Gram-Negative and Fastidious Bloodstream Isolates

Gram-negative cocci: Neisseria

Characteristics. Neisseria species are nonmotile, catalase- and oxidase-positive gram-negative cocci, typically paired with flattened adjacent sides that give a kidney-bean or coffee-bean appearance. Young cultures may also show tetrads. Many strains require blood, serum, cholesterol, or oleic acid supplementation to counteract inhibitory fatty acids in the medium, and N. gonorrhoeae and N. meningitidis generally require added CO₂ for primary isolation, a requirement that varies by strain and often fades on subculture.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Clinical significance. N. meningitidis colonizes the upper respiratory mucosa. Most carriers develop group-specific bactericidal antibody within 7 to 10 days and remain well. A minority progress to meningococcemia and meningitis, sometimes with pleuritis, pericarditis, or arthritis. The organism's lipopolysaccharide endotoxin activates the clotting cascade, and the resulting microvascular fibrin deposition, hemorrhage, and shock produce the petechial-to-ecchymotic rash of meningococcemia. A blood or cerebrospinal-fluid recovery of N. meningitidis is a critical result. Call it under your laboratory's urgent-notification procedure.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

N. gonorrhoeae is considered pathogenic in every specimen, with or without symptoms. Urethral infection in men is usually symptomatic. Endocervical infection in women is commonly asymptomatic until ascending infection has produced pelvic inflammatory disease, with tubal scarring that predisposes to infertility or ectopic pregnancy.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.} Antigenically variable type IV pili mediate adherence and are a principal gonococcal virulence factor.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.} Genital-tract collection and the combined gonorrhea and chlamydia molecular workflow are taught with genital-tract bacteriology.

Specimen handling and media. Pathogenic Neisseria are damaged by drying, temperature extremes, and delay, so inoculate material directly to warmed media whenever possible and incubate it promptly at 35 to 36.5 °C in about 5% CO₂, supplied by a CO₂ incubator, a candle-extinction jar, or a CO₂-generating tablet system. Incubate media bound for a reference laboratory overnight first, so growth is established before transport.^{44 Papp JR, et al. Recommendations for the laboratory-based detection of Chlamydia… 2014.} Selective chocolate-based media for N. gonorrhoeae combine an inhibitor of gram-positive organisms (vancomycin in modified Thayer-Martin and Martin-Lewis media, lincomycin in some New York City-type formulations), colistin against other gram-negative organisms, an antifungal agent (nystatin, anisomycin, or amphotericin B), and trimethoprim to suppress swarming Proteus. Some gonococcal strains are inhibited by the vancomycin in selective media, so inoculate a nonselective chocolate agar plate in parallel when the specimen comes from a sterile site or recovery is critical.^{44 Papp JR, et al. Recommendations for the laboratory-based detection of Chlamydia… 2014.}

Identification. A typical colony on selective medium with a positive oxidase reaction and gram-negative diplococci on smear supports a presumptive identification of N. gonorrhoeae, which the laboratory can report as a preliminary result. Confirmatory testing by a method with a different principle is required before the isolate is reported as N. gonorrhoeae, because other Neisseria species grow on the same media. In suspected sexual assault or abuse, the isolate must be confirmed by two tests of different principles and preserved for possible retesting.^{44 Papp JR, et al. Recommendations for the laboratory-based detection of Chlamydia… 2014.}

Classic confirmation uses acid production from carbohydrates in cystine trypticase agar (CTA) base medium (glucose, maltose, lactose, sucrose, and fructose, with a carbohydrate-free control), read at 24-hour intervals for up to 72 hours. Commercial enzyme-substrate and rapid carbohydrate-utilization kits give the same identifications in 1 to 4 hours.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Gram-negative nonfermenting bacilli

