© INSTITUTPASTEUR/ELSEVIER Paris 1990

Res. Virol. i990, 141, 625-635

MORPHOLOGY OF S T A P H Y L O C O C C U S S A P R O P H Y T I C U S BACTERIOPHAGES

M. Bes (1), H.-W. Ackermann (2), Itr. Brun (3) and J. Fleurette (3)

(1) Centre d'Etudes et de Recherches Bact~riologiques, Institut Pasteur de Lyon, et Facultd de Mddecine A. Carrel, 69372 Lyon Cedex 08 (France), (2) Centre de Rdfdrence pour Virus bactdriens F~lix d'Hdrelle, D~partement de Microbiologie, Facultd de Mddecine, Universitd Laval, Qudbec, Qua. (Canada) GIK 7P4, and (3) Centre national de Rdf~rence des Staphylocoques, Facultd de Mddecine Alexis Carrel, 69372 Lyon Cedex 08 (France)

SUMMARY

The morphology of 6 bacteriophages isolated from Staphylococcus saprophyticus was studied by electron microscopy. Phages had isometric heads and non-contractile tails, thus belonging to the Siphoviridae family. They were subdivided into two morphological groups based on head diameter and tail length. Type I, represented by phages 1154A and 1405, and characterized by an unusually small capsid, may be a new species. KEY-WORDS"Staphylococcus saprophyticus, Siphoviridae, Bacteriophage; Ultrastructure, Types, New species.

INTRODUCTION After Escherichia coli, Staphylococcus saprophyticus is the most frequent bacterial agent of urinary tract infections in young women (Hovelius and Mardh, 1984; Mardh, 1986). This species is now easily identified by biochemical micromethods but epidemiological markers la.ck the ability to identify the different strains efficiently.

Submitted November 28, 1989, accepted September 12, 1990.

M. BES E T A L .

626

Several bacteriophages for S. epidermidis have been used as epidemiological markers (Parisi et al., 1986; De Saxe et al., !981 ; Staal et ai., 1981). Some of these phages can be used to type strains belongit~g to species other than S. epidermidis (Schumacher-Perdreau et al., 1988). In 1985, Fournet et al. isolated new bacteriophages from S. saprophy:icus in an attempt to establish a phage-typin~ system for this species. The present study describes the morphology of these new phages. MATERIALS AND METHODS Bacteriophages and bacteria. The phages studied, 1139, 1154A, 1259, 1314, 1405 and 1563, were obtained after induction of 25 strains of S. saprophyticus by mitomycin C (Fournet et al., 1985). They were purified by 3 successive isolations of single plaques and were propagated on the following bacterial strains: phages 1139, 1259 and 1314 on strain 992; phage 1154A on strain 433; phage 1405 on strain 1255; and phage 1563 on strain 1562. For each phage respectively, 4 plates of nutrient agar (Difco) were flooded with a suspension of the propagating strain which had been grown for 18 h at 37°C in nutrient broth (Difco). Excess fluid was discarded and the phage conveniently diluted in nutrient broth was added in the same manner. Plates were incubated for 18 h at 37°C, then flooded with 10 ml of TAM buffer (10 mM Tris, 10 mM sodium azide, 1 mM MgCI2, pH 6.0) and kept for 30 min at room temperature. The suspension was filtered through 0.45-ttm membranes (HAWG, Millipore, Bedford, MA) and stored at 4°C before ultracentrifugation. Electron microscopy. Ph~o~

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ultracentrifuge using a "SW-55Ti" rotor, and washed twice in TAM buffer. They were then deposited on copper grids with carbon-coated "Formvar" film and stained with 2 % phosphotungstate (PT, pH 7.2) or 2 % uranyl acetate (UA, pH 3.8). Specimens were observed in a "Philips EM 300" electron microscope at 60 kV. Magnification was monitored with catalase crystals (Luftig, 1967). Phage particles were measured at a final magnification of × 297,000.

RESULTS

All 6 S. saprophyticus bacteriophages were tailed and had isometric heads. The tails were long and non-contractile. The phages were divided into two morphological groups based on head diameter and tail length. The dimensions of phages (negatively stained particles only) are listed in table I. Type I was represented by two phages, 1154A and 1405 (fig. I to 7). These phages had a small head, only 46 nm in diameter, and a long tail, 266 nm

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in length with about 63 cross-striations. The presence of capsids with hexagonal and pentagonal outlines indicated that the heads were icosahedral (fig. 6). Undefined terminal structures were sometimes observed on the tails. Preparations of phage 1154A contained a few aberrant structures (fig. 4 and 5), namely particles with a relatively large head of 64 nm in diameter and a non-contractile tail of 280 nm in length, and one unusual particle with an abnormally long tail (430 nm in length). Isolated tail sheaths were also observed (fig. 7). Type II was represented by 4 phages, 1139, 1259, 1314 and 1563 (fig. 8 to 13). Their capsids, 64 nm in diameter, were larger than those of type I. The icosahedral structure of the head was confirmed by the presence of pentagonal capsids (fig. 11) and the apparent lateral insertion of some tails (fig. 8 and 12). Tails were 313 nm in length with about 67 cross-striations. Poorly defined terminal structures were observed. With UA-stained preparations, tails sometimes showed spikes (phage 1259, fig. 8) or disk-like structures (phage 1563, fig. 13). All UA-stained bacteriophage preparations contained negatively and positively stained particles (fig. 3, 9 and 10). Positively stained capsids were surrounded by a halo which increased in size upon exposure to irradiation (fig. 9).

DISCUSSION

The icosahedral capsids and the long non-contractile tails indicate that the 6 phages described here belong to the Siphoviridae family (Matthews, 1982) or Bradley's morphological type B (Bradley, 1967) and to the subgroup B1 defined by Ackermann and Eisenstark (1974). Only negatively stained particles were measured, but differences in size were observed between PT- and UA-stained bacteriophages (table I). For example, positively stained phages with shrunken capsids (due to the affinity of UA for doub!e-stranded DNA) and negatively stained phages with thickened tails were commonly observed (Ackermann and Dubow, 1987a). Halos

TABLEI.

-- Phage dimensions

Phage

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PT

UA

Type

Members

N

Head

Tail

N

Head

Tail

I

1154A, 1405

20

46

266

20

44

268

II

1139, 1314

20

64

313

20

58

287

1259, 1563 N = number of particles measured. Negatively stained particles only; head diameters are between opposite apices.

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