Tech Com:
Microbiology
Aglycone Tests Determine
Hydrolysis of Arbutin, Esculin,
and Salicin by Nonfermentative
Gram-Negative Bacteria
by Sandra K. Frank, M.S., MT(ASCP) SM, and
V. Lyle von Riesen, Ph.D.
Sandra K. Frank,
M.S., MT(ASCP)
SM, is Supervisor
(BacteriologyMycology),
Veterinary
Center,
Diagnostic
University of
Nebraska Lincoln, East
Campus,
Lincoln,
Nebraska. V, Lyle
von Riesen, Ph.D.
is Professor of
Medical
Microbiology,
Department
of
Medical
Microbiology,
University of
Nebraska
Medical
Center, Omaha.
48
Most conventional biochemical determinations used to identify nonfermentative bacilli originally were developed for use with glucose fermenters
(Enterobacteriaceae). Whether the use of
such determinations is applicable to
differentiation of nonfermentative bacilli
is questionable if speciation is not
achieved; therefore, the search for more
appropriate tests must continue.
Because the source of many opportunistic bacteria is environmental, it may
be important to investigate the abilities
of individual isolates to metabolize
widely distributed, naturally occurring
compounds. Glycosides like arbutin,
esculin, and salicin occur in low concentrations in all plants, especially in fruits,
seeds, roots, bark, and leaves. 2 Therefore, they are present in spices, vegetable dyes, and drugs. 3
Structurally, glycosides are sugar
ethers in which the reducing or potential
aldehyde group at carbon one of the
sugar p o r t i o n , the glycone, is condensed
with a hydroxyl group of a nonsugar
component, the aglycone or genin.
Enzymic hydrolysis of the glycosidic
linkage (alpha or beta) results in liberation of these constituents.
As early as 1928, Buchanan and Fulmer 4
suggested that glycosides might be
useful differential substrates. The most
extensive study of the abilities of gramnegative bacteria to hydrolyze glycosides, however, was based upon acid
production from liberated glycone by
fermentative species. 3 The only aglycone-detecting tests thus far developed
are those for determining hydrolysis of
amygdalin," esculin, and the synthetically
prepared beta galactoside ONPG. The
aglycone test for esculin hydrolysis,
often described previously, is traditionally performed in basal media used
for fermentative species.
Since information regarding the abilities of nonfermentative bacilli to hydrolyze the three most c o m m o n l y used beta
glucosides (arbutin, esculin, and salicin)
was incomplete, we elected to develop
aglycone tests for use with these substrates and to screen our nonfermentative bacilli with t h e m . Potassium hydroxide (KOH), used in the performance
of Voges-Proskauer tests, was used to
detect h y d r o q u i n o n e released f r o m
arbutin. Ferric a m m o n i u m citrate was
used to detect esculetin liberated f r o m
esculin. A ferric chloride reagent was
used to detect saligenin released f r o m
salicin.
0007-5027/78/0800/0048 $00.60 © American Society of Clinical Pathologists
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The identification of many nonfermentative gram-negative, opportunistic
agents of infection has become a difficult
task for even the most knowledgeable
referees. Despite continued efforts at
characterization of a large collection of
nonfermentative bacilli assembled by the
late Elizabeth O. King and her successors
at the Center for Disease C o n t r o l , many
strains are still unnamed. 1 Biochemical
tests that w o u l d enable differentiation
of many species and groups are still
needed.
Materials and Methods
Test Organisms
BasaJ Media
A growth medium to which the
glucosides could be added was
formulated to resemble the OF
medium of Hugh and Leifson; 7 it
contained 0.2% Trypticase (BBL),
0.5% sodium chloride, and 0.03%
dipotassium phosphate. The original OF medium was modified by
omitting agar to obtain a liquid
medium (basal broth) or by adding
agar (1.5%) to obtain a solid
medium (basal agar); additionally,
the p H indicator was deleted.
GJucosides
Arbutin was purchased from
Sigma Chemical Company, St.
Louis, M o . Esculin and salicin
were obtained from Difco Laboratories, Detroit, M i c h . The arbutin
(0.5%) and esculin (0.1%) were
added t o basal b r o t h ; ferric
ammonium citrate (0.05%) was
incorporated into the esculin
medium. The arbutin and esculin
broths were dispensed as 1-ml
aliquots into 13 by 100 mm screwcapped tubes. Salicin (0.5%) was
added t o basal agar and t h e
medium was dispensed as 2-ml
aliquots. All three media were
sterilized by autoclaving. The
her designation
Species
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Strains
acidovorans
alcaligenes
alcaligenes
cepacia
cepacia
diminuta
diminuta
fluorescens
fluorescens
maltophilia
maltophilia
putrefaciens
putrefaciens
No. 2306
No. 2110
ATCC 14909
No. 2106
ATCC 10856
No. 2101
ATCC 11568
No. 2335
ATCC 13525
No. 2107
ATCC 13637
No. 2149
ATCC 8071
salicin agar was allowed to solidify
in slanted position.
