A Biotype of Enterobacteriaceae Intermediate between
Citrobacter and Enterobacter
HERBERT BRAUNSTEIN, M.D., MICHAEL TOMASULO, B.A. M.T.(ASCP), SUSAN SCOTT, B.A., M.T.(ASCP),
AND M. PATRICIA CHADWICK, B.S., M.T. (ASCP)
Braunstein, Herbert, Tomasulo, Michael, Scott, Susan, and
Chadwick, M. Patricia: A biotype of enterobacteriaceae intermediate between Citrobacter and Enterobacter. Am J Clin
Pathol 73:114-116,1980. The authors isolated two strains of an
unnamed bacterial biotype with characteristics intermediate
between those of Enterobacter and Citrobacter. The organisms
did not produce acetyl-methyl carbinol, but decarboxylated
lysine. Apart from the latter trait, they most closely resemble
H2S-negative Citrobacter freundii. They differ biochemically
from all other currently accepted species of enterobacteriaceae.
Their pathogenic significance appears similar to that of the two
genera they most closely resemble. Only by recognition and
study of additional strains can their identity be more definitively
delineated and their significance more fully assessed. (Key
words: Enterobacteriaceae biotype; Citrobacter; Enterobacter.)
Department of Laboratories, San Bernardino County
Medical Center, San Bernardino, California
1) and agglutination studies for Salmonella and Arizona, 5 as well as antibiotic susceptibility testing (Autobac 1®).*
Results
The results of the biochemical study are recorded in
Table 1. Both organisms were identical biotypes. Significant features included: metallic luster on EMB
plates, fermentation of glucose and lactose with gas
production in the butt of both triple-sugar iron (TSI)
and Kiigler's iron agar (KIA), and in the insert tube
of a Durham fermentation tube; utilization of citrate;
production of indole; negative Voges-Proskauer (V-P)
reaction; motility present; decarboxylation of lysine
(LDC) and ornithine (ODC); utilization of malonate;
and failure to ferment adonitol.
Antibiotic susceptibility testing showed both strains
to be resistant to cephalothin and ampicillin, and sensitive to tetracycline, chloramphenicol, kanamycin,
gentamicin, colistin, tobramycin and carbenicillin. Review of the charts of both patients indicated that the
sputum isolate probably represented a contaminant,
the gallbladder specimen a pathogen. Both strains were
forwarded to the National Center for Disease Control
(NCDC), which identified them as "Enteric Group 8"
or "Citrobacter-Enterobacter intermediate."
THE USE of computerized biochemical identification
of Enterobacteriaceae has facilitated the detection of
aberrant strains of recognized species of Enterobacteriaceae. In addition, it has permitted the delineation
of biotypes with characteristics incompatible with
legitimately identified species. Studies of the nucleoproteins and other qualities of a number of these biotypes have resulted in the recommendation that additional species designations be accepted. 3 The present
report describes the characteristics of two strains of
an unnamed biotype with characteristics intermediate
between those of Enterbacter and Citrobacter.
Materials and Methods
Both strains were isolated from patients at the San
Bernardino County Medical Center. One was grown
from the sputum of a 6-year-old boy with pulmonary
tuberculosis; the other, obtained eight months later,
from gallbladder drainage fluid of a 63-year-old woman
with acute pancreatitis.
Each was initially isolated on 5% sheep blood agar,
enriched chocolate agar and eosin-methylene blue
(EMB) agar. Subsequent studies included a series of
biochemical tests employing standard methods 5 (Table
Received February 23, 1979; received revised manuscript and
accepted for publication April 10, 1979.
Address reprint requests to Dr. Braunstein: Department of Laboratories, San Bernardino County Medical Center, 780 East Gilbert
Street, San Bernardino, California 92404.
Discussion
Table 1 delineates the peculiarity of this enteric
biotype that precludes its identification as any of the
legitimately accepted species, 4 " 8 including those newly
recommended by the NCDC. 3 The organisms are
clearly Enterobacteriaceae, being glucose fermenters,
oxidase-negative, and nitrate-positive. They are
lactose fermenters and gas producers, resembling
Escherichia coli and H 2 S-negative Citrobacter species
on the EMB plate and in sugar iron agar slants, in which
* Pfizer and Co., New York, N. Y.
