Journal of Antimicrobial Chemotherapy (1996) 37, 1103-1109
Influence of inoculum, medium and serum on the in-vitro susceptibility of
coagulase-negative staphylococci to teicoplanin and vancomycin
H. F. Kennedyt and D . V. Seal*
Department of Microbiology, Royal Hospital for Sick Children, Yorkhill NHS
Glasgow G3 8SJ, UK
Trust,
The susceptibility to teicoplanin of 100 coagulase negative staphylococci,
predominantly isolated from intravenous catheter tips and exit sites was determined
by agar dilution on IsoSensitest media with and without the addition of 20%
inactivated horse serum using an inoculum of lO'cfu/spot. Incorporation of
serum resulted in a three-fold increase in the geometric mean MIC and a fourfold increase in the geometric mean MBC. Further tests, performed in DST and
IsoSensitest medium supplemented with 20% inactivated horse serum, resulted in a
wide variation in teicoplanin MICs with differences in the geometric mean
MICs being up to 8.8-fold in serum media compared to unsupplemented media,
whereas the MICs of vancomycin were unaffected. Under the various experimental
conditions used, the susceptibility of the coagulase negative staphylococci to
teicoplanin varied from 31-100%, while all were consistently susceptible to
vancomycin. We therefore recommend that teicoplanin MICs be determined in the
presence of serum as these are better related to serum drug levels and reflect more
accurately the conditions in vivo. Trials of teicoplanin as monotherapy are required
to provide further insight into the relationship between its in-vitro antibacterial
activity and its clinical efficacy as the drug is easier to administer, better tolerated
and less toxic than vancomycin which might compensate for its reduced activity in
the presence of serum.
Introduction
Teicoplanin is a complex of high molecular weight glycopeptides produced by the
actinomycete, Actinoplanes teichomyceticus. The five major glycopeptide components
differ in their fatty acid side chains (Borghi et al., 1984). Teicoplanin is active against
aerobic and anaerobic Gram-positive bacteria (Williams & Gruneberg, 1984). The
major mechanism of action of teicoplanin involves binding to the terminal amino
acyl-D-alanyl-D-alanine of bacterial cell wall peptidoglycan precursors, thus inhibiting
cell wall synthesis (Somma, Gastaldo & Corti, 1984).
In serum, 90-95% of teicoplanin is bound to albumin (Assandri & Bernareggi,
1987) and the drug is probably also highly bound to tissue proteins. In contrast,
the glycopeptide antibiotic vancomycin is approximately 55% bound to serum
albumin (Krogstad, Moellering & Greenblatt, 1980). Serum protein binding of
•Present address: Tennent Institute of Ophthalmology, Western Infirmary, Church Street, Glasgow
G i l 6NT, UK.
tCorresponding author: Tel: +44-{141)-201-0417, Fax: +44-{141}-201-0836.
1103
0305-7453/96/061103 + 07 $12.00/0
% 1996 The British Society for Antimicrobial Chemotherapy
1104
H. F. Kennedy and D. V. Seal
antimicrobials can affect tissue distribution, delay elimination and interfere with
biological activity (Barre & Tillement, 1989). If serum protein binding affects
significantly the bactericidal activity of an antibiotic in vitro, it is essential to adopt an
appropriate dosage regimen in clinical therapy to ensure that the concentration of
free active antibiotic in vivo still exceeds the minimum inhibitory concentration (MIC)
against the infecting organism. We investigated the extent to which the presence of
serum influences the MICs and minimum bactericidal concentrations (MBCs) of
teicoplanin against 100 isolates of coagulase-negative staphylococci determined on
IsoSensitest agar. In addition, we assessed the influence of inoculum density and
media upon the MICs and MBCs of both teicoplanin and vancomycin determined in
the presence of horse serum.
Materials and methods
Bacterial strains
The coagulase-negative staphylococci had been isolated over the previous year from
blood cultures of 47 patients, the exit sites of 26 intravenous catheters, 21 catheter tips
and six specimens of cerebrospinal fluid. Each isolate was stored on nutrient agar slopes
as well as in Robertson's cooked meat medium and in liquid nitrogen. All isolates were
identified using the API Staph identification kit (API Laboratory Products Ltd.,
Basingstoke, Hampshire) immediately before determining their susceptibility and 87
isolates were identified as Staphvlococcus epidermidis, three each as Staphylococcus
haemolyticus and Staphylococcus hominis, two each as Staphylococcus saprophyticus and
Staphylococcus xylosus and one each as Staphylococcus cohnii, Staphylococcus simulans
and Staphylococcus warneri.
