J. Vet. Med. B 38,401-410 (1991) 0 1991 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 0931 -1793

Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala, Sweden

Induction of Anti-Phagocytic Surface Properties of Staphylococcus aureus from Bovine Mastitis

by Growth in Milk Whey

WUBSHET MAMO', CHARLOTTE HALL~N SANDGREN', MATS LINDAHL~ and PER JONSSON~

Address of authors: Department of Veterinary Microbiology, Section of Bacteriology and Epizootology, Swedish University of Agricultural Sciences,

Biomedical Center, Box 583, S-75123 Uppsala, Sweden Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences,

Biomedical Center, Box 575, S-75123 Uppsala, Sweden

With 4 figures and 2 tables

(Received for publication October 30, 1990)

Summary The respiratory burst activity of bovine polymorphonuclear (PMN) cells in response to milk

whey- and TSB-grown S. awe&$ strains isolated from bovine mastitis was studied in whole blood chemiluminescence (CL) and in a CL system with purified bovine neutrophils. In both cases milk whey-grown S. aureus strains elicited significantly less CL than homologous strains grown in TSB. Ingestion of milk whey-grown S. aureus strains by bovine neutrophils was also considerably lower than that of the corresponding homologous organisms grown in TSB. Binding of complement factor C3 to serum-opsonized milk whey-grown S. aureus strains was lower compared with TSB-grown homologous organisms. Moreover, 5 of 6 S. aureus strains grown in milk whey were significantly more resistant to in vivo clearance from the peritoneal cavity of mice compared with homologous bacteria grown in TSB.

S. aureus strains grown in TSB exhibited hydrophobic surface properties, whereas homologous strains grown in milk whey were hydrophilic.

Introduction Phagocytosis by polymorphonuclear granulocytes is an important defence mechan-

ism of the bovine udder against invading microorganisms (8, 21). Recognition of bacteria by phagocytic cells, resulting in attachment and subsequent ingestion, is determined by the surface properties of both bacteria and phagocytic cells (5,6,25,29). The ingestion (31) and respiratory burst activity (2) of polymorphonuclear (PMN) leukocytes in response to Staphylococcus aureus have been studied in detail (23,27,31,33). Thus it is well known that phagocytosis of S. aureus by PMN leukocytes is strongly promoted by an adequate opsonization via activation of complement and specific antibodies (12, 30, 33). In several studies it has been proposed that the presence of cell-surface components, such as protein A (22) and capsule material (23, 35), as well as charge and hydrophobic surface properties (25, 28) may affect the opsonization and the recognition of the organism by phagocytic cells.

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402 MAMO, SANDGREN, LINDAHL and JONSSON

It has been demonstrated that S. aureus isolated from human infections and grown in vivo are more resistant to intracellular killing by human PMN leukocytes than organisms grown in vitro (I , 15). This resistance has been attributed to the capsule of S.aureus which is expressed when grown in viwo (11). S. aureus strains from bovine mastitis grown under certain conditions (20, 26) and freshly isolated strains (26, 36) have also been proposed to possess a capsule. We and others have reported that growth of S. aureus strains from bovine mastitis in milk or in milk whey results in an alteration of the surface properties of the organisms (18, 32).

The aim of this study was to investigate the effect of growth of S. aureus strains in milk whey on phagocytosis by bovine PMN leukocytes.

Material and Methods Bacterial Strains

Staphylococcus aurem strains were isolated from milk of cows with clinical mastitis. Isolation and identification were done according to recommendations for examination of milk samples (13) at the National Veterinary Institute, Uppsala, Sweden.

Bacterial Growth Conditions Milk whey was prepared as previously described (19). The bacterial culture was prepared by

inoculating an overnight blood agar culture into 1.0ml milk whey which was incubated at 37°C for 10h. The culture was transferred into lOOml milk whey and further incubated at 37°C for another 10 h in a rotary shaker (50 rpm). Bacteria were sedimented by centrifugation (3,000 x g for 20 min) at 4°C and washed twice in phosphate buffered saline (PBS), (pH7.4). The number of bacteria per milliliter was adjusted after counting under the microscope. For intraperitoneal inoculation, viable count was also performed. In the phagocytosis experiments bacteria were either alive or were heat killed (80"C, for 10 min). Homologous organisms treated as above but grown in tryptic soy broth (TSB, Difco, Detroit, Mich. USA) were used as controls in all assays.

