Growth Responses of Staphylococcus aureus and Streptococcus agalactiae to Corynebacterium bovis Metabolites1

Responses o f Staphylococcus aureus a n d Streptococcus agalactiae Corynebacterium bovis M e t a b o l i t e s t

Growth to

J, S. HOGAN, 2 J. W. PANKEY, and A. H. DUTHIE Department of Animal Sciences Hills Science Building University of Vermont Burlington 05405 ABSTRACT

Staphylococcus aureus and Streptococcus agalactiae growth responses to metabolites of Corynebacterium boris cultured in media containing polyoxyethylenesorbitan monolaurate, monooleate, or trioleate and milk were determined. Filter sterilized metabolites of 48-h C. boris cultures in synthetic media were added to cultures of Staph. aureus and Strep. agalactiae. Staphylococcus aureus and Strep. agalactiae were inoculated into 12-h C. boris milk cultures. Growth responses of Staph. aureus and Strep. agalactiae were not affected by C. boris metabolites of synthetic media. Staphylococcus aureus growth was inhibited during logarithmic and stationary phases in milk containing mixed cultures o f C. boris compared with growth in pure Staph. aureus cultures. Streptococcus agalactiae growth curves were similar in pure and C. boris mixed cultures. F a t t y acid compositions were not different in sterile milk and milk containing bacterial cultures. Growth responses of Staph. aureus and Strep. agalactiae were not related to concentration of C. boris metabolites or fatty acid content of media in which C. boris were cultured. INTRODUCTION

The rate of bovine intramammary infections is a function of the microbial load at the teat end (17). Coagulase-negative staphylococci and

Received July 31, 1986. Accepted February 20, 1987. 1Authors acknowledge support received from Vermont Agricultural Experiment Station. 2Present address: Department Dairy Science, Ohio State University, Ohio Agricultural Research and Development Center, Wooster 44691. 1987 J Dairy Sci 70:1294-1301

Corynebacterium boris were often the bacterial groups predominating on the dermal surfaces of teats (1, 3, 6, 20). The role of these commensal bacteria in control of superinfections by major mastitis pathogens is largely speculative. A model for similar skin microflora interactions was well-established in humans. Lipolytic coryneforms and coagulase-negative staphylococci produced substances that retarded Staphylococcus aureus growth (16, 18, 27) and lipases that cleaved triglycerides of sebaceous origin to release free fatty acids (7, 13, 14, 21, 22, 24). The release of long-chain fatty acids on human skin by commensal organisms was correlated with pathogenesis of hemolytic staphylococcal and streptococcal infectious skin disorders (4, 7, 23, 26). Similar microbial interactions in the bovine teat canal have not been documented. Brooks and Barnum (2) reported quarters shedding C. boris were more resistant to infection than bacteriologically negative quarters following cisternal infusion of Stapb. aureus. Resistance to Streptococcus agalactiae challenge was not observed. Pankey et al. (20) found rate of intramammary infections by Staph. aureus in quarters infected with C. boris were reduced 50% compared with rate of infection in bacteriologically negative quarters when teats were challenged by immersion in broth culture. In contrast, C. boris-infected quarters were eight times more susceptible to superinfection with Strep. agalactiae. Corynebacterium bovis preferentially colonized the stratum corneum in the distal third of the teat canal (1, 20). Three hypotheses for enhanced rate of intramammary infections of Strep. agalactiae in C. Boris-infected quarters were offered: 1) C. boris catabolized teat canal keratin components that were inhibitory to Strep. agalactiae; 2) metabolites of C. boris were antagonistic to Strep. agalactiae inhibitory factors of teat canal keratin; or 3) metabolites of C. boris were stimulatory to Strep, agalactiae

1294

COR YNEBA CTERIUM BO VIS METABOLITES and enhanced multiplication through the teat canal. The purpose of this study was to determine the effects of C. boris metabolites on Stapb. aureus and Strep. agalactiae growth in vitro. MATERIALS AND METHODS Trial 1

