Laboratory Evaluation of the Serum Dilution Test
in Serious Staphylococcal Infection
MARIO J. MARCON, PH.D. AND RAYMOND C. BARTLETT, M.D.
Four test media were studied to determine performance characteristics of serum dilution tests used to monitor antimicrobial
therapy during serious Staphylococcus aureus infection being
treated with highly protein-bound antibiotics. Serum inhibitory
titers and serum bactericidal titers obtained with Mueller-Hinton broth supplemented with calcium and magnesium were 3to 16-fold higher than titers obtained with whole human serum
buffered with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). In cation-supplemented Mueller-Hinton
containing 5% albumin or in cation-supplemented MuellerHinton combined with an equal volume of human serum, titers
were 2- to S-fold higher than in whole human serum buffered
with HEPES. Clinical or animal studies are needed to establish
whether the higher titers observed with patient serum containing highly protein-bound drugs diluted in low protein-content media would foster inadequate dosage regiments. In the
meantime, both infectious disease clinicians and microbiologists should be aware of this potential pitfall. (Key words:
Serum dilution test; S. aureus; Protein binding) Am J Clin
Pathol 1983; 80: 176-181
THE SERUM DILUTION TEST (SDT) is considered
useful for guiding therapy and establishing prognosis in
the treatment of bacterial endocarditis, osteomyelitis,
and other infections.3-6-9101519-20'2528 Logically, the serum
inhibitory titer (SIT) or bactericidal titer (SBT) displayed
by serum obtained from an infected patient during therapy should approximate the results of dividing the serum
concentration of the antimicrobial agent8,21 by the minimum inhibitory concentration (MIC) 27 or minimum
bactericidal concentration (MBC) of the organism to the
agent being administered (concentration/MIC or MBC
ratio).
Higher SDT titers could be anticipated in patients
being treated with drugs that are bound highly to serum
proteins if the patient's serum is serially diluted in broth
instead of a serum-containing medium. The difference
in titers reflects the fact that only free, unbound drug
is available for biologic activity. 5 " 12,26 This could result
in a false sense of security during the treatment of a
patient with a highly protein-bound antimicrobial agent.
While a bactericidal titer of 1:8, generally considered
adequate therapy, might be observed in a SDT performed with serial dilution of the patient's serum in
Received November 2, 1982; accepted for publication December
6, 1982.
Address reprint requests to Dr. Marcon: Laboratory Division, Microbiology Section, Children's Hospital, Columbus, Ohio 43205.
Division of Microbiology, Department of Pathology, Hartford
Hospital, Hartford, Connecticut
broth medium, a titer of only 1:2 might be observed
with serial dilution and incubation in 100% pooled, normal human serum.
In 1974, Pien and Vosti surveyed the performance of
the SDT in 26 laboratories and found substantial variation in performance of the procedure. 13 Only two of
the 26 laboratories diluted patient serum with pooled
human serum instead of broth, and there were variations
of the culture media used, the dilution method, the inoculum density, the time of incubation, the method of
subculture, and definition of the bactericidal endpoint.
Three years later in 1977, Stratton and Reller recommended performance of the SDT in Mueller-Hinton
broth supplemented with calcium and magnesium and
combined with heated, pooled human serum at a final
concentration of 50%.22-23 They found that bacteria grew
better in this medium than in 100% serum, and fluctuations in pH could be controlled because of the presence of buffers in the broth.
There is no present evidence that these recommendations have led to a more standardized approach to the
performance of this test.
We conducted a study of the correlation between the
SDT, the assayed serum concentration, and the MIC
and MBC of the infecting organism in 15 cases of bacterial endocarditis, osteomyelitis, or deep-tissue abscess
caused by Staphylococcus aureus. All patients were
treated with oxacillin, cefazolin, or dicloxacillin, antimicrobial agents with greater than 80% protein binding,
or with cephalexin, an agent with less than 50% protein
binding.5 " We wished to determine whether SDT results would agree with the concentration/MIC or MBC
ratio if the MIC or MBC test were conducted in a medium containing physiologic concentrations of human
serum protein. We wished to investigate also the effect
of using various methods of establishing physiologic protein concentrations in the SDT. Albumin appears to be
the primary binding site for most protein-bound antimicrobial agents, but differences have been reported in
the binding of penicillins to whole serum and albumin. 26
Use of concentrated human serum albumin in the SDT
0002-9173/83/0800/0176 $01.10 © American Society of Clinical Pathologists
176
vol. 80-No. 2
SERUM DILUTION TEST FOR 5. AUREUS
is advantagenous because it could be added to broth
medium to produce a physiologic concentration of albumin approximating that found in human serum,
while maintaining the nutritional and buffering attributes of the broth medium. Dilution of broth with
serum, however, results in a reduction both in the concentration of protein and broth components.
