FEMS Microbiology Letters 48 (1987) 195-200 195 Published by Elsevier

FEM 02990

Effect of milk on surface properties of Staphylococcus aureus from bovine mastitis

Wubshet Mamo a, Ferenc Rozgonyi a,b,, Stellan Hjert~n c and Torkel WadstrSm a . .

Section of Bacteriology and Epizootology, Department of Veterinary Microbiology, College of Veterinary Medicine, Swedish University of Agricultural Sciences, Biomedicum, S-751 23 Uppsala, Sweden; b Department of Microbiology, University Medical School of Debrecen,

H-4012 Debrecen, Hungary and c Institute of Biochemistry, Uppsala University, Biomedicum, S-751 23 Uppsala, Sweden

Received 25 May, 1987 Revision received 2 August 1987

Accepted 6 August 1987

Key words: S. aureus; Surface hydrophobicity; Surface charge; Mastitis; Bovine milk

1. SUMMARY

The surface hydrophobicity of cells of S taphy lococcus aureus strains isolated from bovine mastitis grown on conventional agar and broth media was drastically reduced after incubation with bovine milk. Strains grown in high carbohy- drate-high salt media yielded cells with reduced surface hydrophobicity compared to cells grown in conventional media, and adding bovine milk to minimal medium also yielded cells with reduced surface hydrophobicity, as determined by hydro- phobic interaction chromatography and the salt aggregation test. Incubation of strains in milk and growth in a medium supplemented with bovine milk also significantly changed bacterial surface charge as determined by free-zone electrophoresis. Strains with high or with decreased adsorptive and

Correspondence to: W. Mamo, Section of Bacteriology and Epizootology, Dept. of Veterinary Microbiology, Coll. of Veterinary Medicine, Swedish University of Agricultural Scien- ces, Biomedicum, Box 583, S-75123 Uppsala, Sweden.

* Visiting professor at Biomedicum, Uppsala, Sweden. ** Present address: Department of Medical Microbiology,

University of Lund, Solvegatan 23, S-223 62 Lund, Sweden.

aggregating properties did not produce surface capsule or slime. Heat treatment (60 °C or 80 ° C) of the bacterial suspensions did not significantly change their adsorptive and aggregating proper- ties.

2. INTRODUCTION

S. aureus is a major cause of bovine mastitis and of various pyogenic infections in man and animals [1,2]. Since the physicochemical state of the bacterial cell surface plays a crucial role in the ability of S. aureus to colonize and invade the bovine udder in the interaction of the organism with the host defences [3,4], we have been inter- ested in the possible effects of various growth conditions on the surface properties of S. aureus

strains isolated from bovine mastitis. Recent stud- ies on such strians report on the effect of a num- ber of physical, chemical, enzymatic and biologi- cal factors on the pathogen's cell-adhesive proper- ties in the mammary gland [4,5]. Moreover, previ- ous studies in our laboratory have shown that S. aureus strains isolated from bovine mastitis and

0378-1097/87/$03.50 © 1987 Federation of European Microbiological Societies

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other animal infections, as well as from human bacteremia, commonly have hydrophobic cell surfaces when grown in vitro [6-9]. In the present study, we report how surface properties of bovine mastitis S. aureus strains are influenced by milk.

3. MATERIALS AND METHODS

3.1. Bacterial strains The S. aureus strains from bovine mastitis cases,

included in this study were kindly supplied by Dr. Olof Holmberg, National Veterinary Institute, Uppsala, Sweden. The laboratory reference S. aureus strains Smith diffuse and Smith compact have been described [7]. The strains were stored at - 7 0 °C in Tryptic Soy Broth (Difco) containing 15% (v/v) of glycerol.

3.2. Milk samples Sterile bovine milk was prepared from milk

collected aseptically from a healthy cow and centrifuged (1000 rev./min, 10 min) to remove the fat. The milk aspirated from beneath was auto- claved (121 ° C, 15 min) and stored in aliquots of 10 ml at - 20 °C until needed.