Bacterial Surface Hydrophobicity Bacterial cell-surface hydrophobicity was determined by the Salt Aggregation Test (SAT) (16).

Isolation of Bovine Neutrophils Neutrophils were isolated from fresh citrated bovine blood according to the previously

described method (7) with some modifications. Briefly, a volume of 20ml blood was centrifuged at 200 x g for 10 min. Plasma and buffy coat fractions were discarded. After hypotonic treatment, the remaining leukocytes were sedimented by centrifugation and suspended in 1.0 ml of 0.9 % (w/v) NaCI. The cell suspension was applied to a metrizamide gradient with the following densities, 1.113, 1.119, 1.1215, 1.134g/ml (22°C) and centrifuged (1.2OOxg for 45 min). Neutrophils banding at a density of 1.1215 were collected and suspended in 0.5ml of modified Gey's solution (MGS) (7). Cells were counted in Turk's solution and the purity of neutrophils was 97 f 2 (SD)% as judged by May- Grunewald Giemsa staining.

Serum and Opsonization For opsonization of bacteria, normal fetal calf serum (FCS, GIBCO, Scotland, UK) was used for

whole blood CL, and IgG-poor FCS (< 1 pg IgG/ml) (10) for all other assays. Serum aliquots were stored at - 70 "C until required. Bacterial cells (final concentration, 3 x 109 bact/ml) were incubated with 75 Yo (v/v) serum at 37°C for 30 min. After incubation the bacterial suspensions were washed twice and suspended in MGS to the appropriate concentration.

Preparation of F(ab')2 F(ab')2 fragments were prepared from rabbit IgG anti-bovine C3 serum (Organonteknika,

West Chester, PA, USA) (9). The purity of fragments, tested by sodium dodecyl sulphate-polyacryl- ide gel electrophoresis, was higher than 98 %. Prior to usage, the F(ab')2 fragments (2.8 mg/ml) were absorbed with unopsonized bacteria at 4°C for 18 hr.

Induction of Anti-Phagocytic Surface Properties of Staphylococcus uureus

Chemiluminescence (CL) (i) Whole blood CL: Heparinited whole blood from 3 apparently healthy cows was used as a

source of PMN cells. CL was monitored at 37°C in a luminometer (1251 LKB, Wallac, Turku, Finland) according to the method described by MAGNUSSON and EINARSSON (17). The reaction was started by addition of 100 pl of whole blood to 900 p1 of a bacterial suspension (final concentration 1 x lo9 bactlml) in PBS containing 22 pM luminol. Reaction mixtures without bacteria were used as negative controls. The resulting light output was recorded at intervals of 166 s and the CL activity was evaluated by comparing the maximum peak values obtained from the recorded graphs.

(ii) C L of isolated neutrophils: The assay was monitored at 37°C in a luminometer (Lumac Biocounter, RM 2010, Basel, Switzerland). Neutrophils (1 x lo5 per sample) in MGS containing 0.75 % (w/v) bovine serum albumin (BSA, Sigma, St.Louis, Mo., USA) and luminol (60mg/l) in a total volume of 67 ~1 were prewarmed at 37 "C for 30 s. The reaction was started by the addition of 33 pI opsonized bacteria in MGS (final concentration 1 x 108 bact/ml). The resulting light output was recorded graphically for 10 min. The area under the recorded curve was integrated and represented the total CL produced by the cells expressed in arbitrary units.