Materials and Methods
Culture Media for SDT
(1) Mueller-Hinton broth supplemented with calcium and magnesium (MHB-Cat): This medium was
prepared from dehydrated Mueller-Hinton broth base
(lot 8206) provided by Scott Laboratories, Fiskeville, RI.
It was supplemented with calcium chloride and magnesium chloride to achieve afinalcalcium concentration
of 9.5 mg/dL and magnesium of 2.2 mg/dL. The pH
was adjusted to 7.4.
(2) MHB-Cat containing 5% human serum albumin
(MHB-Cat-HA): This medium was prepared by adding
concentrated normal human serum albumin (American
Red Cross Services, Washington, D.C.) to MHB-Cat to
produce a final albumin concentration of 5 g/dL.
(3) MHB-Cat combined with pooled human serum
(MHB-Cat-HS): This medium was prepared by adding
pooled human serum to MHB-Cat in a 1:1 ratio.
(4) Pooled human serum with HEPES buffer (HSHEPES): Pooled, normal human serum was buffered to
a p\\ of 7.4 at 35°C with the addition of HEPES (N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid, Calbiochem-Behring Corp., La Jolla, CA) in a final concentration of 0.05 M. This buffer was prepared by adding
equal portions of 0.5 M HEPES acid and 0.5 M HEPES
sodium salt, which previously had been filter sterilized
and stored at 4°C, to nine parts of normal pooled human
serum. The final serum concentration of this medium
was 90% of normal whole human serum. HEPES buffer
was selected because of its excellent buffering capacity
in the physiologic pH range and its negligible interaction
with cations.
Normal Pooled Human
177
Staphylococcus aureus Isolates
S. aureus isolates were obtained from specimens submitted to the Microbiology Laboratory from patients in
Hartford Hospital. S. aureus ATCC 25923 (American
Type Culture Collection, Rockville, MD) was used for
control purposes. Isolates were stored at -70°C in tryptic digest casein soy (TS) broth containing 15 g/dL of
glycerol. Fresh subcultures were prepared and maintained for up to 2 weeks on TS agar slants, which were
stored at 4°C. Isolates were prepared for testing by subculture to blood agar with overnight incubation.
Antimicrobial Agents
The antimicrobial agents used in this study included
cefazolin (Smith, Kline and French Laboratories, Philadelphia, PA), cephalexin (Eli Lilly and Co., Indianapolis, IN), oxacillin, and dicloxacillin (Bristol Laboratories, Syracuse, NY). Concentrated stock solutions were
stored at -70°C.
Antimicrobial Assays
Assays of antimicrobial serum concentrations were
performed by the method of Sabath and associates16"18
using a Bacillus subtilis (ATCC 6633) spore suspension2
and Antibiotic Medium #1 (Difco Laboratories, Detroit,
MI). Antimicrobial standards were prepared in human
serum.
Antimicrobial-containing Serum Samples
Two types of serum samples were used: (1) normal
pooled human serum was "spiked" with antimicrobial
agents; or (2) serum samples were obtained from patients
undergoing antimicrobial therapy. The spiked sera contained the following antimicrobial agents at the indicated final concentration; cefazolin (40 jig/mL), cephalexin (40 /ug/mL), oxacillin (20 Mg/mL), and dicloxacillin (20 fig/mL). The antimicrobial agents and
concentrations chosen represent agents displaying varying degrees of protein binding at levels commonly
achieved by parenteral or oral dosage.
Serum
Two sources of normal pooled human serum were
used interchangeably with comparable results. The sera
were obtained commercially (Flow Laboratories, McLean, VA; lot 29301012) or were collected from laboratory volunteers and heated for 30 minutes at 56°C to
inactivate complement activity. It then was clarified by
centrifugation to remove particulate matter and lipids.4
Neither serum pool demonstrated inhibitory activity
against any of the staphylococcal strains used in this
investigation.
