FEMS MicrobiologyLetters 78 (1991) 65-68 © 1991 Federation of European MicrobiologicalSocieties0378-1097/91/$03.50 ADONIS 0378109791001032

65

FEMSLE 04300

8-Like haemolysin produced by Staphylococcus lugdunensis F r a n c o i s V a n d e n e s c h 1, Mich61e J. Storrs 2, Fran~oise P o i t e v i n - L a t e r 1, J 6 r 6 m e E t i e n n e 1, Patrice C o u r v a l i n 2 a n d Jean F l e u r e t t e 1 i Centre National de R~f~rence des Staphylocoques, Lyon, and 2Unit~des Agents Antibact~riens, lnstitut Pasteur, Paris, France

Received 4 October 1990 Accepted 10 October 1990 Key words: Staphylococcus lugdunensis; Hemolysin; D N A - D N A hybridization

1. S U M M A R Y Thirty independent clinical isolates of Staphylococcus lugdunensis were tested for expression of haemolysin characteristics of Staphylococcus aureus and for the presence of DNA sequences with homology to S. aureus toxin genes. Twenty-six strains produced a thermostable haemolysin with greatest activity against rabbit erythrocytes. DNADNA hybridisation was performed with S. lugdunensis DNA and probed with the a, fl, 8 and y toxin gene probes of S. aureus as well as that for coagulase. Hybridisation was found with the 8 probe in 28 strains including 6 which did not express haemolysin. Two strains did not hybridise with any of the probes. These findings suggest that S. lugdunensis strains produce a 8-like toxin which shares homology with the 8 toxin of S. aureus.

coccus [1]. It is an occasional but not rare cause of severe infections, such as infective endocarditis, bacteraemia, osteomyelitis, peritonitis and soft tissue infection [2-4]. S. aureus, regarded as the most pathogenic species, produces a wide range of extracellular and cell bound proteins which are potentially important as virulence factors including four haemolysins (a, fl, 8, y) [6,7]. In addition, a 8-like toxin immunologically-related to that of S. aureus is produced by Staphylococcus epidermidis [8]. Here we report a preliminary investigation of the haemolytic activity of 30 different S. lugdunensis strains and we demonstrate, by D N A - D N A hybridisations and haemolytic profiles, the presence of a haemolysin similar to the 8 haemolysin of S. aureus.

3. MATERIALS A N D M E T H O D S 2. I N T R O D U C T I O N Staphylococcus lugdunensis is a newly identified

pathogenic species of coagulase negative staphylo-

Correspondence to: MA. Storrs. Present address: Department of

Food Microbiology,University CollegeCork, Cork, Ireland.

3.1. Bacterial strains

Thirty epidemiologically-unrelated S. lugdunensis strains were obtained from the French National Reference Center for Staphylococci. They were identified as S. lugdunensis by the following characteristics: clumping factor, detected by the Staphyslide test (bioMrrieux), thermostable

66 DNase (bioM6rieux) and ornithine decarboxylase production. Complete identification as S. lugdunensis was determined according to the characteristic growth and biochemical reactions of the strains [1]. Strain S. lugdunensis ATCC 43809 (No. 23) was used as reference [1]. 3.2. Haemolytic activity on blood plates

Bacteria were grown on trypticase soy (TS) agar plates supplemented with 5% defibrinated sheep or rabbit blood (bioM6rieux), and on TS agarose containing 5% rabbit erythrocytes (bioM6rieux) and were incubated for 24 h at 37°C under aerobic conditions. The following characteristics were used to define haemolysis [5]: a-haemolysin forms a wide zone of complete haemolysis with blurred edges on rabbit erythrocyte agar. flHaemolysin gives a wide zone of partial haemolysis on sheep erythrocyte agar plates with complete haemolysis after further incubation at 4°C for 4 - 6 h. ~,-Haemolysin is shown by enhancement of haemolysis when agar was substituted by agarose in TS plates containing 5% rabbit erythrocytes. 8-Haemolysin forms a narrow zone of complete haemolysis with blurred edges on sheep erythrocyte agar plates and complete clearing of the partial zone of fl-haemolysis formed by an S. interrnedius strain [9].