403

Ingestion of 3[.]-Tbymidine-Labelled S. aureus by Neutrophils The assay was performed as previously described (31) with some modifications. Bacteria were

cultured in lOml of milk whey or TSB containing 0.02mCi '[HI-methyl thymidine (specific activity 5 Ci/mmol, Amersham, England). Bacterial cells were washed (3.000 x g at 4°C for 20 min) in PBS, (pH 7.4) until '[HI activity in the supernatant was equivalent to background value. A 667 pl aliquot of the neutrophil suspension (1.5 x lo6 cells/ml) in MGS-BSA and 333 pl bacterial cell suspension (4 x lo9/ ml) in MGS were added to a plastic tube. Bacterial suspensions without neutrophils were incubated as controls. The tubes were incubated at 37°C for 30 min with continuous rotation and then placed on ice. Extracellular bacteria were lysed by treatment with lysostaphin (10pg/ml) at 37°C for 30 min. Cells were washed twice (200 x g, for 10 min) in ice-cold PBS, solubilized with 2 x 200 pI of 10 % SDS and suspended in scintillation liquid (Ready Safe, Beckman, Fullerton, Ca, USA). The remaining radioactivity was counted in a liquid scintillation counter (Beckman LS 3 800, Turku, Finland). Determination of the fraction of ingested bacteria was done by subtracting background radioactivity of bacterial samples incubated without neutrophils from intracellular radioactivity of neutrophils incubated with bacteria. Data was expressed as percentage of the radioactivity in the bacteria initially added (Fig. 3).

Binding of Complement Factor C3 to Serum-Opsonized S . aureus Sixty pl of serum-opsonited S. aureus (4 x lo9 bact/ml) in PBS containing 0.5 YO (w/v) BSA (PBS-

BSA) was mixed with 8 pI of decreasing concentrations of rabbit anti-Cj F(ab'), in the same buffer and incubated at 37°C for 30 min. Each sample was tested in duplicate. The suspensions were washed three times in PBS-BSA and bacteria were incubated with 60 pI of alkaline phosphatase-conjugated goat anti-rabbit IgG F(ab'), (Sigma) diluted 1 : 500 in PBS containing 0.5 % (w/v) Tween at 37°C for 30 min. After washing as above, 60 p1 of substrate (p-nitrophenylphosphate, 1 mg/ml dissolved in 9.7 YO (v/v) diethylamine, pH 9.8) were added and the suspensions were incubated at room tempera-

Table 1. Relative surface hydrophobicity of S. aureus strains isolated from bovine mastitis after growth in milk whey or in tryptic soy broth (TSB). SAT was scored as the lowest molar concentration

(M) of ammonium sulphate at which bacterial aggregation occurred

S. aureus strains SAT value (M)

Organisms grown in Milk whey TSB

F1440 F1935 mj13229 mj10050 mj10064 mj 15129

1.6 2.0 1.8 1.6 1.6 2.0

0.05 0.05 0.05 0.05 0.1 0.05

Each value represents the mean of 4 experiments.

404

0)

Ip > * CU Q,

a I

n

Fig. 1

400

300

200

100

0

MAMO, SANDGREN, LINDAHL and JONSSON

T

Bacterial

Y 0 v) 0 0

r .-. E

T

strains

li P u) 0 0 .r "-.

E

I

. Chemiluminescence (in arbitrary units) of bovine whole blood in response to (m) milk whey or (0) TSB-grown S. aurew strains from bovine mastitis. Samples without bacteria were used as negative controls. The results were obtained from the maximal peak value of the recorded graph and subtracted from that of the negative controls. Average and standard deviation (SD) were obtained

from two experiments with blood from 3 different cows. = p < 0.01, 'b = p C 0.05

ture for 45 min. Bacteria were sedimented as above and 50p1 of the supernatant were added to microtiter wells. The reaction was stopped by addition of 3M NaOH (12.5 pl) and absorbance was read at 405 nm. The AA405 was calculated by subtraction of the absorbance readings of bacteria opsonized in MGS 1.0 % (w/v) BSA from that of the absorbance readings of serum-opsonized bacteria.

In viva Clearance of S. aureus Evaluated by Intraperitoneal Inoculation of Mice NMRI mice (6-9 weeks old) were inoculated intraperitoneally with 0.1 ml of bacterial

suspension (1 x 105 CFU) prepared from milk whey or TSB cultures. Mice were killed 24 hours post- inoculation and the peritoneal cavity was washed with 3.01111 sterile physiological saline (0.9 % w/v). Peritoneal fluid (3.Oml) was removed and diluted for determination of the total number of bacteria recovered (CFU).