Serum Dilution Test
One hundred microliters of serum was placed in the
first well of each 12-well row of a microdilution plate.
Fifty fiL of each of the four test culture media was added
to the remaining wells in the plates. Sera were diluted
serially with each of the media using a semiautomatic
microdiluting device (Dynatech Laboratories, Inc., Alexandria, VA). An additional 50 /xL of test medium was
added to the wells, yielding a final volume of 100 /iL per
well. Log phase bacterial suspensions of clinical S. au-
MARCON AND BARTLETT
178
Table 1. Concentration of Total Protein, Albumin,
Ca++, and Mg++ in Test Media and
Whole Human Serum
ing transfer of this amount. In practice, the highest dilution demonstrating no observable growth was considered the bactericidal titer. Tests were performed in quadruplicate in all cases. When three of four determinations
agreed, the latter was disregarded. If two pairs of determinations agreed within each pair but not between pairs,
the results were recorded as a range. Tests were repeated
when any other type of disagreement was observed.
Concentration
Medium
Total Protein
(g/dl)
Albumin
(g/dl)
Ca + +
(mg/dl)
Mg ++
(mg/dl)
MHB-Cat*
MHB-Cat-HA
MHB-Cat-HS
HS-HEPES
HSf
0.3
5.0
4.1
6.8
6.8-8.4
<0.01
4.8
2.5
4.9
3.7-5.2
8.5
9.4
8.9
8.6
8.8-10.4
2.3
2.2
2.2
1.8
1.8-2.4
A.J.C.P. • August 1983
MIC and MBC Tests
MICs and MBCs were determined by a standardized
microdilution procedure,7 except that HS-HEPES was
used as the test medium. The MBC was determined by
subculture and colony count assay using a disposable
inoculator and employing the same bactericidal endpoint as was described above.
• See "Materials and Methods" for explanation of abbreviations,
t Reference intervals for human serum. Pathology Laboratory, Hartford Hospital, Hartford,
Connecticut, 1980.
reus isolates or S. aureus ATCC 25923 were prepared,
containing approximately 108 bacteria/mL by visual
comparison with a 0.5 McFarland siandard, and were
diluted in water to a density of 5 X 106/mL. Suspensions
were placed in a microdilution inoculum tray (Dynatech) and 10 /*L (±3 nL) was transferred with a handheld disposable inoculator (Dynatech) to each of 96 wells
in a microdilution plate (Dynatech) containing 100 juL
of medium/well. Final bacterial concentration in the
wells was 2-5 X 105/mL, as determined by subculture
and colony-count assay. Plates were sealed with transparent tape (Linbrow Scientific, Inc., Hamden, CT) and
were incubated at 35°C for 20-24 hours. Inhibitory endpoints were observed with an illuminated viewer (Dynatech). Bactericidal endpoints were determined by agitating plates with a vortex laboratory mixer for 5-10
seconds, removing the sealing tape, and subculturing 10
JIL from each well to Mueller-Hinton agar plates using
disposable hand-held inoculators. Considering a final
inoculum of 2-5 X 105/mL, the generally accepted definition of the bactericidal endpoint of 99.9% killing'
would require recovery of less than 2-5 colonies follow-
Results
Chemical Analysis of Test Media
MHB-Cat contained only a small percentage of the
total protein and albumin found in normal human
serum (Table 1). MHB-Cat-HS contained about onehalf of the normal serum concentration of total protein
and albumin. MHB-Cat-HA and HS-HEPES more
closely resembled normal human serum in total protein
and albumin content. All test media contained physiologic concentrations of calcium and magnesium cations.
Evaluation of "Spiked" Serum Samples
The SIT and SBT against the 12 S. aureus strains
tested were highest in samples containing oxacillin, cefazolin, and dicloxacillin when MHB-CAT was used and
lowest when HS-HEPES was used (Table 2). MHB-CatHA and MHB-Cat-HS produced intermediate results.