3.3. Culture media, growth conditions and bacterial suspensions

Strains were grown on blood agar (5% horse blood), Staphylococcus medium 110 (SM 110, Difco) at 37°C for 18 h and in milk-supple- mented medium (MSM). The MSM consisted of NaCI (5 g/l), NazHPO 4 • 2H20 (5 g/l), KH2PO 4 (5 g/l), Fe(NH4)z(SOa)z.6H20 (0.03 g/l), MgSO4.4H20 (0.01 g/l), MnSO4.7H20 (0.01 g/l) and 10% (w/v) sterile milk. Following auto- claving (121°C, 15 min) the pH of the medium was adjusted to 7.0. Bacterial cultures were centrifuged (3000 × g, 20 min), washed and sus- pended in 0.02 M sodium phosphate buffer (pH 6.8) containing 0.02% (w/v) sodium azide. Bacterial suspensions were standardized to an Alcm ~ 540

of 20 (equivalent to 5 X 10 9 cells/mi) (Model 100-20 spectrophotometer, Hitachi Ltd, Tokyo). Bacterial suspensions of SM 110 were divided into two equal parts (1 ml) one being incubated in sterile milk (100 ~l /ml bacteria = 5 x 10 9 cells) at

37°C for 2 h. For some experiments bacterial suspensions were heated at 60 °C or 80 °C for 30 min in a water bath.

3.4. Hydrophobic interaction chromatography (HIC) The methodology has been detailed elsewhere

[10]. Briefly, bacterial suspensions (100 ffl = 5.9 x 10 9 bacteria) were applied onto Pasteur pipettes plugged with glass wool and packed with Octyl Sepharose (CL-4B, Lot No. 9080) (0.6-1 ml) and washed with 5 ml of 1 M ammonium sulphate buffered with 10 mM sodium phosphate buffer, pH 6.8. The absorbance of the eluents was mea- sured at 540 nm. Adsorption was expressed as % decrease in the number of bacteria in the eluent compared to the amount applied. Strains that showed > 80% adsorption to the gel were defined as HIC-positive, 50-80% as moderate, and less than 50% as negative.

3.5. Salt aggregation test (SA T) Salting out of bacterial cells [11] was performed

according to an improved procedure as described by Rozgonyi et al. [12]. The lowest molar con- centration of ammonium sulphate in which visual bacterial aggregation occurred was recorded as the SAT value.

3.6. Free-zone electrophoresis (FZE) Bacterial mobilities, which are related to the

surface net charge, were determined with 10/~1 of bacterial suspensions in the Hjertrn apparatus at 18°C (800 V) with 0.02 M sodium phosphate buffer, pH 6.8, as electrophoresis medium [13]. A horizontal, rotating (40 rev./min) quartz electro- phoresis tube (dimensions: 3 x 8 x 400 mm) was scanned with a UV-beam at 5 min intervals. From the absorbance recordings, the distance of migra- tion of each peak was measured and plotted against time.

3. 7 Bacterial staining To visualize the capsule in the bacaterial

surface, two staining methods, one using India ink [14]. and the other patent blue V (2.5% solution for lymphography, Guerbet S.A., Paris, France) were applied as described by Rozgonyi and Selt- man [15].

4. R E S U L T S

Table 1 summarizes the effect of growth media and milk on the hydrophobic adsorptive proper- ties as assayed by HIC. Strains grown in medium 110 (high carbohydrate-high salt medium) showed a reduced adsorpt ion to octyl sepharose (giving a mean value of 40% of applied cells retained) as compared to strains f rom blood agar, which ad- sorbed more strongly (80% cells retained). A 2-h incubat ion in medium l l0 -con ta in ing milk also resulted in a reduced adsorpt ion (20% of the cells retained). Strains grown in MSM adsorbed poorly to the octyl sepharose.

