Download aureus and Enterococcus faecalis exposed to

0022-26 1 5/89/0030-0045/$10.00
J . Med. Microbiol. - Vol. 30 (1989), 4 5 4 9
Q 1989 The Pathological Society of Great Britain and Ireland
Sc a nning e Iec t ro nmic roscopy of Staphy o coccus
aureus and Enterococcus faecalis exposed to
daptomycin
LINDA J. WALE, A. P. SHELTON and D. GREENWOOD
Department of Microbiology and PHLS Laboratory, University Hospital, Queen's Medical Centre, Nottingham
NG72UH
Summary. The novel lipopeptide antibiotic, daptomycin, at a concentration of
8 mg/L, caused gross morphological changes in both a methicillin-sensitive and a
methicillin-resistant strain of Staphylococcus aureus and in a strain of Enterococcus
faecalis. The earliest (after 1 h) surface lesion observed was the appearance of bosslike processes randomly distributed on the cell surface. Later, grossly deformed
bacteria were seen and in two of the three bacteria prolonged exposure led to
degeneration of the cells into an amorphous syncytial mass. Omission of calcium
(which i s known to potentiate the activity of daptomycin) from the culture medium
did not affect the morphological response to an inhibitory concentration of the
antibiotic.
Introduction
Daptomycin (formerly known as LY146032) is a
novel lipopeptide related to the antibiotic complex
A21978C of Streptomyces roseosporus. The activity
of daptomycin is restricted to aerobic and anaerobic
gram-positive cocci (Eliopoulos et al., 1986; Ehlert
and Neu, 1987; Greenwood and Palfreyman, 1987)
and requires the presence of calcium for its full
expression (Eliopoulos et al., 1986; Andrew et al.,
1987).
The mechanism of action of daptomycin has not
been completely elucidated. Present evidence favours the view that the antibiotic interferes with an
early stage in the biosynthesis of peptidoglycan,
but the compound also affects the integrity of the
cell membrane (Lakey and Lea, 1986; Allen et al.,
1987). The ability of calcium to potentiate the
activity of daptomycin is probably related to a
selectively increased penetration of the lipid moiety
of the lipopeptide into the phospholipid of the cell
membrane (Lakey and Ptak, 1988).
Scanning electronmicroscopy offers the unique
ability to examine surface structures at relatively
high resolution and has proved particularly useful
in the examination of the effect of antibiotics that
act on the bacterial cell wall (Greenwood and
O'Grady, 1969, 1972; Elliott and Greenwood,
1983). In the present study the technique has been
used to examine the effects of daptomycin on
Received 12 Jan. 1989; accepted 8 Mar. 1989.
selected gram-positive cocci in the presence and
absence of calcium.
Materials and methods
Bacteria
Two strains of Staphylococcus aureus (one methicillinsensitive and one methicillin-resistant) and a strain of
Enterococcusfaecalis were randomly selected from clinical
isolates used in a previous study (Andrew et al., 1987).
Culture medium
Iso-Sensitest Broth (Oxoid) was prepared in deionised
water. For supplementation with calcium, analytical
grade CaC1, - 2 H 2 0 was prepared in deionised water,
sterilised by filtration and added to sterilised broth to
achieve a concentration of 2.5 mM.
Antibiotic
Daptomycin was supplied by Lilly Research, Windlesham, Surrey. Solutions of antibiotic were freshly prepared in sterile deionised water as required and further
diluted in broth.
Exposure to antibiotic
Cultures inoculated into Iso-Sensitest broth were
incubated at 37°C in the light path of a continuous
opacity monitoring device (Watson et al., 1969). Daptomycin was added at a standard point in the exponential
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L. J. WALE, A. P. SHELTON AND D. GREENWOOD
phase of growth at which the bacterial population was c.
lo8 cfu/ml.
Inhibitory activity of daptomycin was estimated by
turbidimetric experiments in which bacteria were exposed to a range of antibiotic concentrations. For
morphological studies, concentrations were chosen that
caused complete inhibition of growth as judged turbidimetrically.
Scanning electronmicroscopy
Samples were removed before, and at intervals after,
exposure to daptomycin and processed for scanning
electronmicroscopy as described by Elliott and Greenwood (1983). Briefly, the samples were fixed for 1 h
(glutaraldehyde 5% in 0.05 M sodium cacodylate containing 10mM MgSO,), washed by centrifugation and
dehydrated in alcohol, followed by acetone. The bacterial
suspension was then critical-point dried, coated with gold
and examined in a Jeol lOOC electronmicroscope.
which dense bacterial populations (c. lo8 cfulml)
were exposed to daptomycin in the presence of
2.5 mM calcium, concentrations of daptomycin
exceeding 8 mg/L caused no further change in the
turbidimetric record with any of the three strains
and for this reason this concentration was chosen
for the morphological studies. The turbidimetric
response of dense populations of the three test
organisms to daptomycin 8 mg/L is shown in fig. 1.