Table 2. Replicate SIT and SBT of Pooled Human Serum Spiked with One of Four Antimicrobials
and Tested against 12 Strains of 5. aureus in Four Media*
SIT and SBT in Four Mediaf
MHB-Cat-HA
MHB-Cat
Antimicrobial and lest
Concentration (jig/mL)
Oxacillin
(20)
Cefazolin
(40)
Dicloxacillin
(20)
Cephalexin
(40)
MHB-Cat-HS
HS-HEPES
SIT
SBT
SIT
SBT
SIT
SBT
SIT
SBT
128
64
32
32
16
16
8
8
64-128
32-64
32
16-32
32
32
16
16
128
64-128
32
16
16
16
8
8
16
8-16
16
8-16
16
8-16
16
• See "Materials and Methods" for explanation of abbreviations.
t Serum inhibitory and bactericidal titers given as reciprocals of serum dilution and rep-
8-16
resent modal values: where no single mode value was apparent, data is given as a two-fold
range of the most frequently occurring values.
SERUM DILUTION TEST FOR 5. AUREUS
Vol. 80 • No. 2
No difference was obtained among the different test
media when serum containing cephalexin was tested
against the S. aureus strains.
Evaluation of Patient Sera
Results similar to those determined with "spiked"
sera were obtained with sera collected from 15 patients
undergoing antimicrobial therapy, and these are displayed in Table 3. The SIT and SBT were highest in
MHB-Cat and lowest in HS-HEPES, with the other
media producing intermediate results. No difference was
observed in testing sera from patients being treated with
cephalexin.
Correlation between Serum Antimicrobial
Concentration/MBC Ratio and SBT
Serum antimicrobial concentrations were determined
on the patient sera displayed in Table 3, and antimicrobial concentration/MBC ratios were computed by
dividing the concentration by the MBC of each patient's
S. aureus isolate as performed in HS-HEPES. The ratios
were correlated with actual SBTs (Table 3), which were
obtained with the four test media. For each antimicrobial tested, there was a good linear relationship between
the concentration/MBC ratio and the SBT performed
in all four media (Fig. 1). When SDTs were performed
179
in MHB-Cat, the SBT greatly overestimated (3- to 16fold) the concentration/MBC ratio for cefazolin, oxacillin, and dicloxacillin, but not cefalexin (panel A). The
SBT also overestimated (2- to 5-fold) the ratio for the
three former drugs when SDTs were performed in MHBCat-HA (panel B) or MHB-Cat-HS (panel C). However,
the concentration/MBC ratio correlated well with SBTs
when the SDTs were performed in HS-HEPES (panel
D). Comparable observations were noted for the concentration/MIC ratio and SIT (data not shown).
Discussion
The serum dilution test generally is accepted as a useful guide to the adequacy of antimicrobial therapy in
serious infections such as bacterial endocarditis and osteomyelitis.36910151920-2528 There has been a lack of
standardization of this test, and it is unclear from review
of reports whether any one test method correlates better
with clinical outcome. This investigation has confirmed
the work of others, which has established that substantially higher SITs and SBTs are obtained on patients
being treated with highly protein-bound antimicrobials
when the SDT is performed in a broth medium in contrast to human serum or.a medium containing a high
concentration of serum or albumin.14,22'23 Performance
of the test in human serum with the addition of HEPES
buffer provides conditions most closely resembling the
Table 3. Comparison of SIT and SBT of Patient Serum Collected While Undergoing Antimicrobial Therapy
and Tested against Patient's S. aureus Isolate in Four Media*
SIT and SBT in Four Mediat
MHB-Cat*
MHB-Cat-HA
MHB-Cat-HS
HS-HEPES
Antimicrobial
Agent
Patient
Serum
SIT
SBT
SIT
SBT
SIT
SBT
SIT
SBT
Oxacillin
1
2
3
4
5
128
256
256
256
128
128
256
256
256
128
64
128
128
128
32-64
64
128
128
64-128
32
32-64
128
64-128
32-64
32
32-64
128
64
32-64
16
16
32
32-64
32
16
16
32
32
32
8
Cefazolin
6A
6B
7
8
9
10A
10B
128-256
32
128-256
32
256
128
16-32
128
16-32
128
32
128
128
16
128
16
64
16
128-256
64
16
64-128
8
64
16
64-128
64
8
64-128
16
64
16
128-256
64
16
64
8-16
64
16
64-128
32-64
8-16
32-64
8
64
8
128
32-64
8
32
8
32-64
8
64
32
4
Dicloxacillin
11A
1 IB
12
16-32
256-512
128
8-16
128-256
64-128
4-8
64-128
16
4-8
64-128
16
4
32
16
4
32
8-16
2
16
4-8
2
8-16
4-8
Cephalexin
13
14
15A
1SB
32
8
16
2-4
16
8
8-16
2
32
8-16
16
2
16
8
8
2
32
16
16
2-4
16
8
8-16
2
32
8
8-16
4
8-16
8
8
2
• See "Materials and Methods" for explanation of abbreviations.
