J. Med. Microbiol. - Vol. 30 (1989), 4549 0022-26 1 5/89/0030-0045/$10.00 Q 1989 The Pathological Society of Great Britain and Ireland Sc a n ni n g e Iec t r o nm i c r osc o py of Staphyo co ccus 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 boss- like 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 is 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 selected gram-positive cocci in the presence and Daptomycin (formerly known as LY146032) is a absence of calcium. novel lipopeptide related to the antibiotic complex A21 978C of Streptomyces roseosporus. The activity of daptomycin is restricted to aerobic and anaerobic Materials and methods gram-positive cocci (Eliopoulos et al., 1986; Ehlert and Neu, 1987; Greenwood and Palfreyman, 1987) Bacteria and requires the presence of calcium for its full Two strains of Staphylococcus aureus (one methicillin- expression (Eliopoulos et al., 1986; Andrew et al., sensitive and one methicillin-resistant) and a strain of 1987). Enterococcusfaecalis were randomly selected from clinical The mechanism of action of daptomycin has not isolates used in a previous study (Andrew et al., 1987). been completely elucidated. Present evidence fa- vours the view that the antibiotic interferes with an Culture medium early stage in the biosynthesis of peptidoglycan, but the compound also affects the integrity of the Iso-Sensitest Broth (Oxoid) was prepared in deionised water. For supplementation with calcium, analytical cell membrane (Lakey and Lea, 1986; Allen et al., grade CaC1, - 2H20 was prepared in deionised water, 1987). The ability of calcium to potentiate the sterilised by filtration and added to sterilised broth to activity of daptomycin is probably related to a achieve a concentration of 2.5 mM. selectively increased penetration of the lipid moiety of the lipopeptide into the phospholipid of the cell membrane (Lakey and Ptak, 1988). Antibiotic Scanning electronmicroscopy offers the unique Daptomycin was supplied by Lilly Research, Windle- ability to examine surface structures at relatively sham, Surrey. Solutions of antibiotic were freshly pre- high resolution and has proved particularly useful pared in sterile deionised water as required and further in the examination of the effect of antibiotics that diluted in broth. act on the bacterial cell wall (Greenwood and O'Grady, 1969, 1972 ; Elliott and Greenwood, Exposure to antibiotic 1983). In the present study the technique has been used to examine the effects of daptomycin on 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). Dapto- Received 12 Jan. 1989; accepted 8 Mar. 1989. mycin was added at a standard point in the exponential

45 46 L. J. WALE, A. P. SHELTON AND D. GREENWOOD phase of growth at which the bacterial population was c. which dense bacterial populations (c. lo8 cfulml) lo8 cfu/ml. were exposed to daptomycin in the presence of Inhibitory activity of daptomycin was estimated by 2.5 mM calcium, concentrations of daptomycin turbidimetric experiments in which bacteria were ex- exceeding 8 mg/L caused no further change in the posed to a range of antibiotic concentrations. For turbidimetric record with any of the three strains morphological studies, concentrations were chosen that caused complete inhibition of growth as judged turbidi- and for this reason this concentration was chosen metrically. 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. Scanning electronmicroscopy The marked fall in opacity observed following Samples were removed before, and at intervals after, addition of the antibiotic to cultures of the exposure to daptomycin and processed for scanning methicillin-resistant strain of S. aureus and the electronmicroscopy as described by Elliott and Green- strain of E.faecalis was less marked in the case of wood (1983). Briefly, the samples were fixed for 1 h the methicillin-sensitive strain of S. aureus. (glutaraldehyde 5% in 0.05 M sodium cacodylate contain- ing 10mM MgSO,), washed by centrifugation and dehydrated in alcohol, followed by acetone. The bacterial Morphological studies suspension was then critical-point dried, coated with gold The appearance of the methicillin-sensitive and examined in a Jeol lOOC electronmicroscope. 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 60- /------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

Table. Inhibitory activity of daptomycin for the test strains as judged turbidimetrically a Inhibitory concentration (mg/L)

in presence of in absence of + Ca++ Ca+

Test strain MAC MIC MAC MIC

S. aureus (methicillin- 0.12 1 64 >256 sensitive) S. aureus (methicillin- 0.12 1 64 >64 OJ--- I resistant) 2 4 6 8 10 12 14 16 E.faecalis 0.12 2 128 >128 Time (h) MAC = minimum antibacterial concentration (concentration Fig. 1. Continuous opacity records of (A) S. aureus (methicillin- causing a deviation from normal growth); MIC =minimum sensitive); (B) S. aureus (methicillin-resistant); (C)E. faecalis. inhibitory concentration (concentration causing suppression of Daptomycin was added at arrow to achieve a concentration of growth during overnight incubation). 8 mg/L. SEM OF MORPHOLOGICAL EFFECTS OF DAPTOMYCIN 47