Bacterial Media. Chemically defined growth media for S. aureus (BSB), S. agalactiae (AGM), and C. boris (CBM) were described by Hogan et al. (11). Polyoxyethylenesorbitan monolaurate, monooleate, and trioleate were purchased. 3 Test substrates were each medium supplemented with 2.6 g/L of polyoxyethylenesorbitan monolaurate (BSB20, AGM20, CBM20), monooleate (BSB80, AGM80, CBM80), or trioleate (BSB85, AGM85,CBM85), and filtrates of 48 h C. boris cultures (CB20F, CB80F, CB85F). Bacterial Cultures. Bacterial strains utilized were S. aureus ATCC 28740, Strep. agalactiae ATCC 27956, and C. boris Buzzy 1 (20). Staphylococcus aureus and Strep. agalactiae were cultured and prepared for inoculation into assays as outlined by Hogan et al. (11). Corynebacterium bovis were serially subcultured twice on nutrient agar 4 containing 1% polyoxyethylenesorbitan monooleate (NPM). One colony from terminal subcultures was used to inoculate tubes containing 6 ml of CBM20, CBM80, or CBM85 and incubated 18 h at 37°C. One milliliter of each primary broth subculture was inoculated into 50 ml of CBM20, CBM80, or CBM85 and incubated at 37°C on a gyratory shaker at 200 rpm for 48 h. Cultures were filter sterilized through a .45-/a pore size membrane, and filtrate was retained. Each culture was in duplicate. Microassay o f Bacterial Growtb. Staphylococcus aureus and Strep. agalactiae growth responses were measured by a modification of

3Tweens 20, 80, and 85, Sigma Chemical Co., St. Louis, MO 63178. 4Difco Laboratories, Detroit, MI. s Coming Glass Works, Corning, NY 14831. 6Farm Best, Dairymen Inc., UHT Division, Savannah, GA 31048. Gibco Laboratories, Chagrin Falls, OH 44022.

1295

the assay described by Nonnecke and Smith (19). A single 96-well tissue culture plate s facilitated duplicate assays on effects of four substrates at five concentrations on growth of two bacterial strains. A total of 200 of each 300-/A capacity well was allotted to chemically defined medium and 50 /~I to bacterial inoculum of 20 x 104 cfu/ml. Ten microliters of test substrate were added for final well volume of 260/J1. Controls for Stapb. aureus and Strep. agalactiae were 10 /A BSB and AGM, respectively. Procedures for incubation, plating, and determination of growth responses are described by Hogan et al. (11). Statistical Analysis. Homogeneity of variance and normality of growth response data were confirmed by Bartlett's test for homogenicity and G-test for goodness of fit. Differences between means were measured by Tukey's method for testing all possible comparisons (25). Trial 2

Bacterial Cultures. Bacterial strains were those used in Trial 1. Corynebacterium boris were serially subcultured twice on NPM. One colony from a terminal subculture was inoculated into 50 ml sterile milk 6 and incubated at 37°C on a gyratory shaker at 200 rpm for 12 h. Aliquots of 10, 1, and .1 ml of this culture were used to inoculate quadruplicate flasks containing 50 ml sterile milk. Cultures were designated CBH, CBI, and CBL for high, intermediate, and low initial C. boris concentrations. Flask cultures were incubated for 12 h as described. Staphylococcus aureus and Strep. agalactiae were serially subcultured on trypticase 5% sheep-blood agar 7 (TBA). One colony of both bacterial strains was inoculated into separate 5-ml aliquots of sterile milk and incubated 12 h at 37°C. Twelve-hour CBH, CBI, and CBL cultures were each inoculated with .2 ml of either Stapb. aureus or Strep. agalactiae subcultures. Controls were pure cultures of Stapb. aureus. Strep. agalactiae, and C. boris. An uninoculated sterite milk control was included. Cultures and uninoculated controls were incubated for 72 h at 200 rpm on a gyratory shaker at 37°C. Bacterial populations were quantitated on TBA (15). Diluent was .1% proteose-peptone containing 1% polyoxyethylJournal of Dairy Science Vol. 70, No. 6, 1987

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TABLE 1. Mean growth responses of Staphylococcus aureus, after 12 h incubation, to media containing polyoxyethylenesorbitan monolaurate (PES 20), monooleate (PES 80), and trioleate (PES 85) and mean growth responses to filter-sterilized metabolities of Corynebacterium boris cultured in synthetic media. Growth response 1 Media