0002-9173/80/0100/0114 $00.60 © American Society of Clinical Pathologists
114
115
BRIEF SCIENTIFIC REPORTS
Vol. 73 • No. I
Table I. Characteristics of Citrobacter-Enterobacter Intermediate and Similar Organisms
Characteristic
Oxidase
H 2 S, TSI or K1A
Gas-glucose
Lactose
Citrate
Indole
V-P
Urease
Deoxyribonuclease
Motility
LDC
ODC
Arginine dihydrolase
Adonitol
Arabinose
Raffinose
Mulonatc
Beta galactosidase
Inositol
Tartrate
Rapid hydrolysis of
gelatin (48 hr)
CitrobacterEnterobacter
Intermediate
Citrobacier
freundii
Citrobacier
diversus
0
0
+
+*
0
0*
0
0
0
V
V
V
V
0
V
0
+
+
+
+n
+
0
+t
0
V
V
0
+
V
V
+
0
V
0
0
0
0
+
+
0
0
+
+
+t
+
0
Symbols: 0 *3 lOCf positive: + r* 90Cf positive; V •- 10-90'? positive.
* Traits incompatible with proposed Enterobacter f>erxoviae?
they produce an acid slant and gas in the butt. Their
ability to utilize both citrate and malonate appears to
preclude their identification as Escherichia. On the
other hand, the decarboxylation of lysine is clearly incompatible with their designation as Citrobacter. 4-6
They do, however, except for this trait, most closely
resemble H 2 S-negative Citrobacier freundii. Sedlak,
in Bergey's Manual,1 designated a species called
Citrohacter intermedius, some strains of which were
said to produce indole, decarboxylate lysine, and
utilize malonate. Nonetheless, in the United States,
lysine decarboxylation-positive organisms have not
been accepted as Citrobacter species. 3 - 6
Organisms called "Citrobacter intermedium" were
described by others, 1 8 but these failed to decarboxylate
lysine; it appears probable that in the United States
these could be classified currently within the recognized Citrobacter species, 3,6 since their principal distinguishing characteristic was failure to produce H 2 S.
The species Citrobacter amalonaticus
(Table 1),
recently recommended for acceptance in the United
States, 3 is characterized by failure to decarboxylate
lysine or utilize malonate, and thus appears to be
ruled out.
Motility and decarboxylation of ornithine rule out
Klebsiella. The possibility of Enterobacter appears
remote, since Enterobacter aerogenes, the only
Enterobacter species consistently decarboxylating
both ornithine and lysine, is invariably V-P-positive,
+
+
+
Enterohacter
aerogenes
0
0
+
+
+
Escherichia
coli
0
0
+
+
4
Hafnia
0
0
+
V
V
0
V
0
0
V
0
+
+
+
0
+
+
V
0
+
0
0
0
V
V
V
V
0
+
V
0
+
0
V
+
+
+
V
0
+
0
V
V
0
V
0
0
0
0
+
0
+
0
+
0
+
0
0
+
+
+
+
+
+
+
0
0
+
t Traits incompatible with proposed Citrobacier unuilonalicits.
X Traits incompatible with proposed Enterobacter .\akazakii.:'
ferments adonitol, and is rarely indole-positive. Enterobacter gergoviae3 (Table 1) also appears to be ruled
out. Hafnia alvei3 is never indole-positive and rarely
ferments raffinose. The deoxyribonuclease, ODC,
gelatin hydrolysis, indole, malonate and arabinose
reactions appear to preclude the possibility that these
represent one of the two Serratia species.
Many features are highly uncharacteristic of the
genera Edwardsiella, Proteus, Providencia and Shigella.
Arizona appears to be eliminated by the positive indole
reaction, the failure to produce H 2 S, and the agglutination
studies. Although lactose-fermenting, H 2 S-negative
strains of Salmonella species are occasionally encountered, the positive indole reaction and malonate
utilization appear to rule this genus out.
As is the case with newly delineated groups, the
pathogenic significance of these organisms is uncertain.
It would appear from our two cases that they may
resemble closely other enteric organisms, both in the
probability that they will produce disease and with
respect to the types of specimens from which they may
be isolated.
Review of the susceptibility test data does not offer
hard evidence to suggest which of the two possible
genera (Citrobacter or Enterobacter) the organisms
more resemble. If the antibiotic susceptibility pattern
of the two strains proves to be consistent, they would
appear to be closer in these qualities to Enterobacter
than to C. freundii, since our susceptibility test data
A.J.C.P. • January 1980
BRAUNSTEINET/t/..
