Wound, Tissue, and Genital Tract Bacteriology

Surgically obtained tissue recovers more organisms than a swab with limited absorptive capacity. Mince the tissue and, for routine bacterial culture, homogenize it to a suspension before inoculating all necessary media. When a mold infection is suspected, particularly from the Mucorales, mince or dice the tissue and do not grind it, because homogenization destroys fragile hyphae and lowers both culture and direct-microscopy yield. The surgeon, pathologist, and microbiology laboratory divide the material together, so that histopathology and culture each receive what they need.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

Report the wound or tissue Gram stain with quantitated neutrophils and squamous epithelial cells. Many squamous epithelial cells indicate surface contamination. Abundant neutrophils support active infection. Note intracellular organisms specifically, because they support infection by a true pathogen. Reporting "no organisms seen" does not exclude infection, because a direct Gram smear detects roughly 10⁵ organisms per milliliter, about the concentration at which one organism appears per oil-immersion field.^{11 Tille PM. Bailey & Scott's Diagnostic Microbiology. 2025.},^{1212 Biswas D, et al. Clinical profile and spectrum of bacteriuria in patients with… 2022.} Certain morphologies from a normally sterile site require immediate provider notification: gram-positive cocci in clusters suggesting Staphylococcus aureus in a deep abscess, large gram-positive rods suggesting Clostridium in gas gangrene, and gram-negative diplococci in a sterile-site aspirate.^{1,21 Tille PM. Bailey & Scott's Diagnostic Microbiology. 2025.2 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

Organism significance depends on the specimen source. Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), and beta-hemolytic streptococci, especially Streptococcus pyogenes, are important potential pathogens in any wound and receive a full workup when recovered. Whether an isolate represents infection depends on specimen depth and the clinical evidence of infection, because a surface culture from a colonized chronic ulcer can grow either organism without invasion. Pseudomonas aeruginosa and the Enterobacterales are significant from deep tissue but common colonizers of superficial chronic wounds. Anaerobes are expected in mixed infection after mucosal or tissue disruption, particularly in bite wounds and deep abscesses. Coagulase-negative staphylococci and Corynebacterium are usually contaminants from skin but become significant in prosthetic-joint infection or when recovered repeatedly from a normally sterile site. Cutibacterium acnes requires extended incubation, up to 14 days, for recovery from prosthetic-shoulder and prosthetic-joint specimens. Report a polymicrobial superficial culture as mixed flora, with a note that full identification and susceptibility testing of every isolate is not indicated. Deep or normally sterile sources receive a complete workup.^{1,21 Tille PM. Bailey & Scott's Diagnostic Microbiology. 2025.2 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

Necrotizing fasciitis and gas gangrene are surgical emergencies, and the laboratory communicates its findings immediately. A tissue or aspirate Gram stain showing gram-positive cocci in chains suggests S. pyogenes necrotizing fasciitis. Large boxcar gram-positive rods without neutrophils suggest clostridial myonecrosis. Aeromonas and Vibrio vulnificus cause necrotizing infection after water exposure, particularly in liver disease or immunocompromise. Clostridium perfringens grows rapidly, shows double-zone hemolysis on blood agar, and is positive in the reverse CAMP (Christie-Atkins-Munch-Petersen) test. Collect blood cultures in parallel.^{1,21 Tille PM. Bailey & Scott's Diagnostic Microbiology. 2025.2 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

Nocardia morphology, identification, susceptibility testing, and pathogenicity are covered in mycobacteriology. Related aerobic actinomycetes include Rhodococcus equi (opportunistic granulomatous pneumonia, typically in severe immunosuppression; reclassified as Prescottella equi in 2022, though most US laboratories still report it as Rhodococcus equi), Gordonia and Tsukamurella (pathogenic mainly with a foreign body or underlying chronic infection), and Actinomadura and Streptomyces somaliensis (agents of actinomycotic mycetoma, chiefly in barefoot populations of tropical and subtropical regions). Tropheryma whipplei, the agent of Whipple disease, is not recovered by routine bacterial culture. Diagnosis is made from periodic acid-Schiff-positive foamy macrophages in duodenal biopsy, T. whipplei real-time PCR on tissue, blood, synovial fluid, CSF, or valve, and immunohistochemistry on paraffin-embedded tissue.

