Veterinary Microbiology, 31 (1992) 181-189 Elsevier Science Publishers B.V., Amsterdam
181
Species identification and some characteristics of coagulase-negative staphylococci isolated from bovine udders A. Birgersson a, P. J o n s s o n b a n d O. H o l m b e r g a aSwedish University of Agricultural Sciences, College of Veterinary Medicine, Department of Veterinary Microbiology, Section of Clinical Microbiology, Box 7073, S-750 07 Uppsala, Sweden bSwedish University of Agricultural Sciences, College of Veterinary Medicine, Department of Veterinary Microbiology, Section of Bacteriology and Epizootology, Biomedicum, Box 583, S- 751 23 Uppsala, Sweden (Accepted 16 October 1991 )
ABSTRACT Birgersson, A., Jonsson, P. and Holmberg, O., 1992. Species identification and some characteristics of coagulase-negative staphylococci isolated from bovine udders. Vet. Microbiol., 31:181-189. The species of 203 strains of coagulase-negative staphylococci (CNS), isolated from bovine udder quarters was determined; all were tested for hydrophobicity and encapsulation, attributes that may relate to virulence. Twelve species were identified, of which Staphylococcus simulans, (34.5%), S. chromogenes (16.7%), S. epidermidis (13.8%) and S. xylosus (8.9%) were the most frequent. The majority of strains possessed a hydrophilic cell surface. However, strains from two species (S. chromogenes and S. epidermidis) were more hydrophobic than the others. Only five strains were encapsulated (S. xylosus, 3; S. saprophyticus, 1; and S. sciuri, 1 ). Judging from the low frequencies of hydrophobic and encapsulated strains, and comparing with strains isolated from clinical cases, it is suggested that these properties are not major virulence determinants of CNS.
INTRODUCTION
Coagulase-negative staphylococci (CNS) are often isolated from bovine milk samples. Although they are usually regarded as "minor pathogens" that may cause subclinical mastitis, or are isolated from udders lacking inflammatory reactions, i.e. teat canal colonization (du Preez, 1985 ), they may also be responsible for acute clinical mastitis (Holmberg, 1986 ). In Sweden CNS are isolated from 11% of milk samples from cases of clinical mastitis (Anonymous, 1990). During the last 15 years, there has been a remarkable development in classification of staphylococci (Sneath et al., 1986). Today 28 species of the genus Staphylococcus, of which 25 are coagulase-negative, are recognized in the 0378-1135/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved.
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International Journal of Systematic Bacteriology (Skerman et al., 1980; Anonymous, 1985; Moore et al., 1985; Freney et al., 1988; Anonymous, 1988; Varaldo et al., 1988 and Igimi et al., 1989). In several reports either commercially available systems for the identification of CNS to species level, or early schemes of Kloos and Schleifer ( 1975 ) or Devriese ( 1979 ) with only a few recognizable species have been used (e.g. Baba et al., 1980; Devriese and de Keyser, 1980; Rather et al., 1986; Watts and Owens, 1987). We know of no reports published concerning the identification of CNS-strains isolated from bovine milk, following the identification schemes by Devriese (1986) and Schleifer (1986). Similarly there are no studies focusing on the pathogenicity or virulence determinants of the various species of CNS colonizing the bovine udder. Encapsulation of staphylococci may be related to virulence partly because of its antiphagocytic capacity (Wilkinson, 1983) due to encapsulated cells usually having a hydrophilic surface. In contrast, a hydrophobic cell surface is considered to enhance virulence by promoting bacterial adherence to epithelial cells (Hogt et al., 1983), which is thought to be the first step in the pathogenesis of many diseases involving epithelial surfaces (for review see Rosenberg and Kjelleberg, 1986 ). S. aureus strains isolated from bovine mastitic quarters are reported to possess a hydrophobic cell surface (Jonsson and Wadstr6m, 1984, Mamo et al., 1987), and in consequence only a few encapsulated S. aureus strains have been found. The present study was carried out to determine the isolation rates of different CNS species isolated from bovine milk. Two potential virulence determinants related to the bacterial cell surface, i.e. hydrophobicity and capsule production, were also studied. MATERIALS AND METHODS
Definitions
Clinical mastitis was characterized by ( 1 ) an elevated total cell count (corresponding to > 300 000 cells/ml milk) as measured by the California Mastitis Test, CMT, (Schalm and Noorlander, 1957), (2) bacterial growth and (3) the presence of some or all of the following udder symptoms: swelling, heat, redness, pain, disturbed function in the infected udder quarter, or visible changes in the milk. Although there were no clinical symptoms of inflammation subclinical mastitis was defined by isolation of bacteria together with an elevated total cell count (corresponding to > 300 000 cells/ml milk) from the relevant quarter. Quarters without any signs of inflammation and with a normal total cell count (corresponding to < 300 000 cells/ml milk), were defined as non-
IDENTIFICATION AND CHARACTERISTICS OF COAGULASE-NEGATIVESTAPHYLOCOCCI
183
mastitic even if bacterial growth was obtained (latent infection or teat canal colonization ).
