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Gram-negative, fastidious and vector-borne blood isolates

18 min

  • Confirm a gram-negative diplococcus to species before reporting it
  • Distinguish nonfermenting gram-negative rods from enteric fermenters
  • Recognize a HACEK or Haemophilus blood isolate that needs special identification
  • Read vector-borne infection serology against the week of illness and paired titers

Read the full reference

Try first

Try first

A blood culture grows oxidase-positive gram-negative diplococci. Carbohydrate tests show acid from glucose and maltose and none from lactose. Which species fits?

The next section explains it.

The next section explains it.

Right. The next section explains why.

The next section explains it.

The next section explains it.

Get the idea

Confirming Neisseria

A typical colony, a positive oxidase and gram-negative diplococci give a presumptive identification, which may go out as a preliminary result. Other Neisseria species grow on the same media, so a method with a different principle confirms the species before it is reported.1,2

SpeciesGlucoseMaltoseLactose
N. gonorrhoeae+−−
N. meningitidis++−
N. lactamica+++
Moraxella catarrhalis−−−

Read each color change with care, because a misread can report N. meningitidis as N. gonorrhoeae.2 N. meningitidis from blood or CSF is a critical result.1

Nonfermenters

Nonfermenters are gram-negative rods that do not ferment glucose. On triple sugar iron agar they leave an alkaline slant and an unchanged butt. The enteric fermenters, the Enterobacterales, acidify the butt and are oxidase negative.1

  • Pseudomonas aeruginosa is oxidase positive, grows at 42 °C, smells like grapes, and often makes blue-green pigment.
  • Acinetobacter is oxidase negative and nonmotile, and it can decolorize poorly.
  • Stenotrophomonas maltophilia is oxidase negative and DNase positive, and it is intrinsically carbapenem resistant.1

HACEK and Haemophilus

The HACEK organisms (Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, Kingella) signal within the standard 5-day incubation of current systems. Extended incubation and blind subculture add nothing. Species identification, usually by MALDI-TOF, can still take several days.3

Haemophilus needs hemin (X factor), NAD (V factor) or both. Chocolate agar supplies both. Near a streak of Staphylococcus aureus, which releases NAD, V-dependent colonies grow as satellites. A positive porphyrin test marks a species that makes its own heme and needs no X factor.1

Vector-borne infections

Routine bottles recover these organisms poorly, so the result is read against the week of illness.4,5

  • Rickettsia. The immunofluorescence assay (IFA) for IgG is insensitive in the first week. Confirmation needs a fourfold rise between an acute specimen and a convalescent one 2 to 4 weeks later. One titer never confirms, since 5% to 10% of healthy people have reciprocal R. rickettsii titers of 64 or more.4
  • Lyme disease. Serology can be negative for the first 4 to 6 weeks.5
References
  1. Carroll KC, Pfaller MA, Landry ML, McAdam AJ, Karlowsky JA, Patel R, Pritt BS, eds. Manual of Clinical Microbiology. 13th ed. ASM Press; 2023.
  2. Papp JR, Schachter J, Gaydos CA, Van Der Pol B. Recommendations for the laboratory-based detection of Chlamydia trachomatis and Neisseria gonorrhoeae—2014. MMWR Recomm Rep. 2014;63(RR-2):1-19. Accessed September 27, 2026. https://www.cdc.gov/mmwr/preview/mmwrhtml/rr6302a1.htm
  3. Liesman RM, Pritt BS, Maleszewski JJ, Patel R. Laboratory diagnosis of infective endocarditis. J Clin Microbiol. 2017;55(9):2599-2608. doi:10.1128/JCM.00635-17
  4. Biggs HM, Behravesh CB, Bradley KK, et al. Diagnosis and management of tickborne rickettsial diseases: Rocky Mountain spotted fever and other spotted fever group rickettsioses, ehrlichioses, and anaplasmosis—United States. MMWR Recomm Rep. 2016;65(RR-2):1-44. doi:10.15585/mmwr.rr6502a1
  5. Centers for Disease Control and Prevention. Clinical testing and diagnosis for Lyme disease. Accessed September 27, 2026. https://www.cdc.gov/lyme/hcp/diagnosis-testing/index.html

Watch one

Owen Tillery, 46, became ill 5 days after pulling a tick off his leg. Two sera were tested for Rickettsia rickettsii IgG by IFA.

  • Acute serum, day 4 of illness: reciprocal titer 64.
  • Convalescent serum, 3 weeks later: reciprocal titer 512.

What do the two results show?

  1. Place the acute serum in time: day 4 of illness.

    IFA is insensitive in the first week, so an early result can neither confirm nor exclude.

  2. Read the acute titer alone: 64 cannot confirm infection by itself.

    A titer of 64 turns up in 5% to 10% of healthy people.

  3. Check the interval: 3 weeks, inside the 2-to-4-week window.

    The convalescent specimen has to come late enough for antibody to rise.

  4. Compare the titers: 512 ÷ 64 = 8, an eightfold rise.

    Confirmation depends on the size of the change between the paired specimens.

  5. Apply the rule: eightfold meets it.

    The criterion is a fourfold or greater rise.