PES

10/~g/ml

1 ~g/ml

.1 /~g/ml

BSB: CBM3 CBF 4

20 20 20

100.6 101.6

100.8

98.2

100.3

99.2

96.6

100.4 102.4 100.0

97.1 99.5

101.7

BSB CBM CBF

80 80 80

96.6 102.8 101.8

104.3 102.1 100.4

99.1 97.8 98.2

100.4 100.9 93:2

100.3 103.8 99.6

BSB CBM CBF

85 85 85

97.4 97.8 97.3

104.7 101.4 101.7

98.6 103.1 100.6

98.6 100.6 98.4

96.6 98.2

98.7

.01 ~g/ml

.001 #g/ml

99.6

99.2

1 [Growth under experimental conditions (cfU/log/ml)/growth in BSB without PES (cfUlog/ml)] × 100. 2 Bacto-Synthetic Broth (Difco Laboratories, Detroit, MI) supplemented with 10 g/L dextrose. 3Chemically defined growth medium for cultivation of Corynebacterium boris. 4 Filter-sterilized metabolites of 48-h Corynebacterium boris cultures. Filtrate concentrations of PES expressed as initial concentrations prior to Corynebacterium boris cultivation.

enesorbitan m o n o o l e a t e . Cultures, controls, and bacterial counts were in duplicate.

Fatty Acid Analyses and p H Determination. F a t t y acid c o m p o s i t i o n o f m i l k cultures was d e t e r m i n e d at the end o f i n c u b a t i o n periods by m e t h o d s o f Duthie et al. (5) and Wulff et al. (28). The pH o f milk cultures was measured electronically, s Statistical Analysis. Bacterial counts were expressed as log c o l o n y - f o r m i n g units per milliliter. Differences b e t w e e n bacterial counts were d e t e r m i n e d by T u k e y ' s m e t h o d for testing all possible comparisons. F a t t y acid compositional differences were tested by analysis of variance (25). RESULTS AND DISCUSSION Trial 1

G r o w t h responses of Staph. aureus and Strep. agalactiae were n o t affected by C. boris metabolites of chemically defined media (Tables 1 and 2). Significant differences were not observed b e t w e e n growth responses of

SBeckman 44 pH meter, Beckman Instruments Inc., Irvine, CA 92713. Journal of Dairy Science Vol. 70, No. 6, 1987

Staph.

aureus or Strep. agalactiae to polyo x y e t h y l e n e s o r b i t a n derivates, c o n c e n t r a t i o n of p o l y o x y e t h y l e n e s o r b i t a n derivates, or t i m e o f i n c u b a t i o n (P>.05). Differences were n o t d e t e r m i n e d b e t w e e n Staph. aureus growth responses to the three media (P>.05). Mean g r o w t h response o f Strep. agalactiae to CBM (100.3) was greater (P<.05) than to AGM (95.5) and CBF (97.5). The greater growth response to CBM was inferred to be due to m e d i u m constituents that supplemented nutritional r e q u i r e m e n t s of Strep. agalactiae. Results suggested C. boris had catabolized these Strep. agalactiae growth-stimulating constituents in CBF. This was inconsistent with t h e hypothesis t h a t C. boris metabolites m a y enhance Strep. agalactiae multiplication. C o r y n e f o r m s d o m i n a n t on h u m a n epidermis p r o d u c e d lipases that cleaved ester bonds of lipids to release free f a t t y acids that were inhibitory to h e m o l y t i c staphylococci and s t r e p t o c o c c i (4, 7, 23). The inability to measure an influence o f C. boris lipolytic metabolites on Staph. aureus or Strep. agalactiae was interp r e t e d as either: 1) C. boris did n o t produce lipases to release esterified f a t t y acids, or 2) nonesterified f a t t y acids resulting f r o m C. boris lipolytic activity were not in concentrations sufficient to o v e r c o m e neutralizing effects of

CO R YNEBA CTERIUM BO VIS METABOLITES

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TABLE 2. Mean growth responses of Streptococcus agalactiae, after 12 h incubation, to media containing polyoxyethylenesorbitan monolaurate (PES 20), monooleate (PES 80), and trioleate (PES 85) and mean growth responses to filter-sterilized metabolities of Corynebacterium boris cultured in synthetic media. Growth response 1 Media

