10.2 Neisseria gonorrhoeae, Chlamydia trachomatis, GBS, and Mycoplasma

The same II.H outline that covers vaginitis also tests culture and molecular detection of Neisseria gonorrhoeae, Chlamydia trachomatis, Streptococcus agalactiae (group B Streptococcus, GBS), and Mycoplasma spp., plus organism pathogenicity. These organisms are not diagnosed from a routine aerobic vaginal plate. Each has a specific source, a specific method, and—for gonorrhea and chlamydia—a public-health reporting obligation.

Neisseria gonorrhoeae: culture and NAAT

N. gonorrhoeae is a kidney-bean Gram-negative diplococcus. In symptomatic men, intracellular GNDC in neutrophils on a urethral Gram stain is presumptive evidence of gonorrhea. Do not use Gram stain as a stand-alone diagnostic in women: vaginal and endocervical flora include other Gram-negative cocci and coccobacilli that mimic gonococci.

The organism is fastidious and capnophilic. Inoculate modified Thayer-Martin (MTM), Martin-Lewis, or New York City (NYC) agar and incubate at 35–37°C in 3–7% CO2 with humidity. MTM is chocolate agar plus vancomycin (Gram-positive flora), colistin (most Gram-negative rods except pathogenic Neisseria), nystatin (yeast), and trimethoprim (Proteus swarming)—the classic VCNT combination. NYC medium uses similar inhibitors plus amphotericin and can also support genital mycoplasmas. JEMBEC plates generate CO2 with a tablet for transport. Never refrigerate a specimen destined for gonococcal culture; cold kills the organism. If plating will be delayed, use a validated transport (Amies charcoal) and get the specimen onto selective media quickly.

Presumptive identification: growth on selective media, GNDC morphology, and oxidase-positive (and catalase-positive) colonies. Superoxol (30% hydrogen peroxide) produces immediate brisk bubbling with N. gonorrhoeae. CTA carbohydrate utilization: N. gonorrhoeae acidifies glucose only. N. meningitidis acidifies glucose and maltose; N. lactamica acidifies glucose, maltose, and lactose; Moraxella catarrhalis is asaccharolytic. MALDI-TOF MS identifies colonies recovered in culture. NAAT is the preferred screening method on first-void urine, vaginal, endocervical, and other validated sites, including many extragenital specimens. Culture is still required when antimicrobial susceptibility is needed—treatment failure, suspected resistance, sexual-assault workups in some protocols, or a site/test combination that is not NAAT-validated.

Pathogenicity: type IV pili mediate attachment to nonciliated epithelium and undergo antigenic and phase variation. Outer-membrane proteins, especially Opa proteins and the PorB porin, contribute to invasion and immune evasion; Opa expression also phase-varies. Lipooligosaccharide (LOS), not a full LPS O-antigen, can be sialylated. IgA protease cleaves mucosal antibody. Unlike N. meningitidis, N. gonorrhoeae lacks a true polysaccharide capsule. Antigenic variation of pili, Opa, and LOS explains repeat infection and the failure of simple serologic diagnosis.

Chlamydia trachomatis

C. trachomatis is an obligate intracellular pathogen with a biphasic cycle: the elementary body is the infectious extracellular form; the reticulate body replicates inside a cytoplasmic inclusion. It cannot be recovered on cell-free bacteriologic media. Historical diagnosis used McCoy or HeLa cell culture and inclusion staining; those methods are largely reserved for specialized laboratories. NAAT of first-void urine (men—the first 10–20 mL that washes the urethra, not a midstream clean-catch) or a vaginal or endocervical swab (women) is the routine method. Dual gonorrhea/chlamydia NAAT is standard because co-infection is common.

Serovars D–K cause genital infection; L1–L3 cause lymphogranuloma venereum; A–C cause trachoma. Pathogenicity is driven by the major outer-membrane protein (MOMP, ompA) with serovar-level antigenic variation, type III secretion, and intracellular survival. Untreated infection ascends to pelvic inflammatory disease, infertility, and ectopic pregnancy, and it infects neonates (conjunctivitis, pneumonia). C. trachomatis is nationally notifiable.

Streptococcus agalactiae (GBS)

S. agalactiae is a catalase-negative, Gram-positive coccus in chains, usually beta-hemolytic, Lancefield group B. CAMP testing shows arrowhead-enhanced hemolysis with Staphylococcus aureus; hippurate is positive; PYR is negative (enterococci are PYR-positive). Granada or carrot broth produces an orange carotenoid pigment. CDC and ACOG recommend universal prenatal screening at 36 0/7 to 37 6/7 weeks of gestation with a combined lower-vaginal and rectal swab. Direct plating without enrichment misses a substantial fraction of carriers. Inoculate LIM broth (Todd-Hewitt with colistin and nalidixic acid) or carrot broth, incubate overnight, then subculture to blood or chromogenic GBS agar, or perform a validated NAAT from the enrichment broth. Carrot-broth color change is a useful screen but still requires confirmation per laboratory protocol.

A positive screen this pregnancy is an indication for intrapartum antibiotic prophylaxis (IAP), typically intravenous penicillin G or ampicillin during labor. Other IAP indications include GBS bacteriuria during the current pregnancy, a previous infant with invasive GBS disease, and unknown GBS status plus obstetric risk factors (preterm labor, rupture of membranes ≥18 hours, or intrapartum fever). Pathogenicity for the neonate is capsule-mediated (sialylated polysaccharide inhibiting complement) plus beta-hemolysin/cytolysin. Early-onset disease presents as sepsis or pneumonia; late-onset disease presents as bacteremia or meningitis.

Mycoplasma spp. and Ureaplasma

Mycoplasmas lack a cell wall, so they do not Gram-stain, are intrinsically resistant to beta-lactams, and are not seen as the GNDC on the smear. Mycoplasma genitalium is a cause of nongonococcal urethritis, cervicitis, and pelvic inflammatory disease. Clinical diagnosis is by NAAT (FDA-cleared on urine, urethral, vaginal, and endocervical specimens). CDC STI guidelines do not recommend screening asymptomatic people; test persistent or recurrent urethritis or cervicitis and sexual contacts of infected patients. Macrolide resistance is common. M. genitalium uses a flask-shaped tip organelle (MgPa adhesin) and antigenic variation to attach and persist.

Mycoplasma hominis produces fried-egg colonies on A8 or SP4 agar, metabolizes arginine, and can appear as pinpoint colonies on some blood-containing media. It is associated with BV flora, postpartum fever, and occasional pyelonephritis—not with a routine vaginal pathogen culture report. Ureaplasma spp. (U. urealyticum, U. parvum) hydrolyze urea, form tiny T-strain colonies, and are linked to nongonococcal urethritis and some pregnancy complications. NYC medium can support these organisms, which is why NYC appears in both gonorrhea and mycoplasma discussions. Do not treat every genital mycoplasma isolate from a healthy genital tract as disease.

Reportable STIs

N. gonorrhoeae and C. trachomatis infections are nationally notifiable conditions. The laboratory's job is a complete patient identifier, the organism or NAAT result, specimen source, and timely notification to the public-health jurisdiction—not merely an electronic chart result. GBS colonization is not an STI and is not reported as one. M. genitalium is not nationally notifiable. Local lists vary; follow the current state reportable-disease table. Suspected treatment-resistant gonorrhea should be cultured for susceptibility and discussed with public health.

On the exam, a GNDC that is oxidase-positive and glucose-only is gonococcus until proven otherwise; a request to culture chlamydia on blood agar is a distractor; and a prenatal screen collected at 12 weeks without enrichment is the wrong GBS method.