Relative surface hydrophobicit ies, as deter- mined by the SAT and the presence or absence of capsule using India ink or patent blue V staining methods are presented in Table 2. Strains in- cubated for 2 h with milk or grown in M S M showed a uniformly low surface hydrophobici ty, aggregating only at a high concentrat ion of am-

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mon ium sulfate. With the exception of the strains Smith diffuse, 1319 and 139 which were hydro- philic, all strains tested were hydrophobic when grown on blood agar (i.e., they aggregated at low ammonium sulfate concentrations). When grown in medium 110 most strains showed a reduced hydrophobici ty . Thus, we could demonsrate by both methods that the S. a u r e u s strains lost their hydrophobic, adsorptive, and aggregating proper- ties in the presence of milk (Tables 1 and 2).

With the exception of strain 1319 and the laboratory reference strain Smith diffuse, which were both encapsulated under all test conditions, none of the remaining strains showed visible caps- ule under any of the test conditions.

The electrophoretic mobili ty was determined for selected encapsulated and unencapsulated strains grown under different condit ions (Fig. 1). A higher mobili ty (i.e., higher negative charge) was observed for cells grown or incubated in milk than for cells grown on blood agar, confirming

Table 1

Hydrophobic adsorption of Staphylococcus aureus strains under different growth and incubation conditions

S. aureus strain

Adsorption (%) a of bacteria

Grown on Grown on Incubated with blood agar medium 110 milk b

Grown in milk- supplemented medium

Smith diffuse 30 10 20 10 Smith compact 60 40 20 15 F 1440 90 30 20 10 F 1515 90 50 20 10 F 1623 90 40 30 10 F 1728 90 40 25 10 F 1935 90 50 20 10 F 2102 90 60 40 20 F 2080 90 60 30 10 F 1600 90 ND c 20 10 F 1729 80 ND 10 10 F 2073 70 40 20 20 1319 40 20 10 10 139 60 50 10 10

Mean 80 40 20 10

" As determined by hydrophobic interaction chromatography on octyl Sepharose, the % adsorption being calculated from: A540,~. ~ of applied bact.- A540, ~ of unadsorbed bact.

xlO0. As~,~ m of applied bact.

Each value represents the mean of four tests (S.D. + 10%). b Bacterial cells cultured in medium 110 (see MATERIALS AND METHODS). c ND, not determined.

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T a b l e 2

In f luence of mi lk a n d d i f fe ren t g r o w t h a n d i n c u b a t i o n c o n d i t i o n s o n su r f ace h y d r o p h o b i c i t y a n d ce l l - sur face s t ruc tu re of

Staphylococcus aureus as d e t e r m i n e d b y sal t a g g r e g a t i o n test (SAT) a n d bac te r i a l s t a in ing t echn iques

S. aureus S A T value a of b a c t e r i a Visible

s t ra in G r o w n o n G r o w n o n I n c u b a t e d G r o w n in M S M capsu l e b

b l o o d a g a r m e d i u m 110 wi th mi lk

Smi th d i f fuse >_ 1.5 _> 1.5 >- 1.5 >_ 1.5 +

Smi th c o m p a c t 0.5 1.0 1.0 >_ 1.5 -

F1440 0.1 1.0 >_ 1.5 >- 1.5 -

F1515 0.1 0.1 > 1 . 5 >_1.5 -

F1623 0.1 1.0 1.0 >_ 1.5 -

F1728 0.1 0.5 >_ 1.5 _> 1.5 -

F1935 0.1 0.1 1.0 >_ 1.5 -

F2102 0.1 0.5 >- 1.5 >_ 1.5 -

F2080 0.1 1.0 > 1.5 > 1.5 -

F1600 0.1 1.0 > 1.5 >_ 1.5 -

F1729 0.1 1.0 1.0 > 1.5 -

F2073 0.1 >- 1.5 1.0 >_ 1.5 -

1319 1.0 >_ 1.5 >_ 1.5 >_ 1.5 +

139 1.0 >_ 1.5 >_ 1.5 > 1.5 -

a T h e S A T value is expressed as the lowest m o l a r c o n c e n t r a t i o n o f a m m o n i u m sul fa te a t w h i c h b a c t e r i a agg rega t e . Th i s test was

p e r f o r m e d fou r t imes to c o n f i r m the r ep roduc ib i l i t y o f the assay sys t em a n d the lowest r e p r o d u c i b l e m o l a r c o n c e n t r a t i o n has been

taken .