Statistical Methods Statistical evaluation of the data was performed using the two tailed Student's t-test for unpaired

observations.

Results Cell Surface Hydrophobicity

T h e relative surface hydrophobicity of 6 S. aweus strains grown in milk whey or TSB was determined. All strains grown in the presence of milk whey exhibited hydrophilic

Induction of Anti-Phagocytic Surface Properties of Staphylococcus uureus 405

surface properties (SAT value 2 1.5 M). In contrast, all TSB-grown organisms exhibited hydrophobic surface properties (SAT value < 0.1 M) (Table 1).

Chernilurninescence (CL) The respiratory burst activity in response to serum opsonized milk whey-grown

S. aureus strains was studied in the whole blood system. With the exception of one strain (mjl0050), all strains grown in milk whey elicited considerably lower CL (5-48 YO) than that of TSB-grown homologous bacteria (Fig. 1). Furthermore, CL of purified neutrophils in response to 3 selected heat-killed S. aureus strains (F1440, F1935 and mj10064) was studied. It was demonstrated that the CL response of purified bovine neutrophils to milk whey-grown bacteria was lower (10-20 Yo) to that elicited by homologous bacteria grown in TSB (Fig. 2).

Ingestion by Neutrophils Ingestion of two 3[H]-thymidine-labelled and heat-killed S. aureus strains (F1440 and

mj10064) by purified neutrophils was studied. Bacteria grown in milk whey were highly resistant to phagocytosis. Ingestion of these bacteria was only 16 and 4 YO respectively of that with homologous organisms grown in TSB (Fig. 3).

400

300

200

100

0

I-

0 d d

LL 7-

T

1 Bacterial strains

Fig. 2. Total chemiluminescence (in arbitrary units) of purified bovine neutrophils in response to 3 strains of S. aureus from bovine mastitis grown in (m) milk whey or (0) in TSB. Samples without bacteria were used as negative controls. Average and SD were obtained from 3 experiments with

neutrophils from 3 different cows. ' w ~ = p < 0.001, * = p < 0.05

100

75

50

25

0

MAMO, SANDGREN, LLNDAHL and JONSSON

T I

Bacterial strains

Fig. 3. Ingestion of 3[H]-thymidine-label- led S.aureus strains F1440 and mj10064 grown (U) in milk whey or (0) in TSB by purified bovine neutrophils. Ordinate represents percent of bacteria ingested by neutrophils. Average values and SD were obtained from 4 experiments run in tri- plicate with neutrophils from 4 different

cows. *:* = p 6 0.01

Binding of Complement Factor C3 to Serum Opsonized S . aureus The degree of C3 binding to serum-opsonized S. aureus strains was determined. We

found that binding of complement factor C3 to serum-opsoniied S. aureus strains (F1440 and mj10064) grown in milk whey was -30% of that of homologous strains grown in TSB at the lowest dilution of the F(ab’), fragment (Fig. 4 a and b).

In vivo Clearance All strains grown in TSB were recovered in low numbers (log CFU 2.9-5.1) from the

peritoneal cavity of mice. When milk whey-grown homologous bacteria were inoculated 5 out of 6 strains were recovered in high numbers (log CFU 8.6-9.5). S.aureus strain mj10050 grown in either TSB or milk whey was recovered in low numbers (Table 2).

Discussion An effective killing of Xaureus by PMN leukocytes is dependent on the cellular

respiratory burst activities. In order to activate the cellular respiratory burst, the activating prey, i. e. the bacteria, must be recognized and ingested by the phagocytic cells (2, 4, 14).

