boxes, and pouches closed for at least 48 hours, because air reaching a culture during logarithmic growth kills slower-growing species. Plates inside an anaerobic chamber may be examined at 24 hours. Keep primary plates 5 to 7 days when slow growers are a concern.^{2222 Nagy E, et al. How to isolate, identify and determine antimicrobial… 2018.}

C. difficile testing starts with diagnostic stewardship, because asymptomatic carriage is common and no single assay distinguishes carriage from disease. Testing is limited to unexplained new-onset diarrhea, meaning at least three unformed stools in 24 hours, from a patient who has not received a laxative in the previous 48 hours. Formed stool is rejected unless the physician documents ileus or possible toxic megacolon. Infants 12 months of age or younger are not tested, and children between 1 and 2 years are not tested routinely until other infectious and noninfectious causes have been excluded. Where a laboratory has no preagreed submission criteria, use a multistep algorithm that includes a toxin assay: GDH with toxin, GDH with toxin arbitrated by NAAT, or NAAT with toxin. Where such criteria exist, NAAT alone or a multistep algorithm is acceptable. GDH is produced by toxigenic and non-toxigenic strains alike, so a GDH-positive, toxin-negative result is inconclusive, and a NAAT-positive, toxin-negative result often reflects carriage. Toxigenic culture and cell-culture cytotoxicity neutralization remain reference methods. Enterotoxin TcdA and cytotoxin TcdB damage the colonic epithelium and produce antibiotic-associated diarrhea and pseudomembranous colitis. The 2021 Infectious Diseases Society of America (IDSA)/Society for Healthcare Epidemiology of America (SHEA) focused update revised treatment and left this diagnostic approach unchanged.^{2,4,242 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.4 McDonald LC, et al. Clinical practice guidelines for Clostridium difficile infection… 2018.24 Johnson S, et al. Clinical practice guideline by the Infectious Diseases Society… 2021.}

C. perfringens is common fecal flora, so routine enteric culture does not look for it, and public-health enterotoxin testing confirms foodborne illness. Recovered from tissue or an aspirate in a compatible clinical setting, it forms large gray colonies with a double zone of beta-hemolysis, and the cells are boxcar-shaped gram-positive rods that are usually nonmotile, lecithinase-positive, lipase-negative, and reverse CAMP-positive (synergistic arrowhead hemolysis with a group B Streptococcus streak). Isolation from a wound without clinical myonecrosis does not diagnose gas gangrene.^{2,222 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.22 Nagy E, et al. How to isolate, identify and determine antimicrobial… 2018.}

Anaerobic susceptibility testing is not performed on stool isolates. It is considered when a clinically significant anaerobe comes from a normally sterile site, grows in pure culture, follows a treatment failure, or will be treated for a prolonged course, and it matters most for organisms with unpredictable resistance, especially Bacteroides and Parabacteroides, along with selected Prevotella, Fusobacterium, and Clostridium isolates. The CLSI reference method is agar dilution on supplemented Brucella blood agar. Broth microdilution in supplemented Brucella broth with hemin, vitamin K₁, and 5% lysed horse blood is an alternative for Bacteroides and Parabacteroides with selected agents. Gradient minimum inhibitory concentration tests are widely used for individual isolates when validated against the reference method. CLSI does not provide a routine anaerobic disk-diffusion method. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) publishes a separate standardized disk-diffusion method for selected rapidly growing anaerobes. Its media, breakpoints, and categories are not interchangeable with the CLSI methods taught here.^{2626 European Committee on Antimicrobial… Disk diffusion and quality control.} Under CLSI, breakpoints come from the current M100 anaerobic tables. Routine susceptibility testing of C. difficile from stool is not indicated outside public-health surveillance or an extraintestinal isolate.^{13,2513 CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 2026.25 CLSI. Methods for Antimicrobial Susceptibility Testing of Anaerobic… 2018.} At the bench, check each stool request against the specimen, the collection day, and the organisms the requested method detects.