59
Epidemiol. Infect. (1992), 109, 59-68 Printed in Great Britain
Restriction endonuclease fingerprinting of genomic DNA of Staphylococcus species of bovine origin K. R. MATTHEWS, B. M. JAYARAO AND S. P. OLIVER* Department of Animal Science, Institute of Agriculture, University of Tennessee, Knoxville, Tennessee 37901-1071, USA
(Accepted 3 January 1992) SUMMARY
Fifty-one staphylococcal isolates from mammary secretions of cows with subclinical mastitis were examined by antibiograms and DNA restriction endonuclease fingerprinting (REF). DNA REF differentiated closely related strains of each species isolated from mammary secretions of different mammary glands of the same cow and from the same mammary gland at different periods of the lactation cycle. In addition, REF analysis provided evidence concerning persistence of infection in the same or different mammary gland over different periods of the lactation cycle, and occurrence of infection with similar and dissimilar strains of each Staphylococcus species. Antibiograms were of limited value in differentiating closely related strains. The ease by which REF analysis can be performed together with the reproducibility and clarity of REF patterns suggest that this technique is useful for differentiating closely related and unrelated strains of Staphylococcus species isolated from bovine mammary secretions. INTRODUCTION
Staphylococci are isolated frequently from bovine milk. Staphylococcus aureus is probably the most frequent infectious agent in chronic mastitis in the bovine [1]. Coagulase-negative staphylococci (species other than S. aureus, S. intermedius and some strains of S. hyicus) are considered minor mastitis pathogens generally associated with a mild inflammatory response of the bovine mammary gland. Milk somatic cell counts from mammary glands infected with coagulase-negative staphylococci are generally two- to three-fold higher than from uninfected mammary glands. However, clinical mastitis can occur in mammary glands infected with coagulase-negative staphylococci [2]. During a 5-year study, Smith and colleagues [3] reported that coagulase-negative staphylococci were isolated from 9% of milk samples collected from mammary glands with clinical mastitis. From an epidemiological standpoint, it is important to determine the origin of organisms involved in the aetiology of a disease. In epidemiological studies of S. aureus, phage typing, serotyping, biotyping and plasmid pattern analysis have been used [1, 4-6]. Some of these typing schemes have been used successfully to * Address correspondence to: S. l'. Oliver. Department of Animal Science, 105 MeCord Hall, L'niversity, of Teiniessee. Knoxville. TN 37901-1071, USA.
60
K. R. MATTHEWS AND OTHERS
type coagulase-negative staphylococci of human origin [7, 8] and to a limited extent isolates of bovine origin [4, 9, 10]. However, most of these methods resulted in loW typability for staphylococci of bovine origin. DNA restriction endonuclease fingerprinting (REF) has been developed for many pathogens including Bacteroides species [11], campylobacter [12], and moraxella [13]. RecentlV, Jayarao and co-workers [14] reported that DNA REF was an effective method for differentiating closely related and unrelated strains of Streptococcus uberis isolated from bovine mammary secretions. Tlhis is in agreement with previous reports [15, 16]. Restriction endonuclease fingerprinting, which measures DNA restriction fragment length polymorphism. offers perhaps the most specific current method of typing bacteria. This paper describes DNA REF analysis of staphylococci isolated from mammary secretions of cows with subclinical mastitis and assesses its potential as a repeatable and practical technique to distinguish between strains of a species. M1ATERIALS AND IIETHOI)S
Bacteria Staphylococci (n = 51) were isolated from mammary secretions of cows with subclinical mastitis at the University of Tennessee dairy research herd. Lewisburg, TN. Staphylococci were identified by Gram reaction. catalase and coagulase tests and speciated by the API Staph-Trac system (Analytab Inc., Plainview, NY, USA) and conventional biochemical methods as described [17]. Seven strains obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) were used as control organisms: S. aureus ATCC 10832. 8. aureus ATCC 13709, S. chromogenes ATCC 43764, S. hominis ATCC 27844, S. simulans ATCC 27848, S. xylosus ATCC 29971., and S. xylosus ATCC 35663. Before examination, strains were stored at -80 °C in sterile skim milk.