Bacterial strains A total of 203 CNS strains was isolated from milk samples of clinical cases of mastitis or from quarter milk samples of entire herds submitted, for bacteriological examinations, to the National Veterinary Institute, Sweden, during September 1986 to April 1987. All primary cultures of abundant growth, yielding pure culture with only one morphologically homogenous type of CNS were selected. When there was more than one sample from the same herd, with CNS of the same morphological type, only one of these strains was selected. Primary culturing was done on bovine blood agar plates containing Blood Agar Base No. 2 (Oxoid Ltd., Basingstoke, U K ) , 5% (v/v) bovine blood and 0.05% aesculin. Eighty-six strains from 69 farms were isolated from quarters of cows with clinical mastitis, while 117 strains from 77 different farms were isolated from quarters with subclinical mastitis or from non-mastitic quarters. Identification of strains All strains were originally identified to genus level according to the Scandinavian recommendations on examination of quarter milk samples (Klastrup, 1975). The species determinations and the tests used were based on schemes developed by Devriese (1986) and Schleifer (1986), and were conducted according to Devriese et al. ( 1985 ), with the following minor modifications: Sensitivity to furazolidone was determined on Bacto Mueller Hinton Medium (Difco Laboratories Inc., Detroit, Michigan, USA), ( 1 strain per plate ) with a 100/tg furazolidone disc (BBL Microbiology systems, Becton Dickinson and Co, Cockeysville, USA) according to Baker (1986) for the differentiation of staphylococci and micrococci. Novobiocin resistance was determined on P-agar plates [10 g peptone (Difco), 5 g yeast extract (Difco), 5 g sodium chloride (Merck, Merck-Schuchardt, Darmstadt, Germany), 1 g glucose (Mallinckrodt GMD, Grossostheim, Germany), 5 g agar (Lab M), distilled water 1000 ml] with 5/zg novobiocin discs (Biodisk AB, Solna, Sweden) as described by Kloos and Schleifer (1975). Production of protease was measured exclusively on Calcium Caseinate Agar (Merck). Salt aggregation test The Salt aggregation test (SAT) was performed as described by Lindahl et al. (1981 ), and hydrophobicity was evaluated according to Ljungh et al. (1985). Growth on media, described below (see presence of capsule) was
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used to assess hydrophobic properties. S. aureus strain Cowan 1 (hydrophobic, SAT group I ) and S. aureus strain Wood 46 (hydrophilic, SAT group IV) served as control strains.
Presence of capsule The presence of a true capsule was tested using the Wet India Ink technique described by Duguid ( 1951 ), with slight modifications. All strains were grown on bovine blood agar plates containing Blood Agar Base (Lab M ) and 5% (v~ v) bovine blood. In addition, 48 strains, representing 10 different species, were grown on plates containing only Blood Agar Base (Lab M ). These strains were also grown on Bacto Staphylococcus M e d i u m No. 110 (Difco) supplemented with 1% (v/v) bovine serum. A small portion of a single colony was suspended in a drop of physiological saline on a glass slide. Approximately 5 al ink was placed a few millimeters to the side of the suspension, and a cover-glass was placed on top. The bacterial suspension and ink were allowed to mix for at least 30 s. The S. aureus strain Smith diffuse (originally obtained from Professor B. Wilkinson, Illinois, State University, Normal, IL, USA) served as a positive control, and the unencapsulated variant, Smith compact (Wilkinson), served as the negative control. Statistical analysis Chi-squared analysis was used on the SAT-results. Analysis was performed on a 3 × 5 contingency table, with the categories shown in Table 3. S. simulans, S. chromogenes, S. epidermidis and S. xylosus respectively, were then tested against all the others in three 3 X 2 contingency tables, in order to compare SAT-values of these species, one at the time, to those of all the other species. RESULTS
