Vol. 17, No. 1
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1983, p. 97-99
Copyright C 1983, American Society for Microbiology
Rapid Identification of Staphylococcus aureus and
Streptococcus pneumoniae from Blood Cultures
R. G. KNIGHT* AND D. M. SHLAES
Laboratory and Medical Services, Veterans Administration Medical Center, Cleveland, Ohio 44106
Received 28 June 1982/Accepted 28 September 1982
Simultaneous application of the lysostaphin sensitivity test for identification of
Staphylococcus aureus and the deoxycholate test for the identification of Strepto-
coccus pneumoniae was evaluated for reliability in rapid identification (1 h) of
these organisms from blood cultures by using BACTEC 6B and 7C bottles. The
procedure was applied to 127 cultures, 74 lysostaphin tests and 53 deoxycholate
tests. Lysostaphin-tested organisms included 23 S. aureus, 40 Staphylococcus
epidermidis, 1 Listeria sp., 1 Peptococcus sp., 2 Micrococcus sp., 2 diptheroids,
and 5 mixed cultures. Deoxycholate-tested organisms included 14 S. pneumoniae,
32 Streptococcus spp. (not S. pneumoniae), 1 Listeria sp., 1 Peptococcus sp., 2
diptheroids, and 3 mixed cultures. Analysis of the data revealed positive and
negative predictive values of 95.8 and 94%, respectively, for the lysostaphin test
and 100 and 97.4%, respectively, for the deoxycholate test. The combined tests
provide rapid reliable identification or elimination of S. aureus and S. pneumoniae
in blood cultures.
Rapid techniques for the identification of en-
teric gram-negative bacilli from blood and body
fluid cultures have been developed and are valu-
able aids to accurate therapy for those patients.
Similar techniques for gram-positive organisms
cross-linking pentapeptides in the cell wall of
staphylococci and attacks glycyl linkages more
avidly than other amino acid moieties (1, 6). This
has formed the basis ofthe lysostaphin solubility
test distinguishing staphylococci from micrococ-
ci and, by decreasing the concentration of en-
zyme, has also allowed rapid distinction of
Staphylococcus aureus from Staphylococcus
epidermidis (5, 7). Severance et al. have shown
this to be a reliable and rapid test by using 12
different commercial blood culture media, in-
cluding BACTEC (7, 8).
The deoxycholate solubility test is used rou-
tinely by many laboratories for identification of
adapted this technique for the identification ofS.
pneumoniae from blood cultures (4, 9). We have
studied a modification of these procedures
which yields reliable results within 1 h.
(2). Others have
MATERIALS AND METHODS
Reagents. Lysostaphin (Sigma Chemical Co., St.
Louis, Mo.) was obtained, prepared, and stored as
previously described by Severance et al. (7). Deoxy-
cholate (10%) was obtained from BBL Microbiology
Systems, Cockeysville, Md., and refrigerated at 2 to
8°C until needed.
erythrocytes were used to simulate blood when pre-
served strains were being tested. Three milliliters of
the reconstituted blood was aseptically injected into
each set (6B and 7C) of BACTEC bottles (Johnston
Laboratories, Cockeysville, Md.) before inoculation
with known stock strains of organisms.
Incubation and growth detection. Simulated blood
culture bottles inoculated with known strains of orga-
nisms were incubated at 35 to 37°C for 18 to 24 h. The
aerobic bottle was placed on a model R-2 reciprocator
(New Brunswick Scientific Co., Edison, N.J.) at 250
to 260 rpm, whereas the anaerobic bottles were not
agitated. All simulated cultures were checked for
growth by using a BACTEC 460 instrument (Johnston
Laboratories) before performing the lysostaphin and
deoxycholate procedures. Blood cultures positive for
gram-positive cocci were tested as soon as possible
Test procedure. The lysostaphin and deoxycholate
tests were done simultaneously, with the whole proce-
dure requiring approximately 1 h. Both tests were
done with sterile 13 by 100-mm screw-cap tubes. The
protocol was as follows. (i) Lysostaphin. One milliliter
of blood culture medium was removed from positive
bottles, with 0.5 ml being added to each of two test
tubes containing 4.5 ml of brain heart infusion broth,
which gave a 1:10 dilution (7). A solution of lysostaph-
in containing 57.4 U/ml was added to one tube in the
amount of 0.05 ml; the other tube was used as a
control. Each tube was blended in a Vortex mixer and
placed on the shaker used for BACTEC bottles inside
a 35 to 37°C incubator for 30 min.