Antibiotic susceptibility patterns of staphylococci to antibiotics was evaluated as described by Susceptibility Bauer and co-workers [18] using antibiotic impregnated disks (BBL Microbiology Systems. Cockeysville, MD, USA). All isolates were tested for susceptibility to: ampicillin (10,ug), bacitracin (10 U), cephalothin (30,ug), erythromycin (15 fig), gentamicin (10,ag). kanamycin (30,ug), methicillin (5ufg), novobiocin (30 ,ig), penicillin (10 U), streptomycin (10 fig) and tetracycline (30 fig). Zones of inhibition around disks were recorded as susceptible or resistant. Isolated colonies within a zone of growth inhibition were considered resistant. Preparation of genomic I)NA A modification of the procedure for isolating DNA described by Burnie and Lee [19] was employed. A dense suspension of each isolate was made by transferring bacterial growth from blood agar plates into 1 ml of 10 mm-Tris-hydroxymethylaminomethane (TE), pH 8, in Eppendorf tubes (Brinkmann Instruments Inc., Westbury, NY, USA). Cells were washed once in 1 ml TE, and 500,fl of fresh TE containing 100 fIl of 1 mg/ml lysostaphin (ICN Biochemicals, Cleveland, OH,
DNA fingerprinting of staphylococci
61
USA) were added to each pellet. The mixture was vortexed gently and incubated at 37 'C in a block heater for 1 h. A solution (500 ltl) containing 0 05 m-EDTA (pH 85) and 2 mg/ml sodium dodecyl sulphate was added, vortex mixed and incubated at 68 °C for 30 min. The solution was centrifuged for 5 min and the supernatant pipetted into 1 ml cold ethanol. This was vortex mixed, left at -20 °C for 30 min, clarified by centrifugation (5 min) and the supernatant discarded. The precipitate was washed with 1 ml cold 70% ethanol and the ethanol decanted prior to vacuum drying (10 min). The precipitate was resuspended in 100 Ial TE containing 12 ,al RNase (Ribonuclease-A from Bovine Pancrease Type 1-AS, Sigma, St Louis, MO, USA) which had been treated previously by boiling for 10 min in a TE solution (10 mg/ml). The solution was incubated 30 min at 37 'C. A 3 M sodium acetate (10 ,al, pH 5 5) solution and two volumes (200 ,ul) of cold absolute ethanol were added and kept at -20 'C overnight. The solution was centrifuged, the precipitate washed with 70 % ethanol. and vacuum dried and resuspended in 90 ,al TE. DNA in TE was dissolved bv incubation at 55 'C for 30-60 min and stored at -20 'C. Prior to restriction endonuclease digestion, DNA samples were electrophoresed overnight at 50 V (constant voltage) in gels containing 0 8 % agarose to screen for presence of plasmids and shearing of chromosomal DNA. Strains containing plasmids were eliminated from the study. New DNA samples were prepared if shearing was detected. Restriction endonuclease digestion of DNA Ten microlitres of Eco RI (10 x ) reaction buffer and 2 1A1 Eco RI enzyme (Gibco BRL, Gaithersburg, MD, USA) containing 100 U were added to 90 ,al of DNA in TE. Digestion of DNA was completed after incubation at 37 'C for 3-5 h. The mixture was heated to 70 'C for 10 min to stop digestion. DNA from a strain was prepared at least twice and subsequently digested to establish reproducibility of the method. Hind III digested A-DNA (Gibco BRL) (2 ,dl; 500 ,ug/803 ,ll) was used as the DNA marker. Three microlitres of bromophenol blue loading dye (50% glycerol. 1OO mm-EDTA. 1 % SDS, 01 0% bromophenol blue and 01 % xylene eyanol) were added to 12 pl of digested DNA sample just prior to loading wells. Gel electrophoresis and photography Digested DNA samples were electrophoresed overnight at 50 V (constant voltage) in a horizontal gel containing 0 8 % agarose with Trisborate-EDTA buffer described previously. After electrophoresis, the gel was stained with ethidium bromide (1 ,ug/ml) and visualized under u.v. light and photographed with Polaroid type 55 film. Evaluation of D;\NA REF The polaroid negative was scanned along each track using a computer integrated laser densitometer (Ultroscan XL. LKB Produkter AB. Bromma. Sweden). The Gelscan XL version 2.0 software package (Pharmacia, LKB Biotechnology. Uppsala, Sweden) was used to store scans for comparison and evaluation. Two REF's were evaluated at one time to determine similarities and dissimilarities. Each REF was designated as a distinct pattern if differences in number of fragments and size (in kilobase pairs) were observed. Results obtained
62
K. R. MATTHEWS AND