Twelve species of CNS were identified among the 203 tested strains. Two strains could not be identified. The frequency of different species are shown in Table 1. There was no significant difference in frequency, for most species, between any of the three categories: clinical mastitis, subclinical mastitis and non-mastitic quarters. Nevertheless S. simulans was isolated more frequently from mastitic quarters (91.4% ) than from non-mastitic ones (8.6%) (Table 2). S. chromogenes and S. epidermidis were also isolated more often from inflamed udder quarters than from noninflamed ones. S. hyicus and S. saprophyticus, were only isolated from quarters with clinical or subclinical mastitis, while S. gallinarum was isolated only from non-mastitic quarters. The hydrophobicity of the various CNS strains is presented in Table 3. There was a significant relationship between S A T - v a l u e s and species ( P < 0.001 ); S. chromogenes and S. epidermidis were significantly more hydrophobic than
IDENTIFICATIONAND CHARACTERISTICSOF COAGULASE-NEGATIVESTAPHYLOCOCCI
185
TABLE 1 Frequency of coagulase-negative staphylococcal species from clinical and subclinical cases of bovine mastitis and from non-mastitic quarters Species
S. simulans S. chromogenes S. epidermidis S. xylosus S. warneri S. haemolyticus S. hyicus S. sciuri S. cohnii S. gallinarum S. saprophyticus S. hominis Unidentified Total
Clinical cases
Subclinical cases
Non-mastitic quarters
No. (%)
No. (%)
No. (%)
31 17 11 9 5 2 3 3 1 0 1 1 2 86
33 8 8 7 6 4 5 2 0 0 2 1 0 76
6 9 9 2 3 3 0 2 4 3 0 0 0 4
(36.0) (19.8) (12.8) (10.5) (5.8) (2.3) (3.5) (3.5) (1.2) (0.0) (1.2) (1.2) (2.3) (100)
(43.4) (10.5) (10.5) (9.2) (7.9) (5.3) (6.6) (2.6) (0.0) (0.0) (2.6) (1.3) (0.0) (100)
Total number isolated (%)
(14.6) (22.0) (22.0) (4.9) (7.3) (7.3) (0.0) (4.9) (9.8) (7.3) (0.0) (0.0) (0.0) (100)
70 34 28 18 14 9 8 7 5 3 3 2 2 203
(34.5) (16.7) (13.8) (8.9) (6.9) (4.4) (3.9) (3.4) (2.5) (1.5) ( 1.5 ) (1.0) (1.0) (100)
TABLE 2 Distribution according to udder status for the four most commonly isolated coagulase-negative staphylococci Species
Strains from clinical cases
Strains from subclinical cases
Strains from nonmastitic quarters
44.3 50.5 39.3 50.0 42.4
47.1 23.5 28.6 38.9 37.4
8.6 26.5 32.1 11.1 20.2
(%)
S. simulans S. chromogenes S. epidermidis S. xylosus All cases
(%)
(%)
TABLE 3 Hydrophobicity as revealed by the salt aggregation test (SAT), of 203 coagulase-negative staphylococcal strains Species
I a + II b
IlI c
IV d
No. strains
(%)
No. strains
(%)
S. simulans S. chromogenes S. epidermidis S. xylosus Other species
4 12 2 0 9
(5.7) (35.3) (7.1) (0.0) (17.0)
18 4 14 3 13
(25.7) (11.8) (50.0) (16.7) (24.5)
No. strains 48 18 12 15 31
(%) (68.6) (52.9) (42.9) (83.3) (58.5)
Total
27
(13.3)
52
(25.6)
124
(61.1)
aSAT-values < 0.1 M (hydrophobic), bSAT-values 0.1-0.9 M (hydrophobic), CSAT-values 0.9-1.5 M, dSAT-values > 1.5 M (hydrophilic).
186
a. BIRGERSSONETAL.
those of other species ( P < 0.001 and P < 0.01 respectively). Growth on the three different media did not significantly influence the SAT values for any of the tested strains. Five of the 203 strains were capsulogenic. Three of these strains were identified as S. xylosus and the remaining two as S. saprophyticus and S. sciuri. Growth on the three different media did not influence capsule production. DISCUSSION
The distribution of CNS-species isolated from abnormal and normal bovine milk seems to vary according to reports from many countries. In the present study S. simulans, S. chromogenes, S. epidermidis and S. xylosus were the most common species in both abnormal and normal milk. S. similans was more frequently found in milk from quarters with clinical and subclinical mastitis than in milk from non-mastitic quarters. Baba et al. (1980), only found S. epidermidis, S. haemolyticus, S. xylosus and S. capitis in abnormal milk. Devriese and de Keyser (1980), found S. epidermidis, S. chromogenes, S. simulans and a group of CNS called M (S. warneri) in relatively high numbers in milk samples. These organisms were also frequently isolated in samples from quarters with elevated CMT scores. Rather et al. ( 1986 ) isolated S. simulans, S. xylosus, S. epidermidis and S. saprophyticus most frequently from milk, whereas Watts and Owens ( 1987 ), reported S. epidermidis, S. hyicus and S. chromogenes to be the predominant species from bovine udders. In contrast with our and other investigations Jarp (1991) found no S.