Fig. 2. Scanning electronmicrographs of S. aureus (methicillin- sensitive) 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 grape- daptomycin for 1 h a few cells exhibited roughened like clusters. After exposure to daptomycin 8 mg/L surfaces with occasional extrusions that were for 1 h, the surface of the bacteria appeared commonly associated with incipient sites of divi- roughened with occasional boss-like protuberances, sion, or with the poles of the cell; exposure for 4 h and after 4 h most of the bacteria exhibited these did not appear to lead to further gross alterations, processes (fig. 2B). After exposure to daptomycin but after overnight incubation the bacteria were for 24 h affected cells exhibited grossly altered barely recognisable as streptococci (fig. 4B). surfaces and very prominent cleavage sites (fig. The methicillin-sensitive strain of S. aureus was 2C). also examined for its response to daptomycin in the The effects observed with the methicillin-resist- absence of calcium. In these experiments the ant strain of S. aureus were somewhat different: bacteria were exposed to daptomycin 64 mg/L, unexposed cells commonly displayed small surface which caused a similar turbidimetric response to projections (fig. 3A); cells exposed to daptomycin that obtained with cultures exposed to 8 mg/L in for 1 or 4 h (fig. 3B) exhibited bizarre defects that the presence of calcium. The morphological effects appeared to be particularly associated with cleavage observed were very similar to those found in the sites; and overnight exposure reduced the bacteria presence of calcium : small, randomly distributed to an amorphous syncytium (fig. 3C). lesions were visible after exposure for 1-4 h with The characteristic elongated diplococci of E. severe disruption of the cell wall on prolonged faecalis are shown in fig. 4A. After exposure to exposure to daptomycin. 48 L. J. WALE, A. P. SHELTON AND D. GREENWOOD

Fig. 3. Scanning electronmicrographs of S. auteus (methicillin- resistant) 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 surface so that the cells were reduced to a formless These results clearly demonstrate that daptomy- mass. In contrast, grossly abnormal, but recognisa- ble cells remained after overnight exposure of the cin causes gross morphological alterations to the methicillin-sensitive strains to daptomycin. Such surface of gram-positive cocci. The results are strain variation in response to daptomycin may consistent with the postulated mechanism of action explain differences in the rate of killing of staphy- of the antibiotic in interfering with the biosynthesis lococci and enterococci reported by various authors of peptidoglycan (Allen et al., 1987). The earliest change induced by daptomycin (Ehlert and Neu, 1987; Jorgensen et al., 1987; appears to be the production of small surface Machka and Braveny, 1987; Verbist, 1987). projections not unlike those seen in staphylococci Omission of calcium from the culture medium exposed to penicillins (Greenwood and O’Grady, did not appear to affect the morphological response 1969). However, the gross abnormalities observed to daptomycin, so that the morphological conse- quences of the antibiotic’s activity are evidently in the methicillin-sensitive strain of S. aureus after prolonged exposure (fig. 2C) and in the methicillin- not dependent on the cation. These results, there- resistant strain in the earlier stages of drug exposure fore, support earlier speculation that calcium aids (fig. 3B) are more suggestive of the continued intracellular penetration of the antibiotic to its production of defective cell-wall material, and target site (Lakey and Lea, 1986; Allen et al., 1987; continued, but aberrant septation. Lakey and Ptak, 1988). Overnight exposure to daptomycin of the methi- cillin-resistant strain of S. aureus and the strain of We thank Lilly Research Centre Ltd for financial support, E. faecalis caused total distortion of the bacterial and A. Pawley for photographic assistance. 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 staphylococcal agents. Journal of General Microbiology 70 : 263-270. Allen N E, Hobbs J N, Alborn W E 1987 Inhibition of Greenwood D, Palfreyman J 1987 Comparative activity of peptidoglycan biosynthesis in gram-positive bacteria by LY 146032 against anaerobic cocci. European Journal of LY 146032. Antimicrobial Agents and Chemotherapy 31 : Clinical Microbiology 6 ; 682-684. 1093-1099. Jorgensen J H, Maher L A, Redding J S 1987 In vitro activity of Andrew J H, Wale M C J, Wale L J, Greenwood D 1987 The LY 146032 (daptomycin) against selected aerobic bacteria. effect of cultural conditions on the activity of LY146032 European Journal of Clinical Microbiology 6 : 91-96. against staphylococci and streptococci. Journal of Anti- Lakey J H, Ptak M 1988 Fluorescence indicates a calcium- microbial Chemotherapy 20: 21 3-221. dependent interaction between the lipopeptide antibiotic Ehlert F, Neu H C 1987 In vitro activity of LY146032 LY 146032 and phospholipid membranes. Biochemistry 27 : (daptomycin), a new peptolide. European Journal of Clinical 46 39464 5. Microbiology 6 ; 84-90. Lakey J H, Lea E J A 1986 The role of acyl chain character and Eliopoulos G M, Willey S, Reiszner E, Spitzer P G, Caputo G, other determinants on the bilayer activity of A21978C an Moellering R C 1986 In vitro and in vivo activity of acidic lipopeptide antibiotic. Biochimica et Biophysica Acta LY 146032,a new cyclic lipopeptide antibiotic. Antimicrobial 859 : 2 19-226. Agents and Chemotherapy 30; 532-535. Machka K, Braveny I 1987 Comparative in vitro activity of Elliott T S J, Greenwood D 1983 The response of Pseudomonas LY 146032 (daptomycin) against gram-positive cocci. Eu- aeruginosa to azlocillin, ticarcillin and cefsulodin. Journal ropean Journal of Clinical Microbiology 6 ; 96-99. of’Medica1Microbiology 16 35 1-362. Verbist L 1987 In vitro activity of LY146032, a new lipopeptide Greenwood D, O’Grady F 1969 Antibiotic-induced surface antibiotic, against gram-positive cocci. Antimicrobial Agents changes in microorganisms demonstrated by scanning and Chemotherapy 31 ; 340-342. electron microscopy. Science 163: 1076-1078. Watson B W, Gauci C L, Blache L, O’Grady F 1969 A simple Greenwood D, O’Grady F 1972 Scanning electron microscopy turbidity cell for continuously monitoring the growth of of Staphylococcus aureus exposed to some common anti- bacteria. Physics in Medicine and Biology 14: 555-558.