Blood Cultures Showing Aberrant
Forms of Bacteria
VICTOR LORIAN, M.D.,
AND ALEXANDRA WALUSCHKA,
M.D.
Division of Microbiology and Epidemiology, Department of Pathology,
The Bronx-Lebanon Hospital Center, Bronx, N. Y. 10456
ABSTRACT
Lorian, Victor, and Waluschka, Alexandra: Blood cultures showing aberrant
forms of bacteria. Am. J. Clin. Pathol. 57: 406-409, 1972. Aberrant forms of
bacteria were observed in blood cultures obtained from four patients. One
Enterobacter cloacae, one E. coli and two Klebsiella pneumoniae were isolated
at 48 hr. from blood cultures. All four patients had received penicillin, ampicillin, or cephalothin shortly before the blood cultures were obtained.
the discovery of penicillin,
Gardner 6 described abnormal and filamentous forms of Gram negative bacilli produced when grown in the presence of subinhibitory concentrations of penicillin.
Other authors, in a specific search for Lforms of bacteria, using penicillin in their
media to stimulate L-form formation, produced aberrant and filamentous forms of
Gram-negative bacilli. 8 - 5 ' 7 - 8 Aberrant forms
of Proteus mirabilis have been observed in
urines of patients receiving penicillin therapy,2 and L-forms of E. coli, Klebsiella, and
Proteus have been recovered from urine
after treatment with ampicillin or cephalothin. 9 In this paper, we describe four patients in whose blood cultures aberrant
forms of bacteria were observed.
SHORTLY AFTER
Material and Method
All blood cultures were incubated at
37 C. in bottles of Thiol broth (DIFCO)
and Liquoid trypticase soy broth (Roche).
Received February 25, 1971; received revised
manuscript April 23, 1971; accepted for publication May 3, 1971.
Address reprint requests to: Victor Lorian, M.D.,
Division of Microbiology and Epidemiology, The
Bronx-Lebanon Hospital Center, 1276 Fulton Avenue, Bronx, N. Y. 10456.
The bottles were examined macroscopically
every day for 14 days. At the slightest suspicion of growth, two slides, one stained with
Gram stain and one with Wright's stain
were prepared, and aliquots were subcultured on blood agar, EMB, and trypticase
soy broth. The organisms isolated were
identified by routine procedures and confirmed according to the CDC manual for
identification of enterobacteriaceae. 4 Sensitivity tests were done according to the
Kirby-Bauer method. 1
A series of twofold trypticase soy broth
dilutions was prepared with ampicillin (500
;ug. per ml. to 0.5 y.g. per ml.) and inoculated with 0.05 ml. of a 24-hour broth
culture of the Enterobacter cloacae strain
isolated from Case 1. Another series of twofold trypticase soy broth dilutions was prepared with penicillin (1,000 jug. per ml. to
5 jag. per ml.) and inoculated with 0.05 ml.
of a 24-hr. broth culture of a Klebsiella
strain isolated from Case 2. The tubes
were incubated at 37 C. for 20 hours and
Gram-stained smears were made from all
tubes showing visible growth. The MIC
was determined as the antibiotic concentration in the first tube showing no visible
growth.
406
March
1972
ABERRANT BACTERIAL FORMS IN BLOOD CULTURES
407
FIG. 1. Filamentous forms of Enterobacler cloacae from Case 1.
N
f
Reports of Cases and Results
Case 1. A 42-year-old woman was admitted for high fever, weakness, and intense headaches. Her temperature was
102.5 F. and the leukocyte count was
18,000. She was immediately started on ampicillin, 1 Gm. every 4 hr., intravenously.
A few hours later, blood for culture was
drawn. The blood culture became positive
at 48 hr. and Entcrobactcr cloacae sensitive
to ampicillin was isolated.
The Gram stain revealed Gram-negative
filaments 20 to 100 microns long. The
Wright stain gave a much better tinctorial
contrast than the Gram stain (Fig. 1).
Case 2. A 66-year-old man was hospitalized with a bleeding peptic ulcer and diabetes mellitus. The leukocyte count was
2,800 and his temperature was 99 F. On
the 4 succeeding days he became febrile
(temperatures 100.2 F. to 102.8 F.) and the
•?' AP*
\
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leukocyte count rose to 11,300. Sputum,
urine, and blood cultures were ordered on
the fourth day of admission. The blood
culture was obtained at approximately 11
P.M. the same day, but 1 hour prior to the
time that blood culture was taken, 600,000
U. of penicillin G had been administered.
No other antibiotics had been given.