(haemolytic units per ml) were expressed as the reciprocal of the highest dilution which showed 50% haemolysis. Haemolytic activity was also measured after heating culture supernatants at 60°C for 30 min. Culture supernatants from S. aureus strain Wood 46 and N C T C 10345, known to produce a- and ~-haemolysin respectively, were used as controls. 3.4. D N A - D N A hybridisation

DNA was extracted as previously described [11]. The purified restriction fragments used as probes were labelled in vitro with [a-32p]dATP (Amersham, France). The 17-mer 8 toxin probe was 5' labelled with [3,-32p]dATP (Amersham, France) using T4 polynucleotide kinase (Amersham, France) as described by Maniatis et al. [12]. One /~g of total DNA was transferred to nylon sheets (Schleicher and Schuell) and hybridised with probes for ct-, fl-, 8-, and 3,-haemolysin genes of S. aureus and for coagulase (Table 1). Hybridisations were performed under stringent and nonstringent conditions according standard procedures [12,13].

4. RESULTS

3.3. Haemolytic activity in liquid cultures

4.1. Haemolytic activity None of the S. lugdunensis strains investigated

Strains were grown in 125 ml Todd-Hewitt broth (Difco Laboratories) in 250-ml Erlenmeyer flasks at 150 rpm for 24 h at 37°C. The culture supernatants were titrated for haemolytic activity against rabbit (bioM6rieux), sheep (bioM6rieux) and human erythrocytes in microdilution plates as previously described [10]. Haemolytic titres

produced a-, fl- or T-like haemolysins. Eight of the 30 strains were non-haemolytic while 22 exhibited larger zones of haemolysis on rabbit erythrocyte compared to sheep erythrocyte agar plates. This correlated with an activity which had a synergistic effect with the fl lysin of S. intermedius. Haemolytic titration of culture super-

Table 1 DNA probes used in hybridisation experiments Toxin

Gene

,, B 8

my bib big hid

Coagulase

coa

Probe 0.5-kb Clal fragment from pDU1212 0.8-kb DdeI fragment from pDC007 6-kb HindlII fragment from pJC09 17-mer oligonucleotide (5'-TATCGACACAGTGAAC-3') 0.9-kb EcoRI-Hindlll fragment from pCOA5

Ref. 117] [18] [19] S. Projan, Public Health Research Institute, New York [20]

67 12

[] [] []

10

8

01,llt0

6

E

== Z

4

2

0 2

0

human rabbit sheep

4

8

16

32

64

128

256

Haemolytic units/ml Fig. 1. Haemolytic triers of the 22 haemolytic isolates of S. lugdunensis. Titers were expressed as the reciprocal of the highest dilution which showed 50% haemolysis as assessed visually. natants with human, sheep and rabbit erythrocytes detected a thermostable haemolysin, mainly active against rabbit erythrocytes (Fig. 1). This activity was not enhanced by further incubation at 4°C, suggesting that a fl-like haemolysin was not involved. 4.2. Homology with S. aureus haemolysin probes No hybridisation occurred between the 30 S. lugdunensis strains tested and the gene probes for a, fl and y lysins or for coagulase even under

1

AB

C





2



3 4



5 0

• •

7



DE











• •



I

• •



FGH

K

L

• •







• •



J

• •





Fig. 2. Analysis of DNA by dot blot hybridisation. Total DNA of S. lugdunensis strains was transferred to a nylon sheet and hybridised to the 8 toxin gene 17-met probe labelled with 32p in vitro. Wells L2, I3, F4, H4, E5, I5 correspond to phenotypically 8-negative S. lugdunensis strains. Wells A6 and A7 correspond to S. aureus Wood46 and NCTC1035, respectively.

non-stringent conditions. However, hybridisation occurred between the 8 toxin gene probe and all haemolytic strains, and also with 6 of the 8 nonhaemolytic strains (Fig. 2). These findings suggest that S. lugdunensis D N A has a sequence which is homologous with the 8 toxin structural gene of S. aureus.