Nonfermenters are gram-negative bacilli that fail to ferment glucose or other carbohydrates, using oxidative pathways or none at all. They account for roughly 15% of gram-negative bacilli recovered in clinical laboratories and live in soil, water, and moist hospital environments, appearing on patients mainly as health-care-associated colonizers. Pseudomonas aeruginosa is the most frequently recovered clinically significant nonfermenter. Most of the remainder are Acinetobacter species, Stenotrophomonas maltophilia, and the Burkholderia cepacia complex.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Pseudomonas aeruginosa. An aerobic, catalase- and oxidase-positive gram-negative bacillus with oxidative metabolism, motile by polar flagella. It thrives in nearly any moist reservoir, including medical equipment, disinfectant solutions, and soaps, and colonization increases with length of hospitalization. Serious infection follows burns, surgical wounds, urinary instrumentation, hematologic and immunologic disease, cystic fibrosis, and mechanical ventilation. Its virulence factors include a slime polysaccharide, endotoxin, complement-inactivating proteases, and exotoxin A, which ADP-ribosylates elongation factor 2, shutting down host protein synthesis and producing local tissue necrosis.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Presumptive recognition is based on a grape-like or corn-tortilla odor, rough or ground-glass colonies on blood agar, and pigment: blue-green pyocyanin, yellow-green fluorescent pyoverdin, or both, often with a metallic colony sheen. A positive oxidase reaction, growth at 42 °C, and an alkaline slant with no change in the butt on triple sugar iron agar complete the presumptive identification.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.} Clinically significant isolates receive susceptibility testing. P. aeruginosa is intrinsically resistant to amoxicillin-clavulanate, ceftriaxone, trimethoprim-sulfamethoxazole, the tetracyclines, ertapenem, and tigecycline, so suppress an apparently susceptible result for one of these agents under the intrinsic-resistance rules of your laboratory's adopted Clinical and Laboratory Standards Institute (CLSI) M100 edition.^{66 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. 2026.}

Acinetobacter. Short rod-shaped to coccoid, nonmotile, oxidase-negative, strictly aerobic organisms that decolorize poorly on Gram stain and can be mistaken for gram-positive cocci. They inhabit soil and water and occasionally healthy skin, and they cause health-care-associated bacteremia, pneumonia, bacteriuria, and wound infection. A negative oxidase reaction, nonmotility, and failure to reduce nitrate separate the genus from Pseudomonas. Purplish colonies on MacConkey agar from crystal violet uptake, without lactose fermentation, are characteristic.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.} Carbapenem-resistant A. baumannii (CRAB) is a major treatment problem. Current Infectious Diseases Society of America (IDSA) treatment guidance depends on the species and the carbapenem result, so accurate species-level identification and carbapenem results directly affect treatment.^{77 Tamma PD, et al. IDSA 2026 Guidance on the Treatment of Antimicrobial-Resistant… 2026.}

Burkholderia cepacia complex. Aerobic, non-spore-forming, catalase-positive gram-negative rods, motile by polar flagella. B. pseudomallei and nonmotile B. mallei form the separate B. pseudomallei complex. Both are Tier 1 select agents, and an isolate suggesting either is handled under the rules in Sentinel Recognition and Referral of Select Agents and is never worked up as a routine nonfermenter. The complex, at least 20 closely related species, is an important health-care-associated pathogen in cystic fibrosis and chronic granulomatous disease, where chronic infection accelerates decline. Complex members are phenotypically nearly indistinguishable from one another and from B. gladioli, Ralstonia, Cupriavidus, and Pandoraea, so species-level confirmation is molecular or by validated mass spectrometry.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.} Isolates of the complex are characteristically resistant to the aminoglycosides and the polymyxins. Most Burkholderia carry a modified lipopolysaccharide that confers intrinsic polymyxin resistance, and a restrictive outer membrane limits aminoglycoside entry. CLSI no longer lists intrinsic-resistance entries for the complex, because the previous entries were found unreliable. Disk diffusion breakpoints were removed in 2024 and minimum inhibitory concentration (MIC) breakpoints in 2025, so the current M100 publishes no breakpoints for the complex and recommends against routine susceptibility testing. When testing is performed on request, frozen reference broth microdilution is the only method CLSI calls reproducible for the complex. Report the result with the caution that correlation of MIC values with clinical outcome is not known.^{66 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. 2026.},^{2323 Rhodes KA, et al. Antibiotic resistance in Burkholderia species. 2016.},^{2424 Huse H, et al. A clinical case of Burkholderia cepacia complex and changes to… 2025.}

Stenotrophomonas maltophilia. An opportunistic health-care-associated pathogen favored by mechanical ventilation, broad-spectrum antibiotic exposure, catheters, and neutropenia, and a respiratory colonizer in cystic fibrosis. Recovery alone does not establish infection. Distinguishing bench features are a negative oxidase reaction paired with positive DNase activity, lavender-green colonies with an ammonia-like odor on blood and MacConkey agar, motility, and oxidative utilization of glucose and maltose.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.} The organism is intrinsically resistant to the carbapenems, so carbapenem therapy aimed at a presumed Pseudomonas or Acinetobacter infection leaves S. maltophilia untreated. Current IDSA guidance treats ceftazidime as inactive regardless of the in vitro result, so follow the laboratory's reporting rules for this organism.^{6,76 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. 2026.7 Tamma PD, et al. IDSA 2026 Guidance on the Treatment of Antimicrobial-Resistant… 2026.}