At 48 hr. the blood culture showed
Klebsiella pneumoniae. The Gram stain as
well as the Wright stain showed numerous
filaments 50 to 200 microns long and encapsulated bacilli of normal size (Fig. 2).
Case 3. A 71-year-old woman was admitted with arteriosclerotic heart disease, hypertension, and a possible cerebral embolus. On admission, the leukocyte count was
19,400 and her temperature was 99 F. One
day later, the leukocyte count was 15,500
and her temperature was 100.2 F. Three
blood cultures drawn at that time showed
408
LORIAN AND WALUSCHKA
^
no growth. A week after admission, the
leukocyte count was down to 9,600, but
her temperature had risen to 104 F., and
a blood culture at that time yielded Klebsiella pneumoniae sensitive to cephalothin.
The patient was started on cephalothin, 2
Gm. intravenously every 4 hr. Two days
after the first positive blood culture was
obtained, and in spite of the treatment
with cephalothin, her temperature rose to
102.4 F. Another blood culture was drawn
which, in the next 48 hr., again grew Klebsiella pneumoniae sensitive to cephalothin.
The Gram stain showed Gram-negative
filaments 20 to 30 p long, together with
normal-sized bacilli. The filaments were
better demonstrated in a Wright's stain
preparation.
Case 4. An 84-year-old man was hospitalized because of complete heart block. Three
days after admission, he developed multiple infections from which six different species of bacteria were cultured repeatedly.
A.J.C.P.—Vol. 57
FIG. 2. Filamentous forms of Klebsiella pneumoniae and a few encapsulated normal-sized bacilli from Case 2.
The sites of infection were a decubitus
ulcer, the respiratory system, the urinary
tract, and finally the blood. A blood culture taken 9 days after admission grew
Streptococcus faecalis. Another blood culture taken 18 days after admission grew
Herellea which was resistant to ampicillin
and E. coli which was sensitive to it. On
Gram stain, Gram-negative filamentous
forms 50 to 100 ^ in length were observed,
mixed with very few normal Gram-negative bacilli.
Antibiotic treatment for the 10 days preceding this blood culture consisted of
chloramphenicol, 3 Gm. daily for 7 days,
followed by penicillin, 3,000,000 U. daily,
and ampicillin, 6 Gm. daily. Only the last
antibiotic was being given at the time the
blood culture was obtained.
The samples from the tubes containing
the twofold dilutions of antibiotics showed
the following: the enterobacter from Case
1 showed filaments at 30 ^.g. ampicillin per
March 1972
ABERRANT BACTERIAL FORMS IN BLOOD CULTURES
ml., representing one-eighth of its MIC and
the Klebsiella from Case 2 produced short
filaments at 15 /tg. penicillin per ml., representing one-eighth of its MIC.
Discussion
In all four cases, the organisms isolated
belonged to the group of Enterobacteriaceae. It is generally recommended that
blood for cultures be taken before the patient receives antibacterial therapy, but
this recommendation was not followed in
the four cases presented. However, in all
four cases organisms sensitive to the antibiotic being administered grew out on the
blood cultures in spite of the fact that the
patients had received treatment in average
therapeutic dosages. As already mentioned,
antibiotics which affect cell wall synthesis
can produce abnormal forms of Gram-negative bacilli. Because all four patients,
when the blood specimens for cultures
were taken, were receiving treatment with
penicillin, ampicillin, or cephalothin, we
assume that small amounts of those antibiotics were carried over with the blood
into the bottles for blood cultures and
were the cause of these aberrant forms.
The fact that we obtained filamentous
forms of Enterobacter cloacae with subinhibitory concentrations of ampicillin and
filamentous forms with Klebsiella pneumoniae with subinhibitory concentrations
of penicillin strengthens the basis of this
409
assumption. Wright's stain gave better tinctorial contrast and illustrated the presence
of these abnormal forms of bacteria better
than Gram stain. We suggest, therefore,
that Wright's stain be used every time
there is a suspicion of abnormal forms in
a blood culture.
Acknowledgment.
Miss Barbara Topf summarized the clinical information of the patients investigated, and Leon Reiner reviewed the manuscript.
References
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