epidermidis. The differences between studies concerning species reported and their isolation rates could be attributable to the use of different identification systems (i.e. rapid systems such as API Staph, Staph-Trac and others, as well as the use of the old schemes from Devriese, 1979 and Kloos and Schleifer, 1985 ), inconsistencies in definitions and differences in milk collecting procedures, as well as real differences between various geographical areas. Unpublished observations by Birgersson and co-workers have shown that a single species can dominate the CNS- flora in a herd. Consequently in this study samples were collected from as many herds as possible to reduce the influence of this source of sampling bias. The majority of CNS-strains tested in this study possessed a hydrophilic cell surface. This concurs with the findings o f M a m o et al. ( 1987 ) concerning CNS from bovine mastitis. However, within S. chromogenes and S. epidermidis there were significantly higher percentages of SAT-group I-III strains, i.e., these two species were generally more hydrophobic than the other CNSspecies. The low frequency ofhydrophobic CNS strains suggest that this prop-
IDENTIFICATION AND CHARACTERISTICS OF COAGULASE-NEGATIVESTAPHYLOCOCCI
187
erty is probably not of major importance in the pathogenesis of mastitis caused by CNS. Encapsulation is another cell surface property suggested to be of importance in determining the pathogenicity of staphylococci (Wilkinson, 1983 ). In the present study a capsule was only detected in five strains (three S. xylosus and one each S. saprophyticus and S. sciuri). This suggests that encapsulation of CNS, i.e. production of a true capsule, is probably not a major virulence determinant in the pathogenicity of CNS-strains causing mastitis. However four of the five encapsulated strains were isolated from clinical cases of bovine mastitis. Thus clearly, there is a need for further studies focusing on interspecies differences in virulence between CNS. ACKNOWLEDGEMENTS
The skilful technical assistance of C. Duvhammar, T. Johansson and K. Zimmerman and the excellent secretarial services of I. Zedendahl are gratefully acknowledged. Dr. L.A. Devriese identified some of the strains and I. Torr~ng assisted in the statistical analysis. This study was supported by grant number 536/84 D 151 from the Swedish Council for Forestry and Agricultural Research.
REFERENCES Anonymous, 1985. Validation of the publication of new names and new combinations previously effectively published outside the IJSB, List No. 17. Int. J. Sys. Bacteriol., 35: 223-225. Anonymous, 1988. Validation of the publication of new names and new combinations previously effectively published outside the IJSB, List No. 26. Int. J. Sys. Bacteriol., 38: 328-329. Anonymous, 1990. Svensk Husdjurssk6tsel, meddelande hr. 161, Djurh~ilsov~rd 88/89, Eskilstuna, Sweden, p. 20 (In Swedish). Baba, E., Fukata, T. and Matsumoto, H., 1980. Ecological studies on coagulase-negative staphylococci in and around bovine udder. Bull. University of Osaka Prefecture, Series B, 32: 6975. Baker, J.S., 1986. Differentiation of micrococci from coagulase-negative staphylococci. In: P.A. M~rdh and K.H. Schleifer (Editors), Coagulase-Negative Staphylococci. Almqvist & Wiksell International, Stockholm, Sweden, pp. 27-33. Devriese, L.A., 1979. Identification of clumping-factor-negative staphylococci isolated from cows' udders. Res. Vet. Sci., 27: 313-320. Devriese, L.A., 1986. Coagulase-negative staphylococci in animals. In: P.-A. M~rdh and K.H. Schleifer (Editors), Coagulase-negative Staphylococci. Almqvist & Wiksell International, Stockholm, Sweden, pp. 51-57. Devriese, L.A. and de Keyser, H., 1980. Prevalence of different species of coagulase-negative staphylococci on teats and in milk samples from dairy cows. J. Dairy Res., 47:155-158. Devriese, L.A., Schleifer, K.H. and Adegoke, G.O., 1985. Identification of coagulase-negative staphylococci from farm animals. J. Appl. Bacteriol., 58: 45-55. Duguid, J.P., 195 I. The demonstration of bacterial capsules and slime. J. Pathol. Bacteriol., 63: 673-685.
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Du Preez, J.H., 1985. Teat canal infections. In: Progress in the Control of Bovine Mastitis. Papers presented at the IDF Seminar 21-24 May 1985, at Kiel, Germany., Kieler Milchwirtschaft. Forschung., 37: 267-273. Freney, J., Brun, Y., Bes, M., Meugnier, H., Grimont, F., Grimont, P.A.D., Nervi, C. and Fleurette, J., 1988. Staphylococcus lugdunensis sp. nov. and Staphylococcus schleiferi sp. nov., two species from human clinical specimens. Int. J. Sys. Bacteriol., 38:168-172. Hogt, A.H., Dankert, J. and Feijen, J., 1983. Encapsulation, slime production and surface hydrophobicity of coagulase-negative staphylococci. FEMS Microbiol. Let., 18:211-215. Holmberg, O., 1986. Coagulase-negative staphylococci in bovine mastitis. In: P.