(ii) Deoxycholate. One milliliter of blood culture
medium was removed from positive bottles, with 0.5
ml being added to each of two 13 by 100-mm sterile
tubes. A solution of 10%o deoxycholate was added to
cultures. Reconstituted packed
KNIGHT AND SHLAES
TABLE 1. Cultures tested
Streptococcus sp. (not
aThese mixed cultures included S. aureus and
group A beta-hemolytic streptococcus; S. aureus, S.
epidermidis, and diphtheroids (one in a 6B bottle and
one in a 7C bottle); S. aureus, Escherichia coli,
Klebsiella sp., Pseudomonas sp., and Clostridium sp.;
and S. epidermidis and S. pneumoniae.
bThese mixed cultures included S. epidermidis and
S. pneumoniae; groupAbeta-hemolytic streptococcus
and S. aureus; and Streptococcus milleri and Strepto-
one tube, and sterile distilled water was added to the
other tube, each in the amount of 0.5 ml. The tubes
were placed on the shaker used for BACTEC bottles
inside a 37°C incubator for 30 min.
(iii) Postincubation. All lysostaphin and deoxycho-
late tubes were removed from the incubator and
centrifuged for 10 min at 1,000 to 1,200 x g. The
supernatant was removed, and the pellet was resus-
pended. One drop from the tip of a Pasteurpipette was
used to make smears from test and control tubes. All
slides were allowed to air dry before being heat fixed
and gram stained.
(iv) Interpretation. The lysostaphin test was consid-
ered positive for S. aureus if greater than 90% lysis
occurred as compared with the control. This criterion
for a positive lysostaphin test was based on the work
of Severance et al. (7). Most positive lysostaphin tests
show 100% lysis, and negative tests have no lysis as
compared with the control. When necessary, percent-
age lysis was determined by comparing the average
number of organisms seen in several fields on the
control with treated cells. The deoxycholate test was
considered positive if organisms were present in the
control tube and absent in the tube containing deoxy-
cholate, as determined by gram stains made from each
Study design and organisms tested. A total of 127
cultures consisting of 92 strains of organisms were
tested. The total number ofcultures exceeded the total
number of strains because some strains were tested
from both the 6B (aerobic) and 7C (anaerobic) bottles.
Tests were performed from simulated cultures by
using 38 6B bottles and 38 7C bottles. Thirty-nine 6B
bottles and 12 7C bottles containing current clinical
isolates were tested. The lysostaphin tests included 41
simulated and 33 clinical cultures. The deoxycholate
test included 35 simulated and 18 clinical cultures. For
simulated cultures, we used organisms previously iso-
lated from blood cultures and frozen in skim milk at
-70°C. A list of cultures tested is shown in Table 1.
Although not shown, all organisms in Table 1 were
tested with both lysostaphin and deoxycholate. We
listed them separately, since interpretive values were
calculated independently for each test. The coagulase
test was used to confirm or rule out S. aureus;
morphological and biochemical tests were used for
other organisms in the lysostaphin test analysis. Orga-
nisms used in the deoxycholate test analysis were
identified by biochemical and serological methods.
Interpretive values. The interpretive values were
calculated by methods described by Griner et al. (3).
The positive predictive values, negative predictive
value, false-negative rate, and false-positive rate were
calculated for both the lysostaphin test and the deoxy-
cholate test independently of each other. The interpre-
tive values were based on comparison of the lyso-
staphin and deoxycholate tests against our standard
The BACTEC 6B and 7C bottles were compa-
rable in both the lysostaphin and deoxycholate
tests; therefore, data derived from 6B and 7C
bottles were combined.
Lysostaphin test. The one false-positive test
was a mixed culture of S. pneumoniae and S.
epidermidis in a 6B BACTEC bottle. This result
was attributed to technique (probable loss of
pellet while decanting), since the test was nega-
tive when repeated, and the same culture was
negative in a 7C BACTEC bottle run simulta-
neously. Other mixed cultures tested by the
lysostaphin test gave correct results (Table 2).