PES

10 #g/ml

1 pg/ml

.1 pg/ml

AGM 2 CBM 3 CBF 4

20 20 20

AGM CBM CBF AGM CBM CBF

.01 pg/ml

.001 #g/ml

110.5 107.9 115.5

92.6 95.1 94.3

92.2 98.8 90.0

90.4 b 102.3 a 102.6 a

101.3 93.4

80 80 80

95.0 103.3 100.7

100.5 102.5 97.8

105.6 105.2 93.3

102.5 106.8 95.9

96.8 b 105.6 a 95.5 b

85 85 85

99.8 b 111.4 a 98.9 b

99.9 103.0 97.7

92.4 97.2 96.5

93.7 94.6 95.9

92.1 92.0 96.1

a'bMeans within concentrations for each PES with differing superscripts differ (P<.05). i [Growth under experimental conditions (cfU/log/ml)/growth in AGM without PES (cfUlog/ml)] X 100. 2 Chemically defined growth medium for cultivation of Streptococcus agalactiae. 3Chemically defined growth medium for cultivation of Coyrnebacterium bovis. 4Filter_sterilized metabolites of 48 h Coynebacterium boris cultures. Filtrate concentrations of PES expressed as initial concentrations prior to Corynebacterium bovis cultivation.

w a t e r - s o l u b l e f a t t y acid esters o n a n t i b a c t e r i a l f a t t y acids. T h e s e i n t e r p r e t a t i o n s were supp o r t e d b y t h e results o f Jayne-Williams a n d S k e r m a n (12) t h a t lipid g r o w t h r e q u i r e m e n t o f C. boris was fulfilled b y p o l y o x y e t h y l e n e s o r b i t a n m o n o l a u r a t e a n d m o n o o l e a t e , b u t att e m p t s t o d e t e c t l i p o l y t i c activity of C. boris o n t h e s e c o m p o u n d s was unsuccessful.

diauxic g r o w t h o f Staph. aureus in m i x e d c u l t u r e s was similar to g r o w t h r e s p o n s e s following induced enzyme synthesis for utilization o f n o n l i m i t i n g s u b s t r a t e s as d e s c r i b e d b y G o t t s c h a l k (8).

10.O"

Trial 2

S t a p h y l o c o c c u s aureus g r o w t h d u r i n g logari t h m i c a n d s t a t i o n a r y p h a s e s was s u p p r e s s e d in m i x e d c u l t u r e s c o m p a r e d w i t h g r o w t h in p u r e c u l t u r e s (Figure 1). S t a p h y l o c o c c u s aureus c o u n t s were g r e a t e r in p u r e c u l t u r e s t h a n in CBH, CBI, a n d CBL a f t e r 4, 8, 12, 16, 20, a n d 24 h i n c u b a t i o n ( P < . 0 5 ) . D i f f e r e n c e s w e r e n o t s i g n i f i c a n t b e t w e e n Stapb. aureus c o u n t s in CBH, CBI, a n d CBL ( P > . 0 5 ) . Pure c u l t u r e s o f Stapb. aureus a p p e a r e d t o b e e n t e r i n g d e a t h p h a s e a f t e r 72 h o f i n c u b a t i o n . S t a p h y l o c o c c u s aureus g r o w t h curves in m i l k c o n t a i n i n g C. boris c u l t u r e s were b i p h a s i c , a n d g r o w t h increased throughout the incubation period. S u p p r e s s i o n o f Stapb. aureus g r o w t h jn CBH, CBI, a n d CBL was p o s s i b l y d u e t o c o m p e t i t i o n b e t w e e n species f o r available substrates. T h e

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Hours Figure 1. Stapbylococcus aureus growth in milk and in milk containing 12-h Corynebacterium boris cultures [initial concentrations of Corynebacterium boris at - 1 2 h were 28 × IO s (CBH), 29 × 104 (CBI), and 22 × 103 (CBL) cfu/mll. Milk ( . . . o . . . ) , CBH (- - -m- - -), CBI (-- - ~ - --), and CBL ( - - ~ - - ) . Journal of Dairy Science Vol. 70, No. 6, 1987

1298

HOGAN ET AL,

9-

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~'~ 7-

i ///

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0

12

24

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48

36

60

72

Hours

Figure 2. Streptococcus agalactiae growth in milk and in milk containing 12-h Corynebacterium boris cultures [initial concentrations of C. boris at - 1 2 h were 30 X lO s (CBH), 32 X 104 (CBI), and 24 X 10 a (CBL) cfu/ml]. Milk ( . . . e . . . ) , CBH (- - - m - - -), CBI ( - - z~ -- -), and CBL ( - - o - - ) .