-A. Mfirdh and K.H. Schleifer (Editors), Coagulase-Negative Staphylococci. Almqvist & Wiksell International, Stockholm, Sweden, pp. 203-211. Igimi, S., Kawamura, S., Takahashi, E. and Mitsuoka, T., 1989. Staphylococcusfelis, a new species from clinical specimens from cats. Int. J. Sys. Bacteriol., 39: 373-377. Jarp, J., 1991. Classification of coagulase-negative staphylococci isolated from bovine clinical and subclinical mastitis. Vet. Microbiol., 27:151-158. Jonsson, P. and Wadstr6m, T., 1984. Cell surface hydrophobicity of Staphylococcus aureus measured by the salt aggregation test (SAT). Curr. Microbiol., 10:203-210. Klastrup, O., 1975. Diagnosis of mastitis at the herd and cow level. In: F.H. Dodd, T.K. Griffin and R.G. Kingwill (Editors), Proc. IDF Seminar on Mastitis Control, Reading University, April 7-11. Bulletin, Document 85, pp. 41-52. Kloos, W.-S. and Schleifer, K.H., 1975. Simplified scheme for routine identification of human Staphylococcus species. J. Clin. Microbiol., 1: 82-88. Lindahl, M., Faris, A., Wadstr6m, T. and Hjert6n, S., 1981. A new test based on "salting out" to measure relative surface hydrophobicity of bacterial cells. Biochim. Biophys. Acta, 677: 471-476. Ljung, A.., Brown, A. and Wadstr6m, T., 1985. Surface hydrophobicity of coagulase-positive and coagulase-negative staphylococci determined by the salt aggregation test (SAT). In: J. Jeljaszewicz (Editor), The Staphylococci. Proc. Vth Int. Syrup. Staphylococci and Staphylococcal Infections, Warsaw, June 26-30, 1984. Zentralbl. Bakteriol. Microbiol. Hyg. I. Abteilung, Suppl. 14, Gustav Fischer Verlag, Stuttgart, New York, pp. 157-161. Mamo, W., Rozgonyi, F., Brown, A., Hjert6n, S. and Wadstr6m, T., 1987. Cell surface hydrophobicity and charge of Staphylococcus aureus and coagulase-negative staphylococci from bovine mastitis. J. Appl. Bacteriol., 62: 241-249. Moore, W.E.C., Cato, E.P. and Moore, L.V.H., 1985. Index of the bacterial and yeast nomenclatural changes published in the International Journal of Systematic Bacteriology since the 1980 approved lists of bacterial names ( 1 January 1980 to 1 January 1985). Int. J. Sys. Bacteriol., 35: 382-407. Rather, P.N., Davis, A.P. and Wilkinson, B.J., 1986. Slime production by bovine milk Staphylococcus aureus and identification of coagulase-negative staphylococcal isolates. J. Clin. Microbiol., 23: 858-862. Rosenberg, M. and Kjelleberg, S., 1986. Hydrophobic interactions: role in bacterial adhesion. In: K.C. Marshall (Editor), Advances in Microbial Ecology, vol. 9. Plenum Press, New York, pp. 353-393. Schalm, O.W and Noorlander, D.O., 1957. Experiments and observations leading to development of the California mastitis test. J. Am. Vet. Med. Assoc., 130: 199-204. Schleifer, K.H., 1986. Taxonomy of coagulase-negative staphylococci. In: P.-A. Mfirdh and K.H. Schleifer (Editors), Coagulase-Negative Staphylococci, Almqvist & Wiksell International, Stockholm, Sweden, pp. 11-26. Skerman, V.B.D., McGowan, V. and Sneath, P.H.A., 1980. Approved lists of bacterial names. Int. J. Sys. Bacteriol., 30: 225-420.
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Sneath, P.H.A., Mair, N.S., Sharpe, M.E. and Holt, J.G., 1986. Bergey's Manual of Systematic Bacteriology, vol. 2. The Williams and Wilkins Company, Baltimore, USA, pp. 1013-1035. Varaldo, P.E., Kilpper-B~ilz, R., Biavasco, F., Satta, G. and Schleifer, K.H., 1988. Staphylococcus delphinii sp. nov., a coagulase-positive species isolated from dolphins. Int. J. Sys. Bact., 38: 436-439. Watts, J.L. and Owens, W.E., 1987. Synergistic hemolysis associated with coagulase-negative staphylococci isolated from bovine mammary glands. J. Clin. Microbiol., 25: 2037-2039. Wilkinson, B.J., 1983. Staphylococcal capsules and slime. In: C.S.F. Easmon and C. Adlam (Editors), Staphylococci and Staphylococcal Infections, vol. 2. Academic Press Inc., London, Ltd., pp. 481-523.
181
Species identification and some characteristics of coagulase-negative staphylococci isolated from bovine udders A. Birgersson a, P. J o n s s o n b a n d O. H o l m b e r g a aSwedish University of Agricultural Sciences, College of Veterinary Medicine, Department of Veterinary Microbiology, Section of Clinical Microbiology, Box 7073, S-750 07 Uppsala, Sweden bSwedish University of Agricultural Sciences, College of Veterinary Medicine, Department of Veterinary Microbiology, Section of Bacteriology and Epizootology, Biomedicum, Box 583, S- 751 23 Uppsala, Sweden (Accepted 16 October 1991 )
ABSTRACT Birgersson, A., Jonsson, P. and Holmberg, O., 1992. Species identification and some characteristics of coagulase-negative staphylococci isolated from bovine udders. Vet. Microbiol., 31:181-189. The species of 203 strains of coagulase-negative staphylococci (CNS), isolated from bovine udder quarters was determined; all were tested for hydrophobicity and encapsulation, attributes that may relate to virulence. Twelve species were identified, of which Staphylococcus simulans, (34.5%), S. chromogenes (16.7%), S. epidermidis (13.8%) and S. xylosus (8.9%) were the most frequent. The majority of strains possessed a hydrophilic cell surface. However, strains from two species (S. chromogenes and S. epidermidis) were more hydrophobic than the others. Only five strains were encapsulated (S. xylosus, 3; S. saprophyticus, 1; and S. sciuri, 1 ). Judging from the low frequencies of hydrophobic and encapsulated strains, and comparing with strains isolated from clinical cases, it is suggested that these properties are not major virulence determinants of CNS.