Antibiotics
Teicoplanin was supplied by Marion Merrell Dow, Lakeside House, Stockley Park,
Uxbridge, Middlesex UB11 1BE and vancomycin by Eli Lilly, Kingsclere Road,
Basingstoke, Hampshire RG21 2XA.
Determination of teicoplanin MICs and MBCs with and without incorporation of 20%
inactivated horse serum
MICs were determined by agar dilution of 0.25-100 mg/L teicoplanin in IsoSensitest
agar (Oxoid) with and without 20% inactivated horse serum (Scottish Biotechnology
Instrumentation, Bonnybridge, UK). The inoculum was prepared by diluting overnight
broth culture to a concentration of 107cfu/mL. Both series of plates were inoculated
using a multipoint inoculator which delivered 1 /iL of inoculum to yield 104cfu/spot.
Staphylococcus aureus NCTC 6571 was used as the control organism. Plates were
incubated for 18 h at 37°C. The MIC was defined by the lowest concentration to inhibit
no visible growth. The MBC was then determined by transferring an imprint of plates,
showing visible growth, using a sterile velvet pad to fresh unsupplemented IsoSensitest
medium. After overnight incubation, the MBC was defined as the lowest concentration
to inhibit visible growth.
1105
In-virro activity of teicoplanin
Determination of teicoplanin and vancomycin MICs and MBCs in the presence of
20% inactivated horse serum using different inoculum sizes and test media
The same procedure detailed above was also used to determine MICs and MBCs of
teicoplanin for the coagulase-negative staphylococci in the presence of 20% inactivated
horse serum using an inoculum of 10* cfu/spot on IsoSensitest agar and inocula of 104
and 10* cfu/spot on Diagnostic Sensitivity Test (DST) agar (Oxoid). For comparison,
the MIC and MBC of vancomycin was determined using inocula of 104 and 10* cfu/spot
on both IsoSensitest and DST agars containing 20% inactivated horse serum.
Results
Teicoplanin MICs and MBCs on IsoSensitest agar with and without serum using an
inoculum of 104 cfujspot
The geometric mean MICs were three-fold higher with the addition of serum. The range
of teicoplanin MICs on IsoSensitest agar supplemented with 20% inactivated horse
serum was 0.25—40 mg/L with a geometric mean MIC of 4.2 mg/L, whereas that
determined in the absence of horse serum was 0.25-5 mg/L with a geometric mean MIC
of 1.4 mg/L (Table I(a)).
There was a four-fold increase in the geometric mean MBCs. The MBCs determined
from IsoSensitest agar supplemented with 20% inactivated horse serum ranged from
0.25-100 mg/L with a geometric mean MBC of 9.4 mg/L. From MICs determined in
the absence of horse serum, the corresponding MBCs ranged from 0.25-8 mg/L with
a geometric mean MBC of 2.3 mg/L. (Table I(b)).
The National Committee for Clinical and Laboratory Standards (NCCLS, 1991)
recommends that an MIC of ^ 8 mg/L is indicative of susceptibility to teicoplanin.