116
reveal that almost half of C. freundii strains are susceptible to ampicillin and cephalothin.2
It is suggested that clinical microbiology laboratory
workers maintain awareness of these organisms, which
appear to represent a homogeneous biotype. Only by
their identification and submission to reference laboratories can sufficient data be gathered to verify their
identity as a new species and assess their significance
as disease-producing agents.
References
1. Booth EV. McDonald S: A new group of enterobacteria,
possibly a new Citrobacter sp. J Med Microbiol 4:329-336,
1971
2. Braunstein H: Unpublished observations
3. Brenner DJ, Farmer JJ III, Hickman FW, et al: Taxonomic
and Nomenclature Changes in Enterobacteriaceae. NCDC
Publication, U. S. Department of Health, Education, and
Welfare, Atlanta, 1977, p 15
Edwards PR, Ewing WH: Identification of Enterobacteriaceae.
Third edition. Minneapolis, Burgess Publishing Co., 1972,
p362
Ewing WH: Differentiation of Enterobacteriaceae by Biochemical Reactions. Revised NCDC Publication, Public
Health Service, United States Department of Health, Education, and Welfare, Atlanta, 1974, p 61
Ewing WH, Davis BR: Biochemical Characterization of Citrobacter freundii and Citrobacter diversus. NCDC Publication.
Public Health Service, United States Department of Health,
Education, and Welfare, Atlanta, 1971, p 26
Sedlak J: Genus III, Citrobacter, Bergey's Manual of Determinative Bacteriology. Eighth edition. Edited by RE Buchanan
and NE Gibbons. Baltimore, Williams and Wilkins. 1974,
pp 297-298
Slifkin M, Engwall C: The clinical significance of Citrobacter
intermedium. Am J Clin Pathol 52:351-355, 1969
Immunofluorescence of Renal Lesions in Paraffinembedded and Fresh-frozen Sections
YOUNG J. CHOI, M.D., AND LEOPOLD REINER, M.D.
Choi, Young J. and Reiner, Leopold: Immunofluorescence
of renal lesions on paraffin-embedded and fresh-frozen sections.
Am J Clin Pathol 73: 116-119, 1980. Immunofluorescent
findings in formalin-fixed and paraffin-embedded sections of
kidneys involved by various nephropathies compared favorably
with those in fresh-frozen sections. In some cases, the findings
in paraffin-embedded sections were complementary. Immunoreactivity with complement could not be demonstrated. (Key
words: Nephropathies; Immunofluorescence of paraffin-embedded sections; Immunoglobulins).
IMMUNOFLUORESCENCE (IF)' plays an important role in the pathologic diagnosis of renal lesions.
Although applied originally to fresh-frozen sections,
recent reports2-4 have indicated that IF can also be
applied to formalin-fixed and paraffin-embedded
tissues.
We conducted an IF study of biopsy and autopsy
tissues to compare the results obtained by use of freshfrozen sections with those obtained by use of sections
prepared from old paraffin blocks. We also investigated
the possibility of applying IF to paraffin-embedded
sections that had been stained with hematoxylin-eosin
(or other stains) at the time of accession and stored
in the departmental files for various periods.
Received October 17, 1978; received revised manuscript and
accepted for publication December 27, 1978.
Address reprint requests to Dr. Choi: The Bronx-Lebanon
Hospital Center, 1276 Fulton Avenue, Bronx, New York 10456.
Department of Pathology, The Bronx-Lebanon
Hospital
Center and Albert Einstein College of Medicine,
Bronx, New York
Materials and Methods
The study material comprised specimens from 21
subjects with various nephropathies. Samples of
kidney tissue were obtained by needle biopsy in 18
cases, by autopsy in two, and by both biopsy and
autopsy in one. All specimens were obtained during the
period from 1972 to 1978. There were nine cases of
lupus nephritis, five of membranous nephropathy,
two of focal sclerosing glomerulonephropathy, and one
each of focal proliferative glomerulonephritis, Berger's
disease (IgG-IgA nephropathy) and Goodpasture's
syndrome. At the time of biopsy (or autopsy) one part
of the specimen was processed for IF and the other for
light microscopy. The procedures employed follow.
Immunofluorescence of fresh-frozen tissue: At the
time of biopsy (or autopsy) renal tissue was rapidly
frozen in liquid nitrogen, cut at 2 /j,m in a cryostat, and
flooded with fluorescein-labelled goat antihuman immunoglobulins (IgG, IgA, IgM, C3, and fibrinogen
—Behring Diagnostics). The sections were mounted in
Elvanol® (pH 6.8) and examined under a Leitz ultraviolet microscope. The intensity of fluorescence was