INTRODUCTION
Coagulase-negative staphylococci (CNS) are often isolated from bovine milk samples. Although they are usually regarded as "minor pathogens" that may cause subclinical mastitis, or are isolated from udders lacking inflammatory reactions, i.e. teat canal colonization (du Preez, 1985 ), they may also be responsible for acute clinical mastitis (Holmberg, 1986 ). In Sweden CNS are isolated from 11% of milk samples from cases of clinical mastitis (Anonymous, 1990). During the last 15 years, there has been a remarkable development in classification of staphylococci (Sneath et al., 1986). Today 28 species of the genus Staphylococcus, of which 25 are coagulase-negative, are recognized in the 0378-1135/92/$05.00
© 1992 Elsevier Science Publishers B.V. All rights reserved.
182
A. B I R G E R S S O N ET AL.
International Journal of Systematic Bacteriology (Skerman et al., 1980; Anonymous, 1985; Moore et al., 1985; Freney et al., 1988; Anonymous, 1988; Varaldo et al., 1988 and Igimi et al., 1989). In several reports either commercially available systems for the identification of CNS to species level, or early schemes of Kloos and Schleifer ( 1975 ) or Devriese ( 1979 ) with only a few recognizable species have been used (e.g. Baba et al., 1980; Devriese and de Keyser, 1980; Rather et al., 1986; Watts and Owens, 1987). We know of no reports published concerning the identification of CNS-strains isolated from bovine milk, following the identification schemes by Devriese (1986) and Schleifer (1986). Similarly there are no studies focusing on the pathogenicity or virulence determinants of the various species of CNS colonizing the bovine udder. Encapsulation of staphylococci may be related to virulence partly because of its antiphagocytic capacity (Wilkinson, 1983) due to encapsulated cells usually having a hydrophilic surface. In contrast, a hydrophobic cell surface is considered to enhance virulence by promoting bacterial adherence to epithelial cells (Hogt et al., 1983), which is thought to be the first step in the pathogenesis of many diseases involving epithelial surfaces (for review see Rosenberg and Kjelleberg, 1986 ). S. aureus strains isolated from bovine mastitic quarters are reported to possess a hydrophobic cell surface (Jonsson and Wadstr6m, 1984, Mamo et al., 1987), and in consequence only a few encapsulated S. aureus strains have been found. The present study was carried out to determine the isolation rates of different CNS species isolated from bovine milk. Two potential virulence determinants related to the bacterial cell surface, i.e. hydrophobicity and capsule production, were also studied. MATERIALS AND METHODS
Definitions
Clinical mastitis was characterized by ( 1 ) an elevated total cell count (corresponding to > 300 000 cells/ml milk) as measured by the California Mastitis Test, CMT, (Schalm and Noorlander, 1957), (2) bacterial growth and (3) the presence of some or all of the following udder symptoms: swelling, heat, redness, pain, disturbed function in the infected udder quarter, or visible changes in the milk. Although there were no clinical symptoms of inflammation subclinical mastitis was defined by isolation of bacteria together with an elevated total cell count (corresponding to > 300 000 cells/ml milk) from the relevant quarter. Quarters without any signs of inflammation and with a normal total cell count (corresponding to < 300 000 cells/ml milk), were defined as non-
IDENTIFICATION AND CHARACTERISTICS OF COAGULASE-NEGATIVESTAPHYLOCOCCI
183
mastitic even if bacterial growth was obtained (latent infection or teat canal colonization ).