Detection of Hydroquinone
Liberated from Arbutin
Arbutin is hydrolyzed to yield
hydroquinone and glucose. Hyd r o q u i n o n e is autooxidized t o a
brown or black product; 2 , 8 it is
also rapidly oxidized by alkaline
solutions. 9 Consequently, it is
possible to use potassium hydroxide, readily available in bacteriology laboratories, to detect
reduced hydroquinone. In performing the test, duplicate tubes
of the water-clear arbutin broth
were inoculated with each test
strain; these and tubes of u n inoculated medium were incubated at 37° C and observed daily
for the appearance of b r o w n color
(autooxidized hydroquinone, a
positive test). After three days and
again after 10 days, tubes of
uninoculated control medium
and tubes of inoculated arbutin
medium that had failed to become
brown were examined for the
presence of reduced hydroquinone, which is colorless. Two
drops of 40% KOH was added to
each t u b e ; they were shaken
vigorously and observed for the
immediate development of b r o w n
color (also a positive test).
Source
G. L Gilardi
G. L, Gilardi
ATCC
G. L. Gilardi
ATCC
G. L. Gilardi
ATCC
G. L Gilardi
ATCC
G. L. Gilardi
ATCC
G. L. Gilardi
ATCC
enables detection of esculetin
released upon hydrolysis of esc u l i n . Tubes i n o c u l a t e d w i t h
each test strain and tubes of
uninoculated esculin broth were
incubated at 37° C and observed
from a few hours to 10 days for
blackening due to formation of
the hypothetical esculetin-ferric
ion chelate. 10
Detection of Saiigenin Liberated
from Saiicin
Salicin hydrolysis yields glucose
and saligenin (salicyl alcohol).
Saligenin, soluble in alcohol, gives
a stable violet color with ferric
chloride, while salicin gives no
color; 1 1 therefore, 10% ferric chloride in anhydrous methanol to
detect saligenin released from
salicin is recommended. Duplicate
salicin slants were inoculated with
each test organism; these and
tubes of uninoculated salicin agar
were incubated at 37° C. After
three days and again after 10 days
of incubation, an uninoculated
slant and slants inoculated with
each test strain were flooded w i t h
four to five drops of ferric chloride
reagent and observed for the
development of a violet-gray t o
purple color in both reagent and
medium (positive test).
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A collection of 390 strains of
nonfermentative, gram-negative
bacteria was tested. The collection
contained organisms obtained
from clinical and hospital environment specimens, organisms submitted t o both clinical and medical
reference laboratories as bacteriology check samples, and several additional reference strains
(Table I). Before inoculation into
glucoside-containing media, the
test strains were reisolated t o
blood agar plates and subcultured
to Trypticase soy agar (Baltimore
Biological Laboratories, Cockeysville, MD.) slants; these were
incubated at 37° C for 24 hours.
Growth from the slants was used
to inoculate test media.
Table I —
Results
Detection of EscuJetin Liberated
from Escuiin
The addition of ferric ammonium
citrate t o our esculin medium
The screening of 390 nonfermentative gram-negative bacterial
strains using the described aglycone tests revealed 44 strains
LABORATORY MEDICINE • VOL. 9, NO. 8, AUGUST 1978
49
Table II—A Comparison of Results Given by Glucosidolytic Nonfermenters
within Three and 10 Days of Incubation
Arbutin
lin
Sal cin
Species or CDC Group
3d
10d
3d
10d
3d
10d
Uninoculated medium
Achromobacter Vd (3)*
Flavobacterium sp. (3)
F. meningosepticum (1)
Pseudomonas cepacia (4)
P. maltophilia (23)
Resembles P. maltophilia (1)
Resembles P. pseudoalcaligenes (1)
Resembles Group Ilk (4)
Resembles Group M-4f (1)
Vibrio extorquens
ot
0
3
1
1
1
19
1
1
4
1
1
0
0
3
1
1
23
1
0
4
1
1
0
1
3
1
1
23
1
1
4
1
1
0
1
0
0
4
21
1
1
4
1
1
0
3
0
0
4
22
1
1
4
1
1
* Number of strains tested.
t Total number of strains which be-
Results of the aglycone determinations on the 44 glucosidolytic
strains are presented in Table II.
Twenty-three of the strains were
P. maltophilia, and one additional
hydrolyzer biochemically resembles that species. Four glucosidolytic strains were P. cepacia and
four were species of Flavobacterium. Flavobacterium
meningosepticum (a single strain) hydrolyzed glucosides, as did four
strains which are biochemically
similar t o , but not identical w i t h
Pseudomonas-like Group Ilk. The
flavobacteria were singularly unable to attack salicin.