HACEK organisms and Haemophilus identification

The HACEK acronym groups fastidious, slow-growing gram-negative organisms that share oral and upper-respiratory carriage and a predilection for subacute bacterial endocarditis: Haemophilus species (particularly H. parainfluenzae), Aggregatibacter species (including A. aphrophilus, formerly Haemophilus aphrophilus, and A. actinomycetemcomitans, formerly Actinobacillus actinomycetemcomitans), Cardiobacterium hominis, Eikenella corrodens, and Kingella species. Historically these organisms were considered hard to recover, and laboratories extended incubation for suspected HACEK endocarditis. With current continuous-monitoring systems that practice is obsolete. HACEK organisms are reliably detected within the standard 5-day incubation, and extended incubation with terminal blind subculture does not improve recovery. Species-level identification, usually by mass spectrometry, can take several days after the bottle signals.^{11 Liesman RM, et al. Laboratory diagnosis of infective endocarditis. 2017.}

Haemophilus growth factors. Haemophilus species are oxidase-positive, facultatively anaerobic, small pleomorphic gram-negative rods or coccobacilli defined at the bench by their requirements for X factor (hemin) and V factor (nicotinamide adenine dinucleotide, NAD). Chocolate agar supplies both factors from heat-lysed blood. On media lacking free NAD, a streak of Staphylococcus aureus releases it, and dependent colonies satellite around the streak. The porphyrin test identifies X-factor-independent species directly. These species convert δ-aminolevulinic acid to porphobilinogen and porphyrins, detected as a red color with Kovac's reagent or red fluorescence under a Wood's lamp. X-dependent species cannot make this conversion.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Bone marrow culture

Bone marrow culture is requested when an intracellular or reticuloendothelial-system pathogen is suspected and peripheral blood cultures have been unrevealing, classically for Brucella species and Salmonella enterica serotype Typhi. Both organisms concentrate in reticuloendothelial tissue, including marrow, so marrow culture can remain positive in chronic or relapsing disease and after antimicrobial exposure has suppressed peripheral bacteremia. In typhoid fever, marrow culture remains positive more often than blood culture once therapy has begun.^{1111 Centers for Disease Control and Prevention. Clinical guidance for typhoid fever.} A marrow specimen for suspected brucellosis is inoculated into a blood-culture bottle or another validated system and incubated under the plan for suspected brucellosis. Because recovery of Brucella is often delayed compared with other bloodstream pathogens, current American Society for Microbiology (ASM) and Association of Public Health Laboratories (APHL) sentinel-laboratory guidance directs extending blood-culture bottle incubation from the routine 5 days to about 10 days, with a blind or terminal subculture considered at 7 or 10 days when brucellosis remains suspected after negative bottles. Brucella is a biosafety level 3 (BSL-3), laboratory-acquired-infection risk organism. The laboratory must be told of suspected brucellosis in advance so that the isolate is handled under biological safety cabinet containment and referred through the public-health sentinel-laboratory procedure.^{22 American Society for Microbiology and… Brucellosis-causing Brucella species (BBS). 2025.} Identification and containment of Brucella and Salmonella Typhi are covered in enteric and select-agent bacteriology.

Vector-borne and zoonotic agents diagnosed from blood or serum

A group of spirochetal, rickettsial, and related intracellular bacteria produce systemic bloodstream illness after an arthropod bite or animal contact, and routine blood-culture bottles recover them poorly or not at all. For these agents, the laboratory's role is to select the right specimen and test (serology, stained blood smear, or nucleic acid amplification) and to interpret the result in relation to the timing of the illness. Serologic method mechanics are covered in Immunology. This section covers organism-level test selection and interpretation.