According to our results, S. aureus strains cultured in milk whey induced a signifi- cantly lower CL response in both bovine whole blood and purified neutrophils compared with homologous organisms cultured in TSB. It is unlikely that products released from bacteria had an inhibitory effect on CL per se, since the results obtained from the whole blood CL system using live bacteria were analogous to the results obtained from the CL studies of purified neutrophils using extensively washed and heat-killed bacteria. Instead, we find it possible that milk whey-grown bacteria are poorly recognized by bovine whole blood phagocytic cells or purified bovine neutrophils. This could be confirmed by a

Induction of Anti-Phagocytic Surface Properties of Staphylococcus arrreus 407

Fig.4. The deposition of Complement factor C3 on the surface of S,aureus strains (a) F1440 and (b) mi10064 grown in milk whey (0) or in TSB (0). The binding of C3 was detected as described on

Material and Methods

In 0 P Q Q

ln 0 P Q Q

- 3 - 2 - 1 0 log conc F(ab’)2

- 3 -2 - 1 0 log conc F(ab ’ )2

reduced uptake of milk whey-grown S. aureus strains by neutrophils compared with TSB- grown organisms. The results show that CL activity of neutrophils was due to ingestion and not merely to recognition or attachment of bacteria to the neutrophils.

Serum opsonic factors predominately complement and IgG are known to support an efficient recognition of bacteria by phagocytic cells resulting in uptake of the microorgan- ism. It has been reported that the low phagocytic activities towards certain human S. aureus strains is related to low levels of complement C3 deposition on the bacteria (33,34). In the present study we demonstrate that upon opsonization with serum, the deposition of complement factor C3 on milk whey-grown bacteria was considerably lower than on homologous organisms grown in TSB. However, compared to the low phagocytic activities of purified neutrophils towards milk whey-grown bacteria, the binding of C3 to these organisms was high. A possible explanation for this could be that the surface structure expressed on milk whey-grown bacteria may permit the penetration of C3 molecules but reduce the accessability of C3 binding sites to the bovine neutrophils. A similar effect on C3 binding to encapsulated human S. aureus strains has been reported (35).

408 MAMO, SANDGREN, LINDAHL and JONSSON

Table 2. The number of S. aureus strains recovered from the peritoneal cavity of mice 24 h post- inoculation. Bacterial cells ( 1 x 105 CFU) grown in milk whey or in TSB were used to inoculate mice.

The total number of bacteria recovered was calculated as described in Material and Methods

No. of bacteria recovered from the peritoneal cavity (log f SEM) Organisms grown in S. aureus strains

Milk whey TSB

F1440 F1935 mj 13229 mj10050 mj10064 mj 15129

9.5 f 0.31 8.6 f 0.35 9.2 f 0.31 4.8 & 0.22 8.7 f 0.40 9.3 f 0.22

5.1 f 0.42 4.1 f 0.50 2.9 f 0.27 4.0 f 0.80 3.4 f 0.67 4.7 f 0.05

Each value represents the mean of five experiments.

The existence of a capsule in bovine S.aureus strains has long been debated (24). SUTRA et al. (26) recently reported the presence of polysaccharide capsule types 5 and 8 in S. uureus strains isolated from mastitic milk. In another study, neither milk whey nor TSB- grown S . aureus strains could be demonstrated to possess a capsule (19) by means of the India ink method (3). However, 5 out of 6 strains grown in milk whey exhibited diffuse type colony morphology in SSA (19). In addition, milk whey-grown bacteria were found to be hydrophilic. The possession of diffuse-type colony morphology coincided with resistance to phagocytosis (data not shown).

Diffuse-type colony morphology in combination with hydrophilic surface properties of bacteria grown in milk whey strongly suggests the existence of a coating substance. This substance seems to have a function similar to that of a capsule in the phagocytic assays (35).

Milk whey-grown S . aureus strains also resisted clearance from the peritoneal cavity of mice, most probably due t o the presence of a coating substance expressed upon growth in milk whey, resulting in poor recognition of the organisms by phagocytic cells.

In conclusion, S. aureus strains isolated from bovine mastitis and then grown in the presence of milk whey resist phagocytosis by bovine blood neutrophils and in viva clearance from the peritoneal cavity of mice. The anti-phagocytic coating substance is heat- resistant, renders the bacterial surface hydrophilic and is demonstrated to impede the binding of complement factor C3. This substance could be an important virulence determinant in the development of S. aweus mastitis in the bovine udder.

Acknowledgements We are grateful for the excellent technical assistance of INGRID LARSSON. This study was

supported by grants from the Swedish Council for Forestry and Agricultural Research, grant No. 536/ 84D/151 to W. M. and 0. HOLMBERG; grant No. 546/88 to C. H. S. and I. BJORK.

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