Three strains of Achromobacter
Group V d , a single strain of
Vibrio extorquens, one strain which
biochemically resembles P. pseudoalcaligenes,
and an organism
which biochemically resembles
Group M-4f also attacked the sub-
0
1
1
0
19
1
0
4
1
1
came posit ve with in des gnated incubation period.
strates. All of the glucosidolytic
strains attacked esculin and/or
salicin.
Discussion
The sporadic use of glycosides
as substrates is reflected in the
work of current collectors 1 2 and
contributors to the " M a n u a l of
Clinical Microbiology." 1 1 3 Although
these workers have used the
glycosides esculin, ONPG, and
(infrequently) salicin to characterize nonfermenters, they fail
to list these compounds together
among substrates. Esculin and
salicin continue to be listed as
carbohydrates. 1 1 3 Esculin is also
found listed randomly, 1 , 1 2 1 3 as is
ONPG. 1 3 The various authors may
not realize that these substrates
are related; still, strains of P. pseudomallei,
P. cepacia, P. putrefaciens, P. maltophilia,
P. vesicularis, Groups Ilk (biotypes 1 and
2) and Ve (biotype 1), F. meningosepticum, Flavobacterium species,
Groups l i d , lie, l l h , and Hi,
Xanthomonas and Achromobacter
species, and Vibrio
extroquens
have been reported to hydrolyze
esculin. 1 1 2 1 3 O n l y strains of P.
pseudomallei
and Group Ilk (biotypes 1 and 2) and Ve (biotype 1)
have been reported to hydrolyze
salicin. 1,13
LABORATORY MEDICINE • VOL. 9, NO. 8, AUGUST 1978
Additional data 14 indicate that
beta glucosides and beta galactosides are the beta glycosides most
frequently hydrolyzed by our nonfermentative bacilli. More of our
strains attack beta glucosides than
split beta galactosides, and all
strains which attack beta galactosides (including ONPG) also hydrolyze beta glucosides. Therefore, glycosidolytic nonfermentative bacilli can be differentiated
from nonglycosidolytic strains by
determining their capabilities of
hydrolyzing beta glucosides. We
have developed four aglycone
tests (more appropriate for use in
the characterization of nonfermentative bacilli) that can be used
to detect glucosidolytic ability of
these organisms. Among these are
tests for detecting the aglycones
of amygdalin, 6 arbutin, esculin,
and salicin.
The phenol glucoside arbutin
has been used by zymologists to
confirm beta-glucosidase activity
of yeasts15 and by Twort 5 to characterize fermentative species of
bacteria. To our knowledge, however, the capabilities of nonfermentative bacilli to split arbutin
have not been determined previously. Forty percent KOH can
be used to determine arbutin
hydrolysis. M e d i u m inoculated
with organisms that release hydroquinone become light brown to
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able to hydrolyze one or more
of the three most c o m m o n l y
used beta glucosides. A m o n g the
organisms which failed to attack
the substrates were strains of
Acinetobacter
calcoaceticus
var.
anitratus,
Acinetobacter
calcoaceticus var. Iwoffii,
Pseudomonas
aeruginosa, P. putida, P. acidovorans, P. stutzeri, P. putrefaciens,
P. diminuta, P. testosteroni,
Alcaligenes spp., and Bordetella bronchiseptica.
50
ESCL
In this study of glucosidolytic
ability, strains of P.
maltophilia,
P. cepacia, P.
pseudoalcaligenes,
Achromobacter and Flavobacterium species, F.
meningosepticum, Vibrio extorquens, and some
thus far unidentifiable strains,
were f o u n d capable of attacking
one or more of the three glucosides. No glucosidolytic strains of
P. pseudomallei,
P.
vesicularis,
Pseudomonas-like Group Ve, Flavobacterium-like Groups Mb, lie,
l l h , and Hi, or Xanthomonas
species were encountered; these
organisms are rarely isolated, and
our collection probably does not
include t h e m .
brown-black d u r i n g incubation
(auto-oxidation) or immediately
following reagent addition and
aeration. Color intensity is variable, suggesting that the depth of
color obtained is dependent u p o n
the quantity of h y d r o q u i n o n e
liberated by individual strains.
It is possible to obtain false
positive esculin hydrolysis tests
from hydrogen sulfide (H 2 S)-producing nonfermentative bacilli such
as P. putrefaciens. However, strains
of this species are rarely isolated
f r o m clinical specimens and these
w o u l d have been observed to
produce H2S in routine screening
media such as triple sugar iron
agar.