Streptococcus agalactiae g r o w t h was n o t i n h i b i t e d in C. boris m i x e d cultures (Figure 2). D i f f e r e n c e s were n o t significant b e t w e e n Strep. agalactiae c o u n t s in p u r e c u l t u r e , CBH, CBI, a n d CBL d u r i n g l o g a r i t h m i c g r o w t h a n d d e a t h phases. Streptococcus agalactiae g r o w t h in p u r e c u l t u r e a n d CBL was g r e a t e r t h a n in CBH a n d

CBI a f t e r 16, 20, a n d 24 h i n c u b a t i o n ( P < . 0 5 ) . In c o n t r a s t t o t h e diauxic g r o w t h of Stapb. aureus in m i x e d cultures, t h e s h a p e of Strep. agalactiae g r o w t h curves in CBH, CBI, a n d CBL was similar t o t h a t in p u r e culture. T h e s e f i n d i n g s suggested t h a t s u b s e q u e n t i n c u b a t i o n o f C. b o a s in m i l k did n o t alterStrep, agalactiae physiological responses for u t i l i z a t i o n o f r a t e - l i m i t i n g substrates. Corynebacterium boris c o u n t s did n o t differ in CBH, CB], or CBL m i x e d a n d p u r e c u l t u r e s d u r i n g early s t a t i o n a r y g r o w t h p h a s e (Figures 3, 4, a n d 5). D i f f e r e n c e s were n o t significant b e t w e e n C. boas c o u n t s o f p u r e a n d m i x e d cult u r e s in e i t h e r CBH, CBI, o r CBL a f t e r 4, 8, 12, 20, 24, or 36 h i n c u b a t i o n ( P > . 0 5 ) . Corynebacterium boris in CBL c o n t a i n i n g Stapb. aureus or Strep. agalactiae a n d CB1 c o n t a i n i n g Stapb. aureus were e n t e r i n g d e a t h phase a f t e r 36 t o 4 8 h i n c u b a t i o n . T h e decrease o f C. boris p o p u l a t i o n s in m i x e d c u l t u r e w i t h Stapb. aureus c o r r e s p o n d e d to t h e s e c o n d l o g a r i t h m i c g r o w t h p h a s e o f Stapb. aureus. C o r r e l a t i o n c o e f f i c i e n t (r) a n d c o e f f i c i e n t of d e t e r m i n a t i o n (r 2) b e t w e e n pH of m i x e d c u l t u r e s a n d C. boris c o u n t s a f t e r 72 h i n c u b a t i o n was .88 and .77 ( P < . 0 5 ) . T h e s e results i n d i c a t e d t h a t acid p r o d u c e d b y Stapb. aureus a n d Strep. agalactiae was i n h i b i t o r y to C. boris p e r s i s t e n c e in stat i o n a r y g r o w t h phase.

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-12

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12

24

36

48

60

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Figure 3. Corynebacterium boris growth in milk cultures with 37 X IO s (CBH), 26 X 104 (CBI), and 19 × 103 (CBL) cfu/ml initial inoculum concentrations; CBH (- - -m- - -), CBI ( - - zx - -), CBL

(--~--).

Journal of Dairy Science Vol. 70, No. 6, 1987

0

12

24

36

48

60

72

Hours

Figure 4. Corynebacterium boris growth in milk cultures with 20 × 10 s (CBH), 30 × 104 (CBI), and 22 X 10 a (CBL) initial concentrations that were inoculated with 25 X 10 s cfu/ml of Staphylococcus aureus at 0 h; CBH ( - - - m - - - ) , CBI ( - - z~ -- .), CBL ( - - 0 - - ) .

COR YNEBA CTERIUM BO VIS METABOLITES 9-1

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--m . . . . . . . . . . . .

ix, ,'/=,

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24

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-12

0

12

48

60

72

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Figure 5. Corynebacterium boris growth in milk cultures with 30 × 10 s (CBH), 32 × 104 (CBI), and 24 × 103 (CBL) cfu/ml initial concentrations that were inoculated with 25 × IO s cfu/ml of Streptococcus agalactlae at 0 h; CBH (- - -l- - -), CBI (-- - zx-- -), CBL ( - - O - - ) .