Actinomyces species are filamentous, gram-positive, fermentative anaerobes that colonize the mouth, tonsillar crypts, and female genital tract. Actinomycosis follows disruption of mucosa, producing a draining sinus or a hard mass in the cervicofacial region, thorax, abdomen, or pelvis, classically with an intrauterine device in place for pelvic disease. Collect tissue or aspirate. A swab of a draining sinus recovers the accompanying mixed flora and may miss the organism. Crush sulfur granules, visible macroscopically as yellow grains about 1 mm across, between slides and Gram-stain them to show branching, filamentous, gram-positive rods radiating from a center (Splendore-Hoeppli material surrounds true granules). Anaerobic culture requires enriched media and 5 to 7 days of incubation, with cultures kept for up to 2 to 3 weeks. Actinomyces is distinguished from Nocardia by modified acid-fast staining (negative versus positive) and anaerobic versus aerobic growth.^{11 Tille PM. Bailey & Scott's Diagnostic Microbiology. 2025.}

Bacterial vaginosis (BV) is a polymicrobial vaginal dysbiosis in which hydrogen-peroxide-producing Lactobacillus species are replaced by high concentrations of Gardnerella species, Prevotella, Mobiluncus, Fannyhessea vaginae (formerly Atopobium vaginae), and uncultivated taxa including BVAB2 and Megasphaera type 1, growing in a polymicrobial biofilm on vaginal epithelial cells. BV is associated with preterm birth, pelvic inflammatory disease (PID), endometritis, and increased acquisition of other sexually transmitted infections. Vulvovaginitis is more often caused by Candida species or Trichomonas vaginalis. BV is the most common cause of vaginal discharge, and treatment is directed at symptomatic disease.^{3,23 Workowski KA, et al. Sexually transmitted infections treatment guidelines, 2021. 2021.2 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

Gram stain with a standardized scoring system (Nugent score) is the reference laboratory method for BV. At 1,000× oil immersion, representative fields are scored for three morphotype categories: large gram-positive rods (Lactobacillus morphotypes) scored 0 to 4 inversely, so abundant Lactobacillus lowers the score; small gram-variable rods and coccobacilli (Gardnerella and Bacteroides morphotypes) scored 0 to 4; and curved gram-variable rods (Mobiluncus morphotypes) scored 0 to 2. Those two scores rise with the abundance of the morphotype. The total ranges from 0 to 10: 0-3 is normal, 4-6 is intermediate, and 7-10 is consistent with BV. Morphotype labels are not species identifications. Intermediate flora is a transitional pattern. In a cohort of 7,918 pregnant women, 32% of those with intermediate flora had acquired BV and 30% had shifted to normal flora at follow-up.^{44 Hillier SL, et al. Characteristics of three vaginal flora patterns assessed by Gram… 1992.} The method is high-complexity and training dependent.^{3,23 Workowski KA, et al. Sexually transmitted infections treatment guidelines, 2021. 2021.2 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

The Amsel clinical criteria require at least 3 of 4 findings: homogeneous thin discharge coating the vaginal walls; clue cells (epithelial cells stippled with adherent bacteria) on wet mount; vaginal pH greater than 4.5; and a fishy amine odor after addition of 10% KOH (whiff test). Amsel sensitivity is 37% to 70% against Nugent, with specificity 94% to 99%, so a negative Amsel assessment does not exclude BV.^{33 Workowski KA, et al. Sexually transmitted infections treatment guidelines, 2021. 2021.}

Molecular panels cleared by the Food and Drug Administration (FDA) now provide the most sensitive class of test among symptomatic women, outperforming clinician assessment, on-site microscopy, and Nugent interpretation, particularly for mixed infections. Performance is analyte- and device-specific. The BD MAX Vaginal Panel targets a BV algorithm (L. crispatus, L. jensenii, Gardnerella vaginalis, F. vaginae, BVAB2, Megasphaera type 1) plus Candida species groups and T. vaginalis. The Cepheid Xpert Xpress MVP targets a BV algorithm (Atopobium species, BVAB2, Megasphaera-1) plus Candida and T. vaginalis. The Hologic Aptima BV and Aptima CV/TV assays cover the same spectrum in two separate tests. A positive molecular result detects target nucleic acid and does not establish that the organism is causing disease, because Candida and BV-associated organisms also colonize asymptomatic women. The panels are intended for symptomatic patients. They are not intended for routine screening.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

Culture of Gardnerella is not a diagnostic test for BV, because the organism is recoverable from approximately half of asymptomatic women. Redirect a request for BV culture to Nugent-scored Gram stain, a complete Amsel assessment, or a validated multi-organism NAAT.^{22 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