The marked fall in opacity observed following
addition of the antibiotic to cultures of the
methicillin-resistant strain of S . aureus and the
strain of E. faecalis was less marked in the case of
the methicillin-sensitive strain of S . aureus.
Morphological studies
The appearance of the methicillin-sensitive
strain of S . aureus in the absence of antibiotics is
shown in fig. 2A. The majority of the bacteria were
smooth and rounded, but not always spherical. The
Results
Turbidirnetric studies
/----
80.
The in-vitro activity of daptomycin against the
test strains, as judged turbidimetrically, is shown
in the table. In the presence of a physiological
concentration of calcium (2.5 mM), daptomycin
0.12 mg/L caused partial inhibition of growth, but
a concentration of 1-2 mg/L was required to
prevent growth during overnight incubation. In the
absence of calcium, much higher concentrations of
daptomycin were required to achieve comparable
degrees of growth suppression. In experiments in
60-
Table. Inhibitory activity of daptomycin for the test
strains as judged turbidimetrically
a
Inhibitory concentration (mg/L)
in presence of
Ca+
in absence of
Ca+
Test strain
MAC
MIC
MAC
MIC
S . aureus (methicillinsensitive)
S . aureus (methicillinresistant)
E .faecalis
0.12
1
64
>256
0.12
1
64
>64
0.12
2
128
>128
+
+
MAC = minimum antibacterial concentration (concentration
causing a deviation from normal growth); MIC =minimum
inhibitory concentration (concentration causing suppression of
growth during overnight incubation).
OJ---
I
2
4
6
8
10
12
14
16
Time (h)
Fig. 1. Continuous opacity records of (A) S . aureus (methicillinsensitive); (B) S . aureus (methicillin-resistant); (C)E. faecalis.
Daptomycin was added at arrow to achieve a concentration of
8 mg/L.
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SEM OF MORPHOLOGICAL EFFECTS OF DAPTOMYCIN
47
Fig. 2. Scanning electronmicrographs of S. aureus (methicillinsensitive) exposed to (A) no antibiotic; (B) daptomycin 8 mg/L
for 4 h; (C) daptomycin 8 mg/L for 24 h. Bar = 1 pm.
organisms were grouped in characteristic grapelike clusters. After exposure to daptomycin 8 mg/L
for 1 h, the surface of the bacteria appeared
roughened with occasionalboss-like protuberances,
and after 4 h most of the bacteria exhibited these
processes (fig. 2B). After exposure to daptomycin
for 24 h affected cells exhibited grossly altered
surfaces and very prominent cleavage sites (fig.
2C).
The effects observed with the methicillin-resistant strain of S. aureus were somewhat different:
unexposed cells commonly displayed small surface
projections (fig. 3A); cells exposed to daptomycin
for 1 or 4 h (fig. 3B) exhibited bizarre defects that
appeared to be particularly associatedwith cleavage
sites; and overnight exposure reduced the bacteria
to an amorphous syncytium (fig. 3C).
The characteristic elongated diplococci of E.
faecalis are shown in fig. 4A. After exposure to
daptomycin for 1 h a few cells exhibited roughened
surfaces with occasional extrusions that were
commonly associated with incipient sites of division, or with the poles of the cell; exposure for 4 h
did not appear to lead to further gross alterations,
but after overnight incubation the bacteria were
barely recognisable as streptococci (fig. 4B).
The methicillin-sensitive strain of S. aureus was
also examined for its response to daptomycin in the
absence of calcium. In these experiments the
bacteria were exposed to daptomycin 64 mg/L,
which caused a similar turbidimetric response to
that obtained with cultures exposed to 8 mg/L in
the presence of calcium. The morphological effects
observed were very similar to those found in the
presence of calcium : small, randomly distributed
lesions were visible after exposure for 1-4 h with
severe disruption of the cell wall on prolonged
exposure to daptomycin.
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48
L. J. WALE, A. P. SHELTON AND D. GREENWOOD
Fig. 3. Scanning electronmicrographs of S . auteus (methicillinresistant) exposed to (A) no antibiotic; (B) daptomycin 8 mg/L
for 1 h; (C) daptomycin 8 mg/L for 24 h. Bar= 1 pm.
Discussion
These results clearly demonstrate that daptomycin causes gross morphological alterations to the
surface of gram-positive cocci. The results are
consistent with the postulated mechanism of action
of the antibiotic in interfering with the biosynthesis
of peptidoglycan (Allen et al., 1987).