Corynebacterium boris were i n c u b a t e d 12 h in m i l k p r i o r t o i n o c u l a t i o n w i t h Stapb. aureus or Strep. agalactiae t o s i m u l a t e in vitro t h e in vivo s u p e r i n f e c t i o n c o n d i t i o n s r e p o r t e d b y P a n k e y et al. (20). Corynebacterium boris were i n o c u l a t e d at t h r e e d i f f e r e n t initial c o n c e n -

1299

t r a t i o n s t o d e t e r m i n e if Staph. aureus a n d Step. agalactiae g r o w t h r e s p o n s e s were d e p e n d e n t u p o n c o n c e n t r a t i o n o f C boris in m i x e d cultures. H o w e v e r , C. boris c o u n t s in CBH, CBI, a n d CBL did n o t differ a f t e r t h e 12 h primary cultivation. Meanlog colony-forming u n i t s (-+ SE) o f C. boris in CBH, CBI, a n d CBL p r i o r t o i n c u b a t i o n was 6.5 -+ .1, 5.5 +- .1 a n d 4 . 4 -+ .1. M e a n l o g c o n c e n t r a t i o n o f C. boris w h e n Staph. aureus a n d Strep. agalactiae were i n o c u l a t e d i n t o CBH, CBI, a n d CBL was 8.4 + .1, 8.3 + .2, a n d 8.0 + .2. ( P > . 0 5 ) . T h e similar g r o w t h r e s p o n s e o f b o t h Staph. aureus a n d Strep. agalactiae in CBH, CBI, a n d C B L was d e d u c e d t o b e d u e t o similar c o n c e n t r a t i o n s o f C. boris in each o f t h e t h r e e m e d i a a f t e r t h e 12 h primary cultivation. N e i t h e r Staph. aureus, Strep. agalactiae, n o r C. boris c a t a b o l i z e d f a t t y acids in m i l k ( T a b l e 3). D i f f e r e n c e s were n o t significant b e t w e e n f a t t y acid c o m p o s i t i o n s in sterile m i l k a n d m i l k c o n t a i n i n g a n y o f t h e b a c t e r i a l c u l t u r e s (P> .05). T h e s e results were i n c o n s i s t e n t w i t h t h e c o n c l u s i o n o f Harrigan (9) t h a t t h e C. boris req u i r e m e n t f o r p r e f o r m e d f a t t y acids m a y e x p l a i n t h e p r e d i l e c t i o n o f t h e species f o r t h e u d d e r . Corynebacterium boris p r e f e r e n t i a l l y c o l o n i z e d t h e t e a t canal region o f t h e u d d e r (1, 20). F a t t y acid analysis o f t e a t canal k e r a t i n

TABLE 3. Mean fatty acid compositions of sterile milk (MLK) and of milk containing pure and mixed cultures of Corynebacterium boris (CB), Staphylococcus aureus (SA), and Streptococcus agalactiae (AG). 1 Cultures Fatty acid

MLK

CB

SA

AG

CBSA 2

CBAG 3

(mg/g lipid) C4 C6 Cs C10 C12 C14 C16 C~8 Cls:~ Cls:~

32.8 20.2 11.0 23.8 26.0 91.8 225.0 101.7 214.7 56.3

33,2 20.4 15.4 21.3 25.5 88.4 248.3 100.5 202.6 34.7

38.6 24.0 11.5 24.2 27.6

96.0 279.0 103.4 215.3 22.9

41.7 28.8 13.6 30.5 34.5 106.7 300.3 125.4

269.9 53.8

36.6 24,5 11.6 26.3 31.6 102.4

274.6 124.4 265.6 48.2

37.3 25.4 11.0 27.4 31.2 108.6 289.5 121.6 191.3 43.4

lStapylococcus aureus and Strep, agalactiae were cultured 72 h at 37°C. Pure and mixed C. boris cultures were incubated 84 h at 37°C.

