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Case Report: Misinterpretation of Gram Stain from

Stationary Growth Phase of Positive Blood Cultures for
Brucella and Acinetobacter Species

Ni Nyoman Nami Arthisari

Clinical Microbiology Resident
Udayana University – Sanglah General Hospital
Supervisor: Dr. dr. Ida Sri Iswari, M.Kes, Sp.MK
ABSTRACT
 Misinterpretation of initial Gram-negative staining from clinical
Introduction sample  misidentification bacteria and delays appropriate
antibiotic treatment.

Brucella :
Acinetobacter baumannii :
• Gram-negative, strictly aerobic, catalase-
positive, oxidase-negative bacteria which
• Log phase  Short, Gram-negative rods
• Stationary phase  Coccoid appearance
• Difficult to destain  Misidentification as
Gram-positive or Gram-variable
• Developed extensive resistance to many
antibiotics (broad-spectrum β-lactams and
carbapenems)
• Gram-negative bacteria which are vulnerable to
misidentification
• Brucellosis is rare in developed countries
• Special concern in Saudi Arabia  significant
importation livestock
• Also one of the common laboratory-acquired
infections worldwide
• Primary Gram stain reporting as Gram-positive or Gram-
variable can lead to misidentification of these slow-
growing Gram-negative coccobacilli and a
misidentification of contaminating Coryneform species.
Click icon to add picture
 History: went to the
desert for the school
CASE 1
vacations, he had contact FEBRUARY 2015
with animals

12-year old boy Blood culture Called back for After one week
admission

• Fever, • Gram • IV Ceftriaxone • Patient

• Doxycycline improved
vomiting, positive
• Rifampin • Ceftriaxone
diarrhea cocci in discontinued
pairs and • Discharged on
chains Doxycycline
and Rifampin
Patient was given IV
fluids, tests were
performed and he was sent
home
 CASE 1 (CON’T)
3 sets
4 days  3 aerobic blood culture vialsof aerobic and anaerobic blood cultures
Pleomorphic Gram-positive bacteria Ranging in shape from round cocci to
were flagged positive by Bactec FX®
were isolated from the blood culture short, thin rods bacilli
system

Preliminary report of “Gram-positive bacilli

Usually interpreted as
Coryneform species was suspected
contaminants of unknown clinical importance

After 24 hour- incubation on blood agar plates

Tiny growth was observed on the primary Further suggesting Coryneform isolates as many
Streaking Coryneform species are characterized by slow growth
CASE 1 (CON’T) Done to rule out Corynebacterium urealyticum, C. jeikeium, and
C. striatum, which can be pathogenic in vulnerable individuals

By 48 hrs, good growth was observed on a blood agar plate

Catalase test positive  commonly exhibited by
both Coryneform species and Brucella species.
Presence of this organism in three consecutive vials prompted further identification by VITEK-2
Compact 60 automated system using the VITEK Anaerobe Identification (ANI) card
After 18 hrs, the blood culture isolate remained unidentified on
ANI by 2 different VITEK 2 system machine

Gram staining was repeated from blood agar plate

Urease test using Urea Agar slant tubes (Saudi
The oxidase test using Remel BactiDrop™ Oxidase
Pleomorphic Gram-variable bacteria Prepared Media Laboratory Co. Ltd)  strongly
ampules (Thermo Scientific)  strongly positive
positive after 3 hour- incubation

Coryneform species to be excluded

and Brucella to be suspected Brucella to be suspected

PCR confirmation of Brucella identification, using real-time PCR Method

DNA was extracted using the MagNA-Pure-Compact-System® (Roche Diagnostics)
Real-Time PCR Method
400 μl of blood was DNA was extracted using
DNA material was eluted
drawn from the blood the MagNA Pure LC DNA
in 50 μl total volume
culture positive bottle Isolation Kit I

Brucella detected by real-

time PCR using the
1 cycle and 94°C for 2 After the activation of the
proprietary Brucella Real
min, 1 cycle enzyme at 37°C for 2 min
Time PCR Kit (Shanghai ZJ
Bio-Tech Co., Ltd)

Amplification signal was

Amplification of PCR 60°C for 30 sec for measured at 60°C using
target was done at 93°C annealing for a total of 40 530 detection fluorescent
for 5 sec for denaturation cycles channel in LightCycler 2.0
instrument
CASE #1 RECOMMENDATION
AND DISCUSSION
Case 1 Recommendation and discussion
Identification of pleomorphic Gram-positive bacilli after initial Gram staining from
blood culture should be done by careful interpretation as both Corynebacterium
and Brucella species have similar slow growth patterns.