Table I. (a) MICs of teicoplanin, in the presence or absence of 20% serum against 100
coagulase-negative staphylococci (inoculum: lO'cfu). (b) MBCs of teicoplanin against 100 CNS
Composition of
assay medium
IsoSensitest with
20% horse serum
IsoSensitest alone
Cumulative No. of strains (%) with stated MIC (mg/L)
0.25 0.5
1.0
2.0 2.5
5
8
10
20
40
1
3
9
32
34
65
8
12
25
66
76
100
83
87
98
100
Geometric
mean MIC
(mg/L)
4.2
1.4
(a)
Composition
of MIC assay
medium
IsoSensitest with
20% horse serum
IsoSensitest alone
Cumulative No. of strains (%) with stated MBC (mg/L)
0.25 0.5 i1.0 2.0 2.5 5 8 10 20 40 50 100
1
1
5
23
28
32 60 68
5
7
14
38
64
94 100
(b)
92
99
99
100
Geometric
mean MBC
(mg/L)
9.4
2.3
1106
H. F. Kennedy and D. V. Seal
Therefore in the absence of serum, all the isolates would be classified as susceptible, but
when the tests were performed in the presence of 20% inactivated horse serum, the
MICs of 17 of the isolates were > 8 mg/L and thus would be considered resistant. When
tested on IsoSensitest agar with 20% serum using an inoculum of 106 cfu, the MICs for
only 31 of the isolates were < 8 mg/L (data not shown) and therefore 69% of isolates
would be considered resistant.
Teicoplanin and vancomycin MICs and MBCs in the presence of 20% inactivated horse
serum using different inoculum sizes and test media
MICs of teicoplanin, determined in the presence of 20% inactivated horse serum, varied
more widely than did those of vancomycin when different inoculum sizes and test
media were used. The greatest difference in geometric mean MICs of teicoplanin was
between those determined on DST agar with an inoculum of Wcfu/spot (1.7 mg/L)
and those determined on IsoSensitest agar with an inoculum of 10* cfu (15 mg/L).
These results represent an 8.8-fold increase in geometric mean MICs. The respective
geometric mean MBCs of teicoplanin were 2.7 mg/L and 22.4 mg/L representing an 8.3
fold increase.
In contrast, there was little difference between geometric mean MICs of vancomycin,
determined in the presence of 20% inactivated horse serum. The greatest difference in
geometric mean MICs for vancomycin was between those determined on DST agar with
an inoculum of lO^fu/spot (1.8 mg/L) and those determined on IsoSensitest agar
with an inoculum size of 10*cfu/spot (3.6 mg/L) which represented only a two-fold
difference. The respective geometric mean MBCs of vancomycin also represented a
two-fold difference.
Discussion
Coagulase-negative staphylococci are now recognised as major nosocomial pathogens
in a variety of clinical situations, with S. epidermidis predominating in intravascular
line-associated infections partly because of its preponderance in the normal skin flora
and its ability to adhere to plastics and to produce extracellular slime material. The
resultant microbial biofilm appears to reduce the organism's susceptibility to both
antimicrobial agents and phagocytosis by the host and aids its adherence to the surface
of the intravascular line allowing the organism to grow in clusters (Raad & Bodey, 1992;
Peters, von Eiff & Herrmann, 1995).
The teicoplanin MIC results against 100 coagulase-negative staphylococci determined
on IsoSensitest agar with an inoculum of lO^cfu demonstrated reduced in-vitro
antimicrobial activity with the incorporation of 20% inactivated horse serum (from
100% to 83% susceptibility). As teicoplanin is very highly bound to serum albumin,
it is reasonable to hypothesise that serum protein binding of antibiotic may be involved
in this phenomenon. The in-vitro activities of other antibiotics, such as certain
penicillins and sulphonamides, are also influenced by their high degree of binding to
serum protein (Anton, 1960; Kunin, 1966). It has also been demonstrated, using a
mouse model, that the use of more highly protein bound isoxazolyl penicillins correlated
with decreased in-vivo activity against 5. aureus (Merrikin, Briant & Rolinson, 1983).
Exactly how protein binding affects antimicrobial activity in vivo is a controversial
subject and further research is required before its precise mechanism can be established.
In-vitro activity of teicoplanln
1107
One hypothesis is that protein binding may adversely affect the antibiotic's ability to
reach sites of deep seated infection (Wise, 1986). From the clinical point of view, of
paramount importance, is the amount of free active antimicrobial in serum and tissues,
which depends on the equilibrium between protein bound and non-protein bound drug.
As determination of serum levels of antibiotic provides an estimate of the amount of
total drug available, and teicoplanin is greater than 90% protein bound, less than 10%
of the measured level will comprise free, active antibiotic. For clinical efficacy, serum
levels of antibiotic should exceed the MIC. We therefore propose that the serum levels
of highly protein bound antibiotics such as teicoplanin should be related to the MIC
determined in the presence of serum.