Corynebacterium boris and Staph. aureus mixed cultures, 3Corynebacteriurn boris and Strep. agalactiae mixed cultures. Journal of Dairy Science Vol. 70, No. 6, 1987

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HOGAN ET AL.

i n d i c a t e d k e r a t i n lipids were at least p a r t i a l l y d e r i v e d f r o m m i l k f a t (10). E v i d e n c e was n o t o b s e r v e d to i n d i c a t e C, boris c o u l d c a t a b o l i z e t h e s e f a t t y acids. CONCLUSIONS

Staphylococcus aureus a n d Strep. agalactiae g r o w t h r e s p o n s e s did n o t seem r e l a t e d to c o n c e n t r a t i o n o f C. boris m e t a b o l i t e s or to f a t t y acid c o m p o s i t i o n o f m e d i a in w h i c h C. boris were c u l t u r e d . G r o w t h r e s p o n s e s were a f u n c t i o n o f availability o f r a t e - l i m i t i n g substrates. Staphylococcus aureus g r o w t h was s u p p r e s s e d in C. boris m i x e d m i l k c u l t u r e s b u t n o t in c u l t u r e s c o n t a i n i n g C. boris met a b o l i t e s o f s y n t h e t i c m e d i a . Staphylococcus aureus a n d C. boris w e r e c o m p e t i t i v e for r a t e - l i m i t i n g s u b s t r a t e s in milk. T h e s h a p e o f Stapb. aureus g r o w t h curves in C. boris m i x e d c u l t u r e s i n d i c a t e d t h a t Stapb. aureus a d a p t e d b y utilizing n o n r a t e - l i m i t i n g s u b s t r a t e s to s u s t a i n g r o w t h . Staphylococcus aureus a n d C. boris were n o t c o m p e t i n g for available subs t r a t e s in t h e c o n d i t i o n s o f Trial 1. Streptococcus agalactiae a n d C. boris did n o t a p p e a r to c o m p e t e for r a t e - l i m i t i n g s u b s t r a t e s as did Stapb. aureus a n d C. boris. G r o w t h r e s p o n s e s o f Strep. agalactiae were n o t significantly altered by either the subsequent cultivation of C. boris in m i l k or C. boris m e t a b o l i t e s of synthetic media. P a n k e y e t al. (20) r e p o r t e d C. boris inf e c t i o n s a l t e r e d t h e h o s t d e f e n s e s to r e d u c e Stapb. aureus a n d e n h a n c e Strep. agalactiae s u p e r i n f e c t i o n s . T h e ability o f C. boris to p r o l i f e r a t e in t h e t e a t canal was a s c r i b e d t o t h e p r e s e n c e o f essential lipids in k e r a t i n (1). E v i d e n c e was n o t p r e s e n t t o i n d i c a t e C. boris c o u l d c a t a b o l i z e k e r a t i n f a t t y acids t o e n h a n c e or a b a t e g r o w t h o f m e t a b o l i c a l l y dissimilar b a c t e r i a . T h e p r i m a r y i n t e r a c t i o n b e t w e e n C. boris a n d o t h e r b a c t e r i a in t h e t e a t canal m a y b e c o m p e t i t i o n for available s u b s t r a t e s , as was o b s e r v e d in vitro. REFERENCES

1 Black, R. T., R. T. Marshall, and C. T. Bourland. 1972. Locus of mammary gland infections of Corynebacterium boris. J. Dairy Sci. 55:413. 2 Brooks, B. W., and D. A. Barnum. 1984. The susceptibility of bovine udder quarters colonized with Corynebacterium boris to experimental infection with Staphylococcus aureus or Strepto-