Bacterial strains A total of 203 CNS strains was isolated from milk samples of clinical cases of mastitis or from quarter milk samples of entire herds submitted, for bacteriological examinations, to the National Veterinary Institute, Sweden, during September 1986 to April 1987. All primary cultures of abundant growth, yielding pure culture with only one morphologically homogenous type of CNS were selected. When there was more than one sample from the same herd, with CNS of the same morphological type, only one of these strains was selected. Primary culturing was done on bovine blood agar plates containing Blood Agar Base No. 2 (Oxoid Ltd., Basingstoke, U K ) , 5% (v/v) bovine blood and 0.05% aesculin. Eighty-six strains from 69 farms were isolated from quarters of cows with clinical mastitis, while 117 strains from 77 different farms were isolated from quarters with subclinical mastitis or from non-mastitic quarters. Identification of strains All strains were originally identified to genus level according to the Scandinavian recommendations on examination of quarter milk samples (Klastrup, 1975). The species determinations and the tests used were based on schemes developed by Devriese (1986) and Schleifer (1986), and were conducted according to Devriese et al. ( 1985 ), with the following minor modifications: Sensitivity to furazolidone was determined on Bacto Mueller Hinton Medium (Difco Laboratories Inc., Detroit, Michigan, USA), ( 1 strain per plate ) with a 100/tg furazolidone disc (BBL Microbiology systems, Becton Dickinson and Co, Cockeysville, USA) according to Baker (1986) for the differentiation of staphylococci and micrococci. Novobiocin resistance was determined on P-agar plates [10 g peptone (Difco), 5 g yeast extract (Difco), 5 g sodium chloride (Merck, Merck-Schuchardt, Darmstadt, Germany), 1 g glucose (Mallinckrodt GMD, Grossostheim, Germany), 5 g agar (Lab M), distilled water 1000 ml] with 5/zg novobiocin discs (Biodisk AB, Solna, Sweden) as described by Kloos and Schleifer (1975). Production of protease was measured exclusively on Calcium Caseinate Agar (Merck). Salt aggregation test The Salt aggregation test (SAT) was performed as described by Lindahl et al. (1981 ), and hydrophobicity was evaluated according to Ljungh et al. (1985). Growth on media, described below (see presence of capsule) was
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A. B I R G E R S S O N ET AL.
used to assess hydrophobic properties. S. aureus strain Cowan 1 (hydrophobic, SAT group I ) and S. aureus strain Wood 46 (hydrophilic, SAT group IV) served as control strains.
Presence of capsule The presence of a true capsule was tested using the Wet India Ink technique described by Duguid ( 1951 ), with slight modifications. All strains were grown on bovine blood agar plates containing Blood Agar Base (Lab M ) and 5% (v~ v) bovine blood. In addition, 48 strains, representing 10 different species, were grown on plates containing only Blood Agar Base (Lab M ). These strains were also grown on Bacto Staphylococcus M e d i u m No. 110 (Difco) supplemented with 1% (v/v) bovine serum. A small portion of a single colony was suspended in a drop of physiological saline on a glass slide. Approximately 5 al ink was placed a few millimeters to the side of the suspension, and a cover-glass was placed on top. The bacterial suspension and ink were allowed to mix for at least 30 s. The S. aureus strain Smith diffuse (originally obtained from Professor B. Wilkinson, Illinois, State University, Normal, IL, USA) served as a positive control, and the unencapsulated variant, Smith compact (Wilkinson), served as the negative control. Statistical analysis Chi-squared analysis was used on the SAT-results. Analysis was performed on a 3 × 5 contingency table, with the categories shown in Table 3. S. simulans, S. chromogenes, S. epidermidis and S. xylosus respectively, were then tested against all the others in three 3 X 2 contingency tables, in order to compare SAT-values of these species, one at the time, to those of all the other species. RESULTS
Twelve species of CNS were identified among the 203 tested strains. Two strains could not be identified. The frequency of different species are shown in Table 1. There was no significant difference in frequency, for most species, between any of the three categories: clinical mastitis, subclinical mastitis and non-mastitic quarters. Nevertheless S. simulans was isolated more frequently from mastitic quarters (91.4% ) than from non-mastitic ones (8.6%) (Table 2). S. chromogenes and S. epidermidis were also isolated more often from inflamed udder quarters than from noninflamed ones. S. hyicus and S. saprophyticus, were only isolated from quarters with clinical or subclinical mastitis, while S. gallinarum was isolated only from non-mastitic quarters. The hydrophobicity of the various CNS strains is presented in Table 3. There was a significant relationship between S A T - v a l u e s and species ( P < 0.001 ); S. chromogenes and S. epidermidis were significantly more hydrophobic than
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185
TABLE 1 Frequency of coagulase-negative staphylococcal species from clinical and subclinical cases of bovine mastitis and from non-mastitic quarters Species
S. simulans S. chromogenes S. epidermidis S. xylosus S. warneri S. haemolyticus S. hyicus S. sciuri S. cohnii S. gallinarum S. saprophyticus S. hominis Unidentified Total
Clinical cases
Subclinical cases
Non-mastitic quarters
No. (%)
No. (%)
No. (%)
31 17 11 9 5 2 3 3 1 0 1 1 2 86
33 8 8 7 6 4 5 2 0 0 2 1 0 76
6 9 9 2 3 3 0 2 4 3 0 0 0 4
(36.0) (19.8) (12.8) (10.5) (5.8) (2.3) (3.5) (3.5) (1.2) (0.0) (1.2) (1.2) (2.3) (100)
(43.4) (10.5) (10.5) (9.2) (7.9) (5.3) (6.6) (2.6) (0.0) (0.0) (2.6) (1.3) (0.0) (100)
Total number isolated (%)
(14.6) (22.0) (22.0) (4.9) (7.3) (7.3) (0.0) (4.9) (9.8) (7.3) (0.0) (0.0) (0.0) (100)
70 34 28 18 14 9 8 7 5 3 3 2 2 203
(34.5) (16.7) (13.8) (8.9) (6.9) (4.4) (3.9) (3.4) (2.5) (1.5) ( 1.5 ) (1.0) (1.0) (100)
TABLE 2 Distribution according to udder status for the four most commonly isolated coagulase-negative staphylococci Species
Strains from clinical cases
Strains from subclinical cases
Strains from nonmastitic quarters
44.3 50.5 39.3 50.0 42.4
47.1 23.5 28.6 38.9 37.4
8.6 26.5 32.1 11.1 20.2
(%)
S. simulans S. chromogenes S. epidermidis S. xylosus All cases
(%)
(%)
TABLE 3 Hydrophobicity as revealed by the salt aggregation test (SAT), of 203 coagulase-negative staphylococcal strains Species
I a + II b
IlI c
IV d
No. strains
(%)
No. strains
(%)
S. simulans S. chromogenes S. epidermidis S. xylosus Other species
4 12 2 0 9
(5.7) (35.3) (7.1) (0.0) (17.0)
18 4 14 3 13
(25.7) (11.8) (50.0) (16.7) (24.5)
No. strains 48 18 12 15 31
(%) (68.6) (52.9) (42.9) (83.3) (58.5)
Total
27
(13.3)
52
(25.6)
124
(61.1)
aSAT-values < 0.1 M (hydrophobic), bSAT-values 0.1-0.9 M (hydrophobic), CSAT-values 0.9-1.5 M, dSAT-values > 1.5 M (hydrophilic).