b Visible capsu le u s ing bac t e r i a l s t a i n i n g t echn iques ; - n o capsu le de tec ted .

that low hydrophobicity is associated with higher mobility. The presence of a capsule further in- creased the electrophoretic mobility of the bacteria. Furthermore, prior heating of the test samples did not affect the results (not shown).

5. DISCUSSION

More than a decade ago, Brock et al. [16] reported that mastitis S. aureus strains grown in raw milk and in high carbohydrate-high salt media, in contrast to bacteria from conventional culture media, produced extracellular factors which cause drastic reactions in the bovine skin. A number of studies after this original observation have ad- dressed the virulence of staphylococci to the bacterial surface structures and extracellular prod- ucts [17,18]. In previous reports we showed that S. aureus strains from acute as well as chronic masti- tis expressed high cell-surface hydrophobicity when grown under a variety of conditions [7-9]. Our present report is an extention of our study on bacterial surface properties which may be im-

portant during the initiation of mammary gland infections and in the interaction of bacterial cells with milk in milk-duct epithelium. These epithelial cells may at later stages of the infection be damaged by bacterial toxins, such as staphylococ- cal a-toxi n [19,20], thereby enhancing bacterial colonization of subepithelial tissues with the help of surface structures binding to fibronectin, col- lagen, laminin and perhaps Other matrix compo- nents [21-24].

In this study we have shown that a short period (2 h) of incubation of S. aureus cells in milk or growing cells in milk-supplemented and high carbohydrate-high salt media, caused a drastic change in surface properties (Tables 1 and 2). Kronvall et al. [25] have proposed that binding of a number of serum proteins to group A strepto- cocci and to S. aureus cells may diminish the host immune response to these invading pathogens. Coating of the surface of these udder pathogens by milk and its components, likely to occur within the mammary gland, may have important conse- quences for the later stages of tissue invasion. Studies on phagocytosis suggest that bacteria with

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0 C

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:s

8.0

6.0

4.0

2.0

0.0

8.0

6.0

4.0

2.0

0.0

a • o

// C

- • o

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5 10 15 20

b

d

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Fig. 1. Electrophoretic mobility of S. aureus strains from different test conditions. Free-zone electrophoresis was performed as described in MATERIALS AND METHODS. The samples consisted of 10 #1 of the bacterial cells from blood agar cultures (e), medium 110 (©), milk-supplemented medium (m), or after incubation in milk (A). Buffer was 0.02 M sodium phosphate, pH 6,8. The steeper the slope of the lines, the larger the net surface charge of the bacteria. (a) strain 1319, (b) strain F 1440, (c) strain Smith diffuse, (d) strain Smith compact.

hydrophobic surface properties are readily phago- cytosed [26]. Thus, surface coating of these udder pathogens may be impor tan t to shield the organism from a rapid uptake by phagocytes in milk and in milk-gland tissues.

A C K N O W L E D G E M E N T S

This study was supported by a grant from the Swedish Counci l for Forestry and Agricultural research (A 5691/4032), the Swedish Natura l Sci- ence Research Counci l and the Scientific Research Council , Hunga r i an Minis t ry of Heal th ( 7 6 - 1 1 1 - 03 ETT-3). We are grateful to Dr. Bruno Reiter for s t imulat ing advice. We thank Kar in Elenbr ing for skilful technical assistance. Part of this study

was reported in a symposium on mastitis control and hygienic product ion of milk, Espoo, F in land , June 10-12, 1986.

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