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coccusCan. agalactiae. J. Comp. Med. 48:146. 3 Cullen, G. A., and C. N. Herbert. 1967. Some ecological observations on micro-orgmaisms inhabiting bovine skin, teat canals and milk. Br. Veto J. 123:14. 4 Davidson, C., C. Rogers, A. E. Taylor, D.F.J. Brown, and G.R.E. Naylor. 1980. Qualitative and quantitative methods of studying the effect of lipids on bacteria grown on the surface of solid culture media. J. Med. Microbiol. 13:469. 5 Duthie, A. H., S. Wulff, and R. L. Clark. 1983. Formic acid trap for GC analysis of fatty acids and their salts. J. Chromatogr. Sci. 21:185. 6 Edwards, S. J., and G. W. Jones. 1966. The distribution and characteristics of coagulase-negative staphylococci of the bovine udder. J. Dairy Res. 33:261. 7 Freinkel, R. K. 1968. The origin of free fatty acids in sebum. 1. Role of coagulase-negative staphylococci. J. Invest. Dermatol. 50:186. 8 Gottschalk, G. 1979. Page 143-155 in Bacterial metabolism. 2nd ed. Springer-Verlag Inc., New York, NY. 9 Harrigan, W. F. 1966. The nutritional requirements and biochemical reactions of Corynebacterium boris. J. Appl. Bacteriol. 29:380. 10 Hogan, J. S., A. H. Duthie, and J. W. Pankey. 1986. Fatty acid composition of bovine teat canal keratin. J. Dairy Sci. 69:2424. 11 Hogan, J. S., J. W. Pankey, and A. H. Duthie. 1987. Growth inhibition of mastitis pathogens by long-chain fatty acids. J. Dairy Sci. 70:927. 12 Jayne-Willimas, D. J., and T. M. Skerman. 1966. Comparative studies on coryneform bacteria from milk and dairy sources. J. Appl. Bacteriol. 29:72. 13 Marples, R. R., A. L. Klingman. L. R. Lantis, and D. T. Downing. 1970. The role of the aerobic microflora in the genesis of fatty acids in human surface lipids. J. Invest. Dermatol. 55 : 173. 14 Marples, R. R., D. T. Downing, and A. L. Klingman. 1971. Control of free fatty acids in human surface lipids by Corynebacteriurn acnes. J. Invest. Dermatol. 56:127. 15 Marth, E. H. 1978. Standard methods for examination of dairy products. 14th ed. Am. Public Health Assoc., Inc., Washington, DC. 16 Milyani, R. M., and S. Selwyn. 1978. Quantitative studies on competitive activities of skin bacteria growing on solid media. J. Med. Microbiol. 11:379. 17 Newbould, F.N.S. 1974. Microbial diseases of the mammary gland. Page 269 in Lactation. Vol. 2. Biosynthesis and secretion of milk/diseases. Academic Press, Inc., New York, NY. 18 Noble, W. C., and J. A. Willie. 1980. Carriage of inhibitor-producing organisms on human skin. J. Med. Microbiol. 13: 329. 19 Nonnecke, B. J., and K. L. Smith. 1984. Inhibition of mastitis bacteria by bovine apo-lactoferrin evaluated by in vitro microassay of bacterial growth. J. Dairy Sci. 67:606. 20 Pankey, J. W., S. C. Nickerson, R. L. Boddie, and J. S. Hogan. 1985. Effects of Corynebacterium boris infection on susceptibility to major pathogens. J. Dairy Sci. 68:2684.

CO R Y N E B A CTERIUM BO VIS METABOLITES

21 Reisner, R. M., and M. Puhvel. 1969. Lipolytic activity of Stapbylococcus albus. J. Invest. Dermatol. 53 : 1. 22 Reisner, R. M., D. Z. Silver, M. Puhvel, and T. G. Sternburg. 1968. Lipolytic activity o f Corynebacterium aches. J. Invest. Dermatol. 51 : 190. 23 Ricketts, C. R., J. R. Squire, E. Topley, and H. A. Lilly. 1951. H u m a n skin lipids with particular reference to t h e self-sterilizing power of the skin. Clin. Sci. 10:803. 24 Scheimann, L. G., G. Knox, D. Sher, and S. Rothman. 1960. The role of bacteria in t h e f o r m a t i o n o f free f a t t y acids on h u m a n skin surface. J. Invest. Dermatol. 34:171.

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25 Sokal, R. R., and F. J. Rohlf. 1981. Biometry. 2nd ed. W. H. F r e e m a n and Co., San Francisco, CA. 26 Stewart, M. E., D. T. Downing, and J. S. Strauss. 1980. The anatomical origin o f skin surface linoleate as indicated b y its distribution a m o n g sebaceous and epidermal lipid classes. Clin. Res. 28: 795A. 27 Wright, P., and C. S. Terry. 1981. A n t a g o n i s m within populations of micro-organisms f r o m normal h u m a n skin. J. Med. Mierobiol. 14:271. 28 Wulff, C. A., A. H. Duthie, and S. Wulff. 1980. Functional analysis of gas chromatographic data for C-4 t h r o u g h C-18:2 f a t t y acids. J. A m . Oil Chem. Soc. 57:34.

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