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a. BIRGERSSONETAL.
those of other species ( P < 0.001 and P < 0.01 respectively). Growth on the three different media did not significantly influence the SAT values for any of the tested strains. Five of the 203 strains were capsulogenic. Three of these strains were identified as S. xylosus and the remaining two as S. saprophyticus and S. sciuri. Growth on the three different media did not influence capsule production. DISCUSSION
The distribution of CNS-species isolated from abnormal and normal bovine milk seems to vary according to reports from many countries. In the present study S. simulans, S. chromogenes, S. epidermidis and S. xylosus were the most common species in both abnormal and normal milk. S. similans was more frequently found in milk from quarters with clinical and subclinical mastitis than in milk from non-mastitic quarters. Baba et al. (1980), only found S. epidermidis, S. haemolyticus, S. xylosus and S. capitis in abnormal milk. Devriese and de Keyser (1980), found S. epidermidis, S. chromogenes, S. simulans and a group of CNS called M (S. warneri) in relatively high numbers in milk samples. These organisms were also frequently isolated in samples from quarters with elevated CMT scores. Rather et al. ( 1986 ) isolated S. simulans, S. xylosus, S. epidermidis and S. saprophyticus most frequently from milk, whereas Watts and Owens ( 1987 ), reported S. epidermidis, S. hyicus and S. chromogenes to be the predominant species from bovine udders. In contrast with our and other investigations Jarp (1991) found no S.
epidermidis. The differences between studies concerning species reported and their isolation rates could be attributable to the use of different identification systems (i.e. rapid systems such as API Staph, Staph-Trac and others, as well as the use of the old schemes from Devriese, 1979 and Kloos and Schleifer, 1985 ), inconsistencies in definitions and differences in milk collecting procedures, as well as real differences between various geographical areas. Unpublished observations by Birgersson and co-workers have shown that a single species can dominate the CNS- flora in a herd. Consequently in this study samples were collected from as many herds as possible to reduce the influence of this source of sampling bias. The majority of CNS-strains tested in this study possessed a hydrophilic cell surface. This concurs with the findings o f M a m o et al. ( 1987 ) concerning CNS from bovine mastitis. However, within S. chromogenes and S. epidermidis there were significantly higher percentages of SAT-group I-III strains, i.e., these two species were generally more hydrophobic than the other CNSspecies. The low frequency ofhydrophobic CNS strains suggest that this prop-
IDENTIFICATION AND CHARACTERISTICS OF COAGULASE-NEGATIVESTAPHYLOCOCCI
187
erty is probably not of major importance in the pathogenesis of mastitis caused by CNS. Encapsulation is another cell surface property suggested to be of importance in determining the pathogenicity of staphylococci (Wilkinson, 1983 ). In the present study a capsule was only detected in five strains (three S. xylosus and one each S. saprophyticus and S. sciuri). This suggests that encapsulation of CNS, i.e. production of a true capsule, is probably not a major virulence determinant in the pathogenicity of CNS-strains causing mastitis. However four of the five encapsulated strains were isolated from clinical cases of bovine mastitis. Thus clearly, there is a need for further studies focusing on interspecies differences in virulence between CNS. ACKNOWLEDGEMENTS
The skilful technical assistance of C. Duvhammar, T. Johansson and K. Zimmerman and the excellent secretarial services of I. Zedendahl are gratefully acknowledged. Dr. L.A. Devriese identified some of the strains and I. Torr~ng assisted in the statistical analysis. This study was supported by grant number 536/84 D 151 from the Swedish Council for Forestry and Agricultural Research.
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