t Serum inhibitory and bactericidal titers given as reciprocals of serum dilution and rep-
resent modal values; where no single mode value was apparent, data is given as a two-fold
range of the most frequently occurring values.
180
MARCON AND BARTLETT
10
100
SERUM BACTERICIDAL TITER
FIG. 1. Correlation of serum antimicrobial concentration/MBC performed in HS-HEPES with serum bactericidal titers for 15 clinical 5.
aureus isolates. Serum concentrations, MBCs, and bactericidal titers
were determined for cefalexin (•), cefazolin (O), oxacillin (•), and
dicloxacillin (D). A. Bactericidal titers were performed in MH-Cat (A),
MH-Cat-HA (B), MH-Cat-HS (C), or HS-HEPES (D), and are expressed as reciprocals of serum dilution. Titers previously recorded as
a range are plotted as arithemetic mean values. Regression lines were
constructed by visual inspection of the data points. The dashed lines
in each panel represent perfect correlation.
protein-binding conditions that occur in vivo, while assuring stabilization of pH during incubation of the test
organism. While there is no clinical data suggesting that
higher titers obtained in broth medium with the SDT
A.J.C.P. • August 1983
in patients being treated with highly protein-bound
drugs has lead to inadequate therapy and treatment failures, this risk certainly appears to exist, on the basis of
our observations and the reports of others.14,22-23
The results of this investigation further demonstrate
that, for highly protein-bound antimicrobial agents (e.g.,
dicloxacillin), the SIT and SBT determined in broth
medium may be 16 times greater than would be predicted based on the concentration/MIC or MBC ratio
when the MIC and MBC test is performed in a medium
most closely resembling human serum (HS-HEPES).
However, both SITs and SBTs showed good correlation
with these ratios when HS-HEPES was used as a diluent
in the SDT.
These data support the recent observations of Jordon
and Kawachi9 and Stratton and co-workers.24 The former investigators confirmed the usefulness of the SDT
as an indicator of successful therapy in bacterial endocarditis but observed that the SBT did not correlate well
with the serum antimicrobial concentration/MBC ratio.
They concluded that this result.probably was obtained
because the MBC was not determined in a serum-containing medium.9 The latter inyestigators showed that
better correlations were found between measured and
predicted SBTs (based on concentration/MBC ratios)
when both the SDT and MIC/MBC determinations were
performed in a supplemented broth-human serum medium.24
The present study has been limited to laboratory evaluation of in vitro methods arid media on S. aureus isolates from patients with serious systemic infection. Although generalized conclusions regarding testing of
other organisms cannot be made, our results have very
practical applications because (1) the SDT is commonly
used to assess adequacy of antistaphylococcal therapy,
and (2) highly protein-bound drugs are most likely to
be used by themselves when treating S. aureus infections.
Human serum buffered with HEPES appears to be
a suitable and desirable medium for evaluation of antistaphylococcal therapy in the SDT. Because human
serum is not a uniform product, new batches or lots of
serum must be screened to insure satisfactory growth of
control S. aureus isolates. It must be emphasized that
HS-HEPES should not be used to evaluate other organisms, whose growth in this medium has not been assessed, or the activity of other drugs (e.g., aminoglycosides) that could be affected by the presence of HEPES.
At this point, it is still unclear as to which SDT result
(in terms of both method of performance and titer) best
correlates with a successful clinical outcome. Indeed,
results may differ in terms of disease entity being treated
and immune status of the patient. Further clinical and
animal studies with highly protein-bound drugs may in-
SERUM DILUTION TEST FOR S. AUREUS
Vol. 80 • No. 2
dicate whether use of certain SDT results may foster
inadequate dosage regimens. Until then, clinicians and
microbiologists alike should be aware of potential pitfalls
in interpretation of SDT results.
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