5. D I S C U S S I O N Our findings demonstrate that haemolysins similar to a-, fl-, or 7-haemolysins of S. aureus were not produced by S. lugdunensis. The haemolysin detected here was a heat-stable 8-like toxin which displayed a synergistic effect with the fl toxin of S. intermedius. 8-Toxin may be distinguished from other S. aureus haemolysins by its heat stability, neutralisation by leicithin and serum and by its pattern of activity towards erythrocytes from various species. 8-Toxin can damage a variety of cells by its detergent-like action on cell membranes [14]. Unlike S. aureus 8 toxin, which displays a wide variety of activity affecting most cell types, the 8-like toxin of S. lugdunensis was preferentially active against rabbit erythrocytes (Fig. 1). D N A - D N A hybridisation results agreed with the phenotypic observations and demonstrated that the 8-like gene of S. lugdunensis had homology with the 8 toxin structural gene ( h i d ) of S. aureus. Interestingly, it was previously shown that the D N A relatedness between S, aureus and S. lugdunensis is 5% [1]. Expression of some extracellular products by S. aureus is dependent on a regulatory locus called arg [15] and recently the hid gene was located adjacent to the agr locus on the chromosome of S. aureus 8325-4 [16]. In the present study, we found sequences related to hid present in some non-haemolytic strains, suggesting that the structural gene was inactive. We found that the majority of S. lugdunensis strains investigated possessed sequences homologous to the 8 toxin of S. aureus and since no other haemolysins were detected, it is possible that the conservation of this toxin in S. lugdunensis is due to association of hid with a regulatory gene and not to its role as a possible virulence determinant. Since S. lugdunensis is a

68 n e w p a t h o g e n i c species of c o a g u l a s e n e g a t i v e s t a p h y l o c o c c u s a n d a & l i k e t o x i n was the sole h a e m o l y s i n d e t e c t e d , it w o u l d b e i n t e r e s t i n g to d e t e r m i n e the role of this t o x i n in the p a t h o g e n e sis o f S. lugdunensis i n f e c t i o n a n d to i n v e s t i g a t e the p o s s i b l e m e c h a n i s m of t o x i n e x p r e s s i o n i n these strains.

ACKNOWLEDGEMENTS W e t h a n k T.J. F o s t e r for the gift o f p r o b e s for a, /3 a n d 3' t o x i n s a n d for c o a g u l a s e a n d for critical r e a d i n g of the m a n u s c r i p t a n d S. P r o j a n for the gift of the ~ t o x i n o l i g o n u c l e o t i d e p r o b e .

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lmmunochemical and Molecular Genetic Analysis of Bacterial Pathogens (Owen, P. and Foster, T.J., eds.), pp. 103-119, Elsevier, Amsterdam. [7] Bramley, A.J., Patel, A.H., O'Reilly, M., Foster, R. and Foster, T.J. (1989) Infect. lmmun. 57, 2489-2494. [81 Turner, W.H. and Pickard, D.K. (1979) Infect. Immun. 23, 910-911. [9] Hrbert, G.A. and Hancock, G.A. (1985) J. Clin. Microbiol. 22, 409-415. [10] Clyne, M., de Azavedo, J., Carlson, E. and Arbuthnott, J.P. (1988) J. Clin. Microbiol. 26, 535-539. [11] Etienne, J., Poitevin-Later, F., Renaud, F. and Flenrette J. (1990) FEMS Microbiol. Lett. 67, 93-98. [12] Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [13] Mabilat, C. and Courvalin, P. (1988) Ann. Microbiol. (Inst. Pasteur) 139, 677-681. [14] Scheifele, D.W., Bjomson, G.L., Dyer, R. and Dimmick, J.A. (1987) Infect. Immun. 55, 2268-2273. [15] Recsei, P., Kreiswirth, B., O'Reilly, M., Schlivert, R., Gruss, A. and Novick, R.P. (1986) Mol. Gen. Genet. 202, 58-61. [16] Janzon, L., Lofdahl, S. and Arvidson, S. (1989) Mol. Gen. Genet. 219, 480-485. [17] O'Reilly, M., de Azavedo, J., Kennedy, S. and Foster, T.J. (1986) Microb. Pathogen. 1, 125-138. [18] Coleman, D.C., Arbuthnott, J.P., Pomeroy, H.M. and Birkbeck, T.H. (1986) Microb. Pathogen. 1, 549-564. [19] Cooney, J., Mulvey, M., Arbuthnott, J.P. and Foster, T.J. (1988) J. Gen. Microbiol. 134, 2179-2188. [20] Phonimdaeng, P., O'Reilly, M., O'Toole, P.W. and Foster, T.J. (1988) J. Gen. Microbiol. 134, 75-83.

The cloning and nucleotide sequence of the serine protease gene (aspA) of Aeromonas salmonicida ssp. salmonicida.

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