The earliest change induced by daptomycin
appears to be the production of small surface
projections not unlike those seen in staphylococci
exposed to penicillins (Greenwood and O’Grady,
1969). However, the gross abnormalities observed
in the methicillin-sensitive strain of S . aureus after
prolonged exposure (fig. 2C) and in the methicillinresistant strain in the earlier stages of drug exposure
(fig. 3B) are more suggestive of the continued
production of defective cell-wall material, and
continued, but aberrant septation.
Overnight exposure to daptomycin of the methicillin-resistant strain of S . aureus and the strain of
E. faecalis caused total distortion of the bacterial
surface so that the cells were reduced to a formless
mass. In contrast, grossly abnormal, but recognisable cells remained after overnight exposure of the
methicillin-sensitive strains to daptomycin. Such
strain variation in response to daptomycin may
explain differences in the rate of killing of staphylococci and enterococci reported by various authors
(Ehlert and Neu, 1987; Jorgensen et al., 1987;
Machka and Braveny, 1987; Verbist, 1987).
Omission of calcium from the culture medium
did not appear to affect the morphological response
to daptomycin, so that the morphological consequences of the antibiotic’s activity are evidently
not dependent on the cation. These results, therefore, support earlier speculation that calcium aids
intracellular penetration of the antibiotic to its
target site (Lakey and Lea, 1986; Allen et al., 1987;
Lakey and Ptak, 1988).
We thank Lilly Research Centre Ltd for financial support,
and A. Pawley for photographic assistance.
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SEM OF MORPHOLOGICAL EFFECTS OF DAPTOMYCIN
49
Fig. 4. Scanning electronmicrographs of E.faecalis exposed to (A) no antibiotic; (B) daptomycin 8 mg/L for 24 h. Bar= 1 pm.
REFERENCES
Allen N E, Hobbs J N, Alborn W E 1987 Inhibition of
peptidoglycan biosynthesis in gram-positive bacteria by
LY 146032. Antimicrobial Agents and Chemotherapy 31 :
1093-1099.
Andrew J H, Wale M C J, Wale L J, Greenwood D 1987 The
effect of cultural conditions on the activity of LY146032
against staphylococci and streptococci. Journal of Antimicrobial Chemotherapy 20: 21 3-221.
Ehlert F, Neu H C 1987 In vitro activity of LY146032
(daptomycin), a new peptolide. European Journal of Clinical
Microbiology6 ; 84-90.
Eliopoulos G M, Willey S, Reiszner E, Spitzer P G, Caputo G ,
Moellering R C 1986 In vitro and in vivo activity of
LY 146032,a new cyclic lipopeptide antibiotic. Antimicrobial
Agents and Chemotherapy 30; 532-535.
Elliott T S J , Greenwood D 1983 The response of Pseudomonas
aeruginosa to azlocillin, ticarcillin and cefsulodin. Journal
of’Medica1Microbiology 16 35 1-362.
Greenwood D, O’Grady F 1969 Antibiotic-induced surface
changes in microorganisms demonstrated by scanning
electron microscopy. Science 163: 1076-1078.
Greenwood D, O’Grady F 1972 Scanning electron microscopy
of Staphylococcus aureus exposed to some common anti-
staphylococcal agents. Journal of General Microbiology 70 :
263-270.
Greenwood D, Palfreyman J 1987 Comparative activity of
LY 146032 against anaerobic cocci. European Journal of
Clinical Microbiology 6 ; 682-684.
Jorgensen J H, Maher L A, Redding J S 1987 In vitro activity of
LY 146032 (daptomycin) against selected aerobic bacteria.
European Journal of Clinical Microbiology 6 : 91-96.
Lakey J H, Ptak M 1988 Fluorescence indicates a calciumdependent interaction between the lipopeptide antibiotic
LY 146032 and phospholipid membranes. Biochemistry 27 :
4639 4 6 45.
Lakey J H, Lea E J A 1986 The role of acyl chain character and
other determinants on the bilayer activity of A21978C an
acidic lipopeptide antibiotic. Biochimica et Biophysica Acta
859 : 2 19-226.
Machka K , Braveny I 1987 Comparative in vitro activity of
LY 146032 (daptomycin) against gram-positive cocci. European Journal of Clinical Microbiology 6 ; 96-99.
Verbist L 1987 In vitro activity of LY146032, a new lipopeptide
antibiotic, against gram-positive cocci. Antimicrobial Agents
and Chemotherapy 31 ; 340-342.
Watson B W, Gauci C L, Blache L, O’Grady F 1969 A simple
turbidity cell for continuously monitoring the growth of
bacteria. Physics in Medicine and Biology 14: 555-558.
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