The alcohol glucoside salicin
has been used extensively in the
characterization of fermentative
microorganisms, yet relatively few
investigators have used this sub-
N o n e of our g l u c o s i d o l y t i c
strains failed to hydrolyze both
esculin and salicin, although some
attacked one glucoside and not
the other. Therefore, the abilities
of nonfermentative bacilli to hydrolyze glucosides should probably be determined using both of
these substrates. Since the flavobacteria were the only glucosidolytic organisms that failed to attack
salicin, their characteristic inability
to hydrolyze the substrate may
have differential significance.
Glucosidolytic organisms could
be subdivided according to their
esculin/salicin hydrolysis patterns
as + / + , + / - , or, perhaps, - / + .
Strains in each subdivision could
then be further characterized
using additional appropriate biochemical tests. Most strains falling
into the + / + subdivision, in our
experience, w o u l d be easily identifiable as P. maltophilia,
one of
the most frequently isolated nonfermentative species. Strains having the + / - pattern might be
presumptively identified as flavobacteria.
References
1. Tatum, H.W., W.H. Ewing and R.E. Weaver,
1974. Miscellaneous gram negative bacteria,
p, 270-294. In Manual of Clinical Microbiology. 2nd edition. Edited by Lennette,
E.H., EH. Spaulding, and J.P. Truant. Washington, D C , American Society for Microbiology.
2. Mcllroy, R.J., 1950. The plant glycosides.
London, Edward Arnold and Co.
3. Claus, E.P., 1970. Pharmacognosy, 6th edition, Philadelphia, Lea and Febiger.
4. Buchanan, RE., and E.I. Fulmer, 1930.
Physiology and Biochemistry of Bacteria.
Baltimore, Williams and Wilkins.
5 Twort, F.W., 1907. The fermentation of glucosides by bacteria of the typhoid-coli group
and the acquisition of new fermenting powers
by Bacillus dysenteriae and other microorganisms. Proc. R. Soc, London, Ser. B:
79:329-336.
6. Frank, S.K. and V.L. von Riesen. Paper strip
test for detecting hydrolysis of amygdalin by
nonfermentative, gram-negative bacilli. Unpublished data.
7. Hugh, R., and E. Leifson, 1953. Thetaxonomic
significance of fermentative versus oxidative
metabolism of carbohydrates by various
gram-negative bacteria. J. Bacteriol. 66:
24-26.
8. Armstrong, E.F., 1924. The carbohydrates and
the glucosides, 4th edition. New York,
Longmans, Green and Co.
9. Plimmer, R.H.A., 1926. Practical Organic and
Biochemistry. New York, Longmans, Green
and Co.
10. Blazevic, D.J. and G.M. Ederer, 1975. Principles of Biochemical Tests in Microbiology.
New York, John Wiley and Sons.
11. Criddle, W.J. and G.P. Ellis, 1967. Qualitative organic chemical analysis. New York,
Plenum Press.
12. Weaver, RE., H.W. Tatum and D.G. Hollis,
1972. The identification of unusual pathogenic
gram negative bacteria (Elizabeth O. King),
preliminary revision, September, 1972. Center
for Disease Control, Atlanta, Ga.
13. Hugh, R., and G.L Gilardi, 1974. Pseudomonas, p. 250-269. In Manual of Clinical
Microbiology. 2nd edition. Edited by Lennette,
E.H., E.H. Spaulding, and J.P. Truant (ed.),
Washington, D.C., American Society for
Microbiology.
14. Frank, S.K., 1977. Masters thesis, University
of Nebraska Medical Center, Omaha,
Nebraska.
15 Lodder, J., 1970. The Yeasts. Amsterdam,
North Holland Publishing Co.
•
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Although reagent addition and
aeration intensifies b r o w n colors
resulting f r o m auto-oxidation, it is
not necessary to add reagent to
determine that such tests are positive. Uninoculated control medium
and arbutin medium containing
nonfermentative bacilli that d o
not hydrolyze arbutin never develop color d u r i n g incubation
or after addition of reagent. Strains
which produce brown pigment
could conceivably give false positive tests; however, pigment production should have become obvious, for example, on media used
in performing susceptibility tests.
strate t o identify nonfermentative
species. O u r results indicate that
the aglycone test for hydrolysis
of salicin can detect saligenin
liberated by nonfermentative bacilli. A l t h o u g h the test procedure
requires addition of a reagent,
it is easily p e r f o r m e d , more economical, possibly more rapid, and
definitely more easily interpreted
than conventional acid-detecting
tests in which this substrate is
used.
•
Happy poly. May others appreciate
that not all differentials are boring.
Submitted by Maggie Diulio, Raymond Felle and Virginia Felle.
•
URL
1: ^,
LABORATORY M E D I C I N E • VOL. 9, NO. 8, AUGUST 1978
51