The results of this study demonstrated how varying the inoculum size and test
medium influenced the MICs of teicoplanin. The geometric mean MIC of teicoplanin
for the isolates was 1.7mg/L when of lO'cfu/spot was inoculated onto DST agar
supplemented with 20% inactivated horse serum and all isolates were susceptible
whereas when IsoSensitest agar with 20% serum was inoculated with 10'cfu/spot,
the geometric mean MIC was 15 mg/L and only 3 1 % of isolates were susceptible (data
not shown). The precise reason for these conflicting results remains unclear. In general,
the MICs of teicoplanin on DST agar containing 20% inactivated horse serum were
also consistently lower than those determined on IsoSensitest agar containing 20%
inactivated horse serum (Table II). This finding is the opposite to that found by
Felmingham et al. (1987) since the MICs of teicoplanin determined in the absence of
serum were higher on DST agar and lower on IsoSensitest agar. In contrast, the MICs
of vancomycin were minimally affected by any variation in either medium composition
or inoculum size and showed all the isolates to be susceptible with MICs < 8 mg/L.
The task of relating in-vitro susceptibility to teicoplanin and clinical response is
complex. To help achieve standardisation, we recommend that serum should be
incorporated into the test medium to permit MICs to be related to serum drug levels.
Although we acknowledge that the choice of medium and inoculum that should be used
Table II. The effect of medium and inoculum on in vitro activities of teicoplanin and vancomycin
against 100 coagulase-negative staphylococci in the presence of 20% serum
Medium
ISO
ISO
DST
DST
Medium
ISO
ISO
DST
DST
Inoculum
(cfu)
104
106
104
10*
Inoculum
(cfu)
104
106
104
10*
Teicoplanin MIC (mg/L)
geometric mean
range
4.2
15.0
1.7
5.8
0.25-^40
1-100
0.5-8
1^0
Vancomycin MIC (mg/L)
geometric mean
range
1.9
3.6
1.8
3.4
0.5-5
2-5
0.5-5
1-8
ISO, IsoSensitest agar; DST, diagnostic sensitivity test agar.
Teicoplanin MBC (mg/L)
geometric mean
range
9.4
22.4
2.7
10.3
0.25-100
1-100
0.5-40
r-ioo
Vancomycin MBC (mg/L)
geometric mean
range
2.2
4.1
2.0
3.8
0.5-5
2-8
0.5-5
1-8
1108
H. F. Kennedy and D. V. Seal
involves a degree of compromise, we concur with the consensus of the Working Party
of the BSAC (1991), that IsoSensitest agar is preferred over other media and recommend
an inoculum of 104 cfu/spot. With a breakpoint of 8 mg/L (NCCLS, 1991), peak serum
levels of 30-40 mg/L of total teicoplanin should be sufficient to ensure a satisfactory
response to treatment of an infection caused by a susceptible species. We also agree with
the recommendation that serum levels of teicoplanin should be monitored to optimise
therapy by ensuring these levels are achieved (MacGowan et al., 1992; Wilson,
Griineberg & Neu, 1993).
While the results of this study demonstrate vancomycin's superior in-vitro activity
against coagulase-negative staphylococci, teicoplanin offers several advantages which
should be taken into account when considering treatment with a glycopeptide
(Felmingham, 1993). These include the drug's lower nephrotoxic potential (Davey &
Williams, 1991), once daily dosing (Rowland, 1990), the ability to administer the drug
by rapid injection intravenously or intramuscularly, and the absence of histamine
release which can result in the 'red-man syndrome' (Smith et al., 1989). However, given
the influence of the test conditions on the susceptibility of coagulase-negative
staphylococci, clinical trials are required to explore further the relationship between the
in-vitro antibacterial activity of teicoplanin and its clinical efficacy to see whether or
not its favourable pharmacological properties might compensate for its reduced activity
in the presence of serum.
Acknowledgements
This study was supported by a grant from Marion Merrell Dow Limited. We are
grateful to S. Robertson for her excellent technical assistance and to I. M. Kennedy
for useful discussion and typing the manuscript.
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{Received 13 February 1995; returned 21 March 1995; revised 7 June 1995;
accepted 10 February 1996)