Lyme borreliosis

Borrelia burgdorferi sensu stricto and, rarely, B. mayonii cause Lyme disease in the United States, the latter in the upper Midwest. B. garinii and B. afzelii are Eurasian genospecies. Ixodes hard ticks transmit the spirochete, and in most cases the tick must be attached for more than 24 hours before transmission occurs.^{1212 Centers for Disease Control and Prevention. How Lyme disease spreads.}

Laboratory diagnosis is based on serology, because spirochete numbers in blood and cerebrospinal fluid are too low for reliable culture or amplification. The two-tiered algorithm of the Centers for Disease Control and Prevention (CDC) begins with a sensitive first-tier enzyme immunoassay. A positive or equivocal result reflexes to a second tier, either an IgM and IgG immunoblot or, in modified two-tiered testing cleared by the Food and Drug Administration (FDA) in 2019, a second, different enzyme immunoassay.^{13,1413 Centers for Disease Control and Prevention. Clinical testing and diagnosis for Lyme disease.14 Mead P, et al. Updated CDC recommendation for serologic diagnosis of Lyme… 2019.} Band-count criteria apply only to immunoblots: an IgG blot is positive with 5 of 10 defined bands, an IgM blot with 2 of 3.^{1515 Centers for Disease Control and Prevention. Recommendations for test performance and interpretation from the… 1995.} Disregard a positive IgM result when the illness has lasted more than 30 days, because by then a true infection has produced IgG. Serology may be falsely negative during the first 4 to 6 weeks of infection, so CDC does not recommend serologic testing when a patient with a plausible exposure has erythema migrans, which is diagnosed clinically. Titers stay elevated long after cure, so serology is not used to monitor treatment response. An antibody index comparing cerebrospinal fluid with serum supports neuroborreliosis, and nucleic acid amplification performs best on synovial fluid in Lyme arthritis, where organism burden is highest.^{1313 Centers for Disease Control and Prevention. Clinical testing and diagnosis for Lyme disease.}

Southern tick-associated rash illness (STARI) follows a lone star tick bite with an erythema migrans-like rash. Its cause is unknown, Lyme serology is negative, and no diagnostic laboratory test exists.^{1616 Centers for Disease Control and Prevention. About Southern tick-associated rash illness.}

Relapsing fever

Louse-borne relapsing fever (B. recurrentis, transmitted by the human body louse from a purely human reservoir) and soft tick relapsing fever (several Borrelia species transmitted by Ornithodoros soft ticks) produce cycles of recurring fever as the spirochete varies its surface antigens, escapes the antibody response, and re-emerges. Spirochetemia peaks during fever, so peripheral blood collected during a febrile episode is the classic specimen. Spirochetes are found by darkfield examination or on a stained smear, with Wright-Giemsa, acridine orange, or fluorescent antibody staining. Polymerase chain reaction (PCR) on whole blood is more sensitive than microscopy and can stay positive between febrile episodes and early in treatment. Many assays cannot separate the relapsing-fever species from one another. Serology is most informative 14 days or more after onset. Antigenic variability and cross-reactivity with the Lyme borreliae limit its usefulness. Assays using the GlpQ antigen, which the Lyme group lacks, are more specific for relapsing fever.^{1717 Centers for Disease Control and Prevention. Clinical guidance for soft tick relapsing fever (STRF).}

Leptospirosis

Leptospira species are tightly coiled spirochetes with hooked ends that give a question-mark shape. Rodents are the principal reservoir, shedding organisms in urine, and infection follows contact with contaminated water or soil. Illness is classically biphasic, a bacteremic phase followed by an immune phase as antibody appears. During the first week of illness, real-time PCR on whole blood provides the early confirmed diagnosis. After the first week, urine becomes the better PCR specimen. The microscopic agglutination test (MAT), performed with live antigen in reference laboratories, is the serologic reference method. Agglutinating antibody appears toward the end of the first week, peaks at 3 to 4 weeks, and can persist for years. Culture is insensitive and slow and is never used as the sole method. Incubate Fletcher or Ellinghausen-McCullough-Johnson-Harris (EMJH) medium at 28 to 30 °C in the dark and examine it weekly by darkfield microscopy, for up to about 13 weeks, for spinning hooked organisms growing below the surface.^{1818 Centers for Disease Control and Prevention. Leptospirosis.}

Rickettsioses and scrub typhus

Rickettsia species are small, obligately intracellular gram-negative bacilli that escape the phagosome with their own phospholipase and replicate free in the cytosol. Spotted-fever-group organisms also use host-cell actin to move from cell to cell. They spread through vascular endothelium and produce vasculitis. Typhus-group organisms accumulate inside the cell until it ruptures. Orientia tsutsugamushi, the agent of scrub typhus, is a related but distinct genus.^{33 Carroll KC, et al. Manual of Clinical Microbiology. 2023.}

Practice