The paired titers show an eightfold rise, which meets the fourfold criterion. The pair gives serologic confirmation of recent infection.

Your turn

Problem 1 of 3

An oxidase-positive colony of gram-negative diplococci grows on selective GC medium from a genital specimen. What is required before the isolate is reported as Neisseria gonorrhoeae?

Correct. Colony, oxidase, and Gram stain give only a presumptive identification, because other Neisseria species grow on the same media. Confirmation by a different principle, such as carbohydrate utilization or an enzyme-substrate kit, precedes the report. In suspected sexual assault or abuse, two tests of different principles are required and the isolate is preserved.

Incorrect. Growth on chocolate agar repeats the colony features already seen. N. meningitidis, N. lactamica, and some N. cinerea also grow on selective GC medium, so a method with a different principle confirms the species.

Incorrect. Nitrocefin detects β-lactamase for surveillance and isolate characterization and carries no information about the species.

Hint
  1. Colony, oxidase and Gram stain are shared by several Neisseria species that grow on the same medium.
  2. Ask whether the tests already done can tell Neisseria species apart.

Review Gram-negative cocci: Neisseria

Problem 2 of 3

A single acute-phase serum gives a reciprocal Rickettsia rickettsii IgG titer of 64 by IFA. How is this result interpreted?

Incorrect. Between 5% and 10% of healthy people carry reciprocal R. rickettsii IgG titers of 64 or more, so one titer cannot confirm acute infection.

Incorrect. IgG by indirect immunofluorescence is the serologic reference for acute rickettsial infection. A single titer cannot place the infection in time. A fourfold rise between paired specimens shows current infection.

Correct. A single elevated titer never confirms acute infection. The indirect immunofluorescence assay (IFA) is the serologic reference standard but is insensitive in the first week, so confirmation is retrospective and rests on a fourfold or greater IgG rise between paired acute and convalescent specimens.

Hint
  1. Ask whether one titer can show when the antibody appeared.
  2. Recall how common a titer of 64 is in healthy people.

Review Rickettsioses and scrub typhus

Problem 3 of 3

A blood isolate is a gram-negative rod with colorless colonies on MacConkey agar. Triple sugar iron agar shows an alkaline slant and an unchanged butt. The oxidase is positive, and the colonies have a blue-green tint and a grape-like odor. Which group fits?

The Enterobacterales ferment glucose and acidify the butt, and they are oxidase negative. This isolate does neither.

Acinetobacter is oxidase negative and makes no pigment.

An unchanged butt shows no glucose fermentation. Oxidase positive with blue-green pigment and a grape odor is the P. aeruginosa pattern, which a validated system confirms.

S. maltophilia is oxidase negative and DNase positive, and its colonies are lavender-green.

Review Gram-negative nonfermenting bacilli

Use it

  • Harold Beckett, 63, has a prosthetic mitral valve and a month of low fevers.
  • Three blood-culture sets are drawn for suspected endocarditis.
  • The clinician asks the laboratory to incubate the bottles for 21 days in case of a HACEK organism.
Decision 1 of 3

How do you answer the request?

Current systems recover HACEK organisms within the standard incubation. Three extra weeks add nothing and delay the final report.

Extended the incubation for suspected HACEK endocarditis

Continuous-monitoring systems recover HACEK organisms within the standard 5-day incubation, so extended incubation and terminal blind subculture add nothing and delay the final report. Species identification, usually by MALDI-TOF, can still take several days after the bottle signals.

Continuous-monitoring systems detect HACEK organisms within about 5 days. The explanation tells the clinician that the negative final covers them.

The laboratory's own bottles and instrument recover these organisms. Referral would only slow the answer.

Review HACEK organisms and Haemophilus identification

Decision 2 of 3

On day 2, two sets signal, and the third signals on day 3. The Gram stain shows small pleomorphic gram-negative coccobacilli. The subculture grows on chocolate agar. On blood agar it grows only as tiny colonies beside a Staphylococcus aureus streak. What does the blood agar show?

S. aureus releases NAD, the V factor. Satellite growth beside the streak shows a V requirement.

Growth only beside the S. aureus streak is satellite growth on released NAD. With chocolate-agar growth, it points to Haemophilus.

The streak was placed on purpose to test for NAD dependence. The tiny colonies depend on it.

Review HACEK organisms and Haemophilus identification

Decision 3 of 3

The porphyrin test is positive. What do you do next?

H. influenzae needs X factor and is porphyrin negative. A positive porphyrin test points away from it.

H. ducreyi needs X factor and no V factor, and it is porphyrin negative. This isolate needs V factor and is porphyrin positive.

Porphyrin positive with a V requirement fits H. parainfluenzae, a HACEK member linked to endocarditis. The HACEK species overlap, so MALDI-TOF or a molecular method makes the call.

Review HACEK organisms and Haemophilus identification

The clue that settled this case is satellite growth beside the S. aureus streak. It showed a V-factor requirement, and the positive porphyrin test narrowed it to an X-independent Haemophilus. MALDI-TOF identified H. parainfluenzae from all three sets on day 4.

Keep

Sources checked