Inappropriate treatment may be prescribed due to such misinterpretations

It is recommended that oxidase test from the plate and urease test from the
culture vial be performed after apparent identification of pleomorphic Gram-
positive bacilli

Brucella infection is of particular concern in

Saudi Arabia, given the high volume of livestock importation
Case 1 Recommendation and Discussion (con’t)

Misidentification due to misleading initial Gram-staining results can

also lead to inadvertent exposure of laboratory staff to Brucella

Earlier detection would be helpful in ensuring subsequent

appropriate handling of the relevant samples in the laboratory

For example, in procedures that can result in aerosolization and

protect other laboratory staff from exposure
Case 2 (March 2015) Medical history  diabetes mellitus,
hypertension, dyslipidemia, chronic renal
insufficiency and ischemic heart disease

Admitted to an outside hospital with AHis79-year-old Saudi

pneumonia requiring intubation and
mechanical ventilation
hospital course was man by
complicated (8septic
March)
Treated extensively with broad
shock, acute renal failure on top of chronic renal
spectrum antibiotics
insufficiency requiring hemodialysis

30 March: transferred to Johns Hopkins Aramco Healthcare required mechanical ventilation

Imipenem and levofloxacin  extubated two days Reintubated because of extensive respiratory
later secretions

Blood Cultures
Identified MDR Acinetobacter Treated with Colistin successfully
Case 2 (con’t)
Blood Cultures
Three sets of aerobic and anaerobic blood cultures were 16 hrs  two aerobic blood culture vials were flagged positive
collected by the Bactec FX® system

Gram Stain
Giant Gram-positive oval cocci from both vials, suggesting
Preliminary report of Gram positive bacilli
Staphylococcus species

Blood Agar and MacConkey Agar after 18 hrs

1-2 mm non-pigmented, domed, and mucoid colonies with
Suggesting the presence of Gram-negative bacilli
smooth surfaces

Gram staining was repeated from both the plate and the vials
Gram-negative rod-shaped organisms and giant Gram-positive oval shaped
Case 2 (Con’t)

VITEK-2 Gram-Negative
Identification test (GNI) card Real-Time PCR Subculture
(bioMérieux)

DNA was extracted as described Columbia agar (Gram-positive

Growth on the blood agar plate above and real-time PCR was media), blood agar, and
was confirmed as A. baumanii carried as described previously MacConkey agar

24 hours later, no growth was

PCR was carried out directly from observed on Colombia media,
the vial and the presence of A. excellent growth was observed
baumanii was confirmed on BA and MAC
CASE #2 RECOMMENDATION
AND DISCUSSION
CASE 2 RECOMMENDATION AND
DISCUSSION

Acinetobacter generally have the form of

short Gram-negative rods
•Become more coccoid in the stationary
phase
They may be misidentified as either
Gram-variable or Gram-positive cocci

Tille, Patricia M. Bailey & Scott’s Diagnostic Microbiology. 14th ed. St. Louis, Missouri: Elsevier; 2017.
 CASE 2 RECOMMENDATION AND
DISCUSSION
In this case:
Preliminary Gram-staining was
misinterpreted
Identification of giant Gram-
positive oval cocci was
erroneously made
MAHON TEXTBOOK
 Acinetobacter organisms can resist
decolonization and retain the crystal violet
stain, leading to misidentification.
 They can appear as gram-positive cocci in
smears made from blood culture bottles
It is recommended that once giant Gram-positive
oval cocci are visualized on preliminary Gram-
staining from blood culture vials, the technologist
should repeat the Gram stain from the plate after 4-
6h
Or add a few drops of the cultured material into
Triple Sugar Iron (TSI) medium and repeat the
Gram stain after 2-4 h incubation at 37°C
 Organisms including Acinetobacter do not
ferment glucose, and are strictly aerobic,
therefore they will not change the color of the
TSI medium
 Staphylococcus which carries out acidic
fermentation
 CASE 2 RECOMMENDATION AND DISCUSSION

MDR- Acinetobacter is on the increase worldwide

Few reports on the MDR properties of A. baumannii in Saudi Arabia, with evidence
pointing to increases in nosocomial MDR- A. baumannii infections.
Misinterpretations of initial Gram-staining results, such as occurred in case 2, could
be avoided
This would in turn help expedite antibiotic susceptibility testing on the A.
baumannii isolates and hence identification of the most appropriate antibiotic
treatment
This recommended procedure would have many benefits for both patients and
healthcare providers, including reductions in morbidity and mortality, fewer
laboratory tests and procedures, fewer and shorter hospital and intensive care unit
stays, and decreased financial costs
REFERENCES
1. Tille, Patricia M. Bailey & Scott’s Diagnostic Microbiology. 14th ed. St. Louis,
Missouri: Elsevier; 2017.
2. Mahon CR, Lechman DC, Manuselis G. Textbook of Diagnostic Microbiology.
6th Edition. Elsevier : 2019.