Three false-negative tests from pure cultures of
S. aureus were positive when repeated, again
suggesting technical error.
Deoxycholate test. The one false-negative test
was from the mixed culture of S. pneumoniae
and S. aureus previously discussed under the
lysostaphin test. The explanation for this result
is obvious, since staphylococci are deoxycho-
late insoluble. A repeat test was not done.
Although the data are not shown in Table 2, all
of the organisms tested with lysostaphin were
also tested by the deoxycholate test and vice
versa. Both tests were 100% reliable in this
respect, since none of the pure cultures used in
the lysostaphin test were positive by the deoxy-
cholate test, and none of the pure cultures used
in the deoxycholate test were positive by the
lysostaphin test. The mixed culture containing
S. pneumoniae and S. epidermidis was the only
exception in that the deoxycholate test was
falsely negative due to the presence of the S.
epidermidis. S. aureus and S. pneumoniae were
correctly identified or ruled out in all other
J. CLIN. MICROBIOL.
IDENTIFICATION OF S. AUREUS AND S. PNEUMONIAE
TABLE 2. Lysostaphin and deoxycholate test results
No. of samples
aRepeat tests were not used in calculating interpretive values. Total lysostaphin tests, 74; total deoxycholate
b The one false-positive lysostaphin test and one false-negative deoxycholate test were both due to a mixed
culture of S. epidermidis and S. pneumoniae.
c The three false-negative lysostaphin tests were positive when repeated.
mixed cultures listed in Table 1. The mixed
culture of S. aureus and group A beta-hemolytic
streptococcus was positive in the lysostaphin
test. Although contrary to expected results, this
was probably due to the relatively small number
of group A beta-hemolytic streptococci present
in this bottle. Results from all other mixed
cultures were consistent with expected results.
A reliable method for rapid identification of
gram-positive cocci from blood cultures has long
been needed. Severance et al. (7) found the
lysostaphin test to be such a method for deter-
mining the presence of S. aureus in cultures of
blood and other body fluids. They also reported
on the effects of various blood culture media on
sensitivity of staphylococci
Their study indicated that BACTEC 6B and 7C
bottles gave excellent results. However, the
study was limited to 10 strains of S. aureus and
10 strains of S. epidermidis for each of these
bottle types. Our study was done with 127
cultures in BACTEC 6B and 7C bottles (Table 1)
for both the lysostaphin and deoxycholate tests.
These bottles were equally satisfactory in our
The deoxycholate test is well established as a
standard procedure for presumptive identifica-
tion of isolated colonies of S. pneumoniae (2).
The previously published applications of this
technique to blood cultures have disadvantages.
The procedure of Wasilauskas and Ellner (9)
requires over 2 h. In our hands, the technique of
Murray (4) was associated with false-positive
identifications due to loss of deoxycholate-treat-
ed material from the slide during staining. Mur-
ray reported that up to 75% lysis could be seen
with streptococci other than S. pneumoniae.
The early detection allowed by the radiometric
method combined with our rapid identification
methods further reduced the time required for
identification of S. aureus and S. pneumoniae
from clinical material.
Mixed cultures of gram-positive cocci are
rarely encountered in true septicemia. However,
our results prove that this could lead to errone-
ous results with either the lysostaphin or the
deoxycholate test (especially if it is a combina-
tion of staphylococci and
Mixed cultures of staphylococci or streptococci
with clearly distinguishable gram-positive bacilli
or gram-negative bacilli present little problem,
since the tests are still interpretable.
In conclusion, the combined lysostaphin and
deoxycholate test provides a reliable method for
identification of S. aureus and S. pneumoniae
within 1 h of detection of growth by the BAC-
TEC radiometric method. Positive predictive
values were 95.8 and 100% for S. aureus and S.
We are grateful to the staff of the Cleveland Veterans
Administration Medical Center Microbiology Laboratory for
their support and to Johnston Laboratories for the BACTEC
bottles used in this study. We thank Ruth Kirk and Elizabeth
Williams for excellent secretarial assistance.
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VOL. 17, 1983