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Gram-negative, fastidious and vector-borne blood isolates

18 min

Skills you'll build4 skills
  • 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

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?

Keep going to see the reasoning.

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

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 carefully, because a misread can lead to N. meningitidis being reported 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) are detected within the standard 5-day incubation of current systems. Extended incubation and blind subculture do not improve recovery. 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 indicates a species that makes its own heme and does not need X factor.1

Vector-borne infections

Routine bottles recover these organisms poorly, so test results are interpreted by the week of illness.4,5

  • Rickettsia. The immunofluorescence assay (IFA) for IgG is insensitive in the first week. Confirmation requires a fourfold rise between an acute specimen and a convalescent one 2 to 4 weeks later. A single titer does not confirm infection, because 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

Follow the bench demonstration

Sources for this step
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

Read each decision and its explanation.

Owen Draper, 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. Note when the acute serum was drawn: day 4 of illness.

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

  2. Interpret the acute titer: a titer of 64 cannot confirm infection.

    A titer of 64 is found in 5% to 10% of healthy people.

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

    The convalescent specimen must be collected 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 criterion: an eightfold rise 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.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

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 gives no information about the species.

Hint
  1. Several Neisseria species that grow on the same medium share the colony appearance, oxidase and Gram stain.
  2. Consider whether the tests already done can distinguish Neisseria species.

Review Gram-negative cocci: Neisseria

Problem 2 of 3 · Hints available

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 have 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 does not confirm acute infection. The indirect immunofluorescence assay (IFA) is the serologic reference standard but is insensitive in the first week, so confirmation is retrospective and requires 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 · On your own

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

Read the case evidence, then make each decision. The step is complete without it.

  • Harold Summers, 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 do not improve recovery 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 do not improve recovery 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 clinician then knows that a negative final report covers them.

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

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. Together with growth on chocolate agar, it suggests Haemophilus.

The streak was placed on purpose to test for NAD dependence. The tiny colonies grow only next to 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 argues against 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 have overlapping reactions, so MALDI-TOF or a molecular method confirms the species.

Review HACEK organisms and Haemophilus identification

The key finding is satellite growth beside the S. aureus streak, which shows a V-factor requirement. The positive porphyrin test then indicates an X-independent Haemophilus. MALDI-TOF identified H. parainfluenzae from all three sets on day 4.

For reference

Sources checked