Classic Gardnerella vaginalis is a small pleomorphic gram-variable coccobacillus, catalase-negative and oxidase-negative, that grows on enriched media at 35 to 37 °C in 5% CO₂ over 24 to 48 hours. The classic presumptive reaction is diffuse beta-hemolysis on human-blood bilayer Tween agar. Sheep blood gives poor growth and little or no visible hemolysis. Beta-lactamase production is not an established identification or reporting characteristic, and no CLSI susceptibility method or breakpoint exists for the genus, so routine antimicrobial susceptibility testing (AST) of a vaginal isolate is not indicated.^{11 Tille PM. Bailey & Scott's Diagnostic Microbiology. 2025.}

Vulvovaginal candidiasis and trichomoniasis

Vulvovaginal candidiasis is supported by budding yeast, hyphae, or pseudohyphae on saline or 10% KOH wet preparation. Vaginal pH is usually below 4.5. Microscopy sensitivity is roughly 40% to 80%, so a negative wet mount does not exclude the diagnosis. Culture remains the reference standard. Obtain it for recurrent, severe, or refractory disease to identify non-albicans yeast. Nakaseomyces glabratus (formerly Candida glabrata) does not form hyphae or pseudohyphae and is easily missed microscopically. Detection alone does not prove disease, because 10% to 20% of women harbor vaginal yeast without symptoms.^{3,23 Workowski KA, et al. Sexually transmitted infections treatment guidelines, 2021. 2021.2 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

T. vaginalis trophozoites are pyriform, 7 to 30 micrometers, with four anterior flagella and one posterior flagellum along an undulating membrane about half the body length. There is no cyst form. A fresh 0.9% saline wet mount is examined immediately for jerky motility. Sensitivity is 44% to 68% versus culture and falls to about 20% within 1 hour. NAAT is the most sensitive test and is now standard. Culture (InPouch TV, examined daily over 5 of 7 days) remains available when a viable isolate is needed for suspected nitroimidazole resistance. The OSOM Trichomonas Rapid Test is an immunochromatographic antigen assay with sensitivity 82% to 95% in women. It is not adequate in men. Wait at least 3 weeks after treatment before repeating a NAAT, because residual nucleic acid may be detected. CDC recommends annual Trichomonas screening for all women with HIV. Morphology details of the trophozoite are covered in the parasitology lessons.^{3,23 Workowski KA, et al. Sexually transmitted infections treatment guidelines, 2021. 2021.2 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

For NAAT screening, the Centers for Disease Control and Prevention (CDC) specifies the optimal urogenital specimens: a vaginal swab for women, which may be self-collected in a clinical setting, and first-catch urine for men. An endocervical swab is an acceptable alternative in women. Swab material, insertion depth, rotation, and the interval since last void are assay-specific and follow each manufacturer's instructions. Several FDA-cleared assays detect N. gonorrhoeae and C. trachomatis simultaneously, and several are also cleared for rectal and pharyngeal swabs, enabling site-based screening in men who have sex with men. A laboratory using an assay at a site or specimen type outside its cleared intended use establishes and documents its own performance specifications under the Clinical Laboratory Improvement Amendments (CLIA; 42 CFR 493.1253(b)(2)). That is a separate obligation from verifying an unmodified cleared test.^{5,25 Workowski KA, et al. Sexually transmitted infections treatment guidelines, 2021. 2021.2 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

NAAT is the recommended test because its sensitivity exceeds that of culture. It is not universally more specific, and some gonococcal NAATs cross-react with commensal Neisseria in oropharyngeal specimens, where the assay's site-specific performance must be checked. Culture is retained to recover an isolate in suspected treatment failure, disseminated infection, conjunctivitis, medicolegal cases, and public-health surveillance.^{5,25 Workowski KA, et al. Sexually transmitted infections treatment guidelines, 2021. 2021.2 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

For gonococcal culture, an endocervical or urethral swab is collected after removing surface mucus, using a rayon, Dacron, or calcium alginate tip on a plastic or wire shaft. Cotton tips and wooden shafts can inhibit the organism. Direct bedside inoculation onto modified Thayer-Martin or Martin-Lewis medium in a CO₂-generating system such as JEMBEC or Gono-Pak is optimal. Sterile-site specimens (blood, synovial fluid, cerebrospinal fluid) are also plated to nonselective chocolate agar, because the vancomycin in selective media inhibits some gonococcal strains. Incubate at 35 to 36.5 °C in approximately 5% CO₂ with humidity and examine at 24 and 48 hours. The organism is fragile. It survives in swab transport media at room temperature for no more than about 1 hour. Never refrigerate it in transit.^{5,25 Workowski KA, et al. Sexually transmitted infections treatment guidelines, 2021. 2021.2 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

Gonococcal identification and susceptibility testing

A urethral Gram stain showing intracellular gram-negative diplococci within neutrophils is presumptively diagnostic in symptomatic men, with sensitivity greater than 95% and specificity greater than 99%. It is inadequate for asymptomatic men and is not recommended for cervical, rectal, or pharyngeal specimens. Colonies are small, gray to pink-brown, oxidase-positive, and do not grow on MacConkey agar. Gram-negative diplococci with a positive oxidase test from selective genital media remain only a presumptive identification, because N. meningitidis, N. lactamica, Kingella denitrificans, and Moraxella catarrhalis share this profile. A strong superoxol reaction (30% hydrogen peroxide) supports the identification. Carbohydrate utilization or a validated commercial or molecular method, including MALDI-TOF, confirms the species. N. meningitidis can also cause urethritis and is indistinguishable from N. gonorrhoeae on Gram stain.

Susceptibility testing uses GC agar base with 1% defined growth supplement, since the organism grows unreliably on Mueller-Hinton agar, incubated at 35 to 36.5 °C in 5% CO₂ for 20 to 24 hours. CLSI M100 (36th edition, 2026) now provides intermediate and resistant categories for ceftriaxone (S less than or equal to 0.12, I 0.25, R greater than or equal to 0.5 micrograms per milliliter) and cefixime (S less than or equal to 0.06, I 0.12, R greater than or equal to 0.25), replacing the earlier susceptible-only interpretive criteria. Disk-diffusion breakpoints for these two agents were removed pending review. Agar dilution is the reference method. Preserve isolates from suspected treatment failure and refer them to the public health laboratory, and report suspected cephalosporin treatment failure to public health within 24 hours.^{6,56 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. 2026.5 Workowski KA, et al. Sexually transmitted infections treatment guidelines, 2021. 2021.}

Gonorrhea is nationally notifiable. Chlamydia is also notifiable in every state. The laboratory reports to the local health jurisdiction according to state law.^{55 Workowski KA, et al. Sexually transmitted infections treatment guidelines, 2021. 2021.}

Treponema pallidum subsp. pallidum causes venereal syphilis, subsp. pertenue causes yaws, and subsp. endemicum causes endemic syphilis (bejel). The related species T. carateum causes pinta. Pathogenic treponemes cannot be cultured on artificial media, so diagnosis depends on direct visualization early and serology thereafter.

Chlamydiae are obligate intracellular bacteria that depend on the host cell for replication. They have been called energy parasites because reticulate bodies primarily scavenge host ATP through ATP/ADP translocases. Elementary bodies generate their own ATP, largely from imported glucose-6-phosphate. Two morphologic forms alternate through the developmental cycle: the infectious, non-dividing but metabolically active elementary body and the intracellular, replicating reticulate body. Under stress such as nutrient deprivation or cell-wall-active antibiotics, reticulate bodies enlarge into non-dividing aberrant bodies, a reversible state called persistence that explains chronic and recurrent disease. Taxonomically, all species have been returned to the single genus Chlamydia after the former Chlamydophila split (1999) was reversed (2015). At least six species have caused documented human infection: C. trachomatis, C. pneumoniae, and the zoonotic species C. psittaci, C. abortus, C. caviae, and C. felis.^{77 Marti H, et al. Zoonotic and other veterinary chlamydiae. 2024.}

C. trachomatis serovars sort into three disease groups: A through C cause trachoma, D through K cause urogenital infection, and L1 through L3 cause lymphogranuloma venereum.

Ureaplasma species and Mycoplasma hominis may colonize the vagina and cervix. Colonization does not indicate disease, but their presence in placental membranes or amniotic fluid is associated with chorioamnionitis, preterm birth, and neonatal pneumonia or sepsis. M. genitalium is a cause of nongonococcal, nonchlamydial urethritis in men and cervicitis in women.

Universal antepartum screening for Streptococcus agalactiae (group B Streptococcus, GBS) colonization is performed at 36 0/7 to 37 6/7 weeks of gestation. A screen is not collected when intrapartum prophylaxis is already indicated because GBS bacteriuria was found in the current pregnancy at any concentration, or because a previous infant had invasive GBS disease. A negative culture is considered valid for 5 weeks. If delivery has not occurred by 41 0/7 weeks, repeating a previously negative screen is reasonable. The specimen is one swab, preferably flocked, that samples the lower vagina near the introitus and then the rectum through the anal sphincter, without a speculum. Two separate swabs are also acceptable, and self-collection after instruction can produce comparable yield. Vaginal sampling alone under-detects colonization. Place the swab in Stuart or Amies transport medium and deliver it within 24 hours. Refrigerate an Amies specimen at 4 to 8 °C if transport is delayed.^{9,109 American College of Obstetricians and… Prevention of group B streptococcal early-onset disease in… 2020.10 Filkins L, et al. Guidelines for the detection and identification of group B… 2021.}

Inoculate the swab into a selective enrichment broth and incubate it 18 to 24 hours at 35 to 37 °C in ambient air or 5% CO₂ before subculture. Lim broth is Todd-Hewitt broth with colistin 10 micrograms per milliliter and nalidixic acid 15 micrograms per milliliter. Trans-Vag broth is a distinct formulation, Todd-Hewitt broth with gentamicin 8 micrograms per milliliter and nalidixic acid 15 micrograms per milliliter. Enrichment roughly doubles recovery compared with direct plating, and ASM 2021 guidance considers direct plating alone unacceptable for the antepartum screen. Abundant Enterococcus faecalis can nevertheless suppress GBS in broth, so direct agar inoculation is an adjunct. Subculture is to sheep blood agar, CNA agar, or a validated chromogenic medium.^{1010 Filkins L, et al. Guidelines for the detection and identification of group B… 2021.}

After enrichment, candidate colonies are usually larger than 0.5 mm at 24 hours, gray to white and translucent, with a narrow zone of beta-hemolysis or no hemolysis. Approximately 5% to 6% of screening isolates are nonhemolytic, so examine plates at 24 hours and again at 48 hours. Pigment-based media such as Granada miss nonhemolytic strains. GBS appears as gram-positive cocci in chains on Gram stain and is catalase-negative. CAMP and hippurate hydrolysis are traditional presumptive tests. The CAMP factor (Cfb) is itself nonhemolytic and synergizes with the beta-lysin of a beta-hemolytic S. aureus strain to produce the arrowhead zone. Current identification uses MALDI-TOF MS or a group B latex method. Latex reagents cross-react with S. pseudoporcinus and S. halichoeri. A PYR test resolves the cross-reaction, because S. agalactiae is PYR-negative. Up to 31% of target-colored colonies on chromogenic media are not GBS, so candidate colonies require confirmation.^{1010 Filkins L, et al. Guidelines for the detection and identification of group B… 2021.}

NAAT on the enriched broth is an acceptable alternative, but direct-specimen NAAT is not recommended for antepartum screening because reported false-negative rates range from 6.3% to 22%.^{1010 Filkins L, et al. Guidelines for the detection and identification of group B… 2021.} The FDA-cleared Cepheid Xpert Xpress GBS assay (510(k) K222638, cleared September 27, 2023) is labeled for direct vaginal/rectal swab testing at intrapartum in term patients whose antepartum screening result is unknown or unavailable and who have no additional risk factors warranting empiric prophylaxis. Its negative result is presumptive and cannot exclude colonization, and it yields no isolate, so culture is still required when susceptibility testing is needed.^{15,1615 U.S. Food and Drug Administration. 510(k) K222638. 2023.16 Cepheid. Xpert Xpress GBS instructions for use, 303-1853 Rev B.}

A positive screen documents maternal colonization and generally indicates intrapartum prophylaxis to prevent early-onset neonatal GBS disease, except with prelabor cesarean delivery and intact membranes. Screening and prophylaxis do not prevent late-onset disease. For a patient with high-risk penicillin allergy (immediate anaphylaxis or a severe rare delayed non-IgE reaction), the isolate is tested for clindamycin and erythromycin susceptibility under current CLSI M100. Erythromycin results are the screen for inducible clindamycin resistance, and when erythromycin is resistant with clindamycin susceptible, an inducible-resistance test is performed and clindamycin is reported resistant if the D-zone is present. Report the clindamycin result. Erythromycin is tested only as the screen for inducible resistance and is not reported for prophylaxis. Routine penicillin or cefazolin AST is not required, because GBS remains predictably susceptible to both. The D-test method is covered with antimicrobial susceptibility testing.^{9,11,109 American College of Obstetricians and… Prevention of group B streptococcal early-onset disease in… 2020.11 Rosa-Fraile M, et al. Reliable detection of group B Streptococcus in the clinical… 2017.10 Filkins L, et al. Guidelines for the detection and identification of group B… 2021.} At the bench, check the specimen source before you interpret an isolate, because the same organism can be a pathogen in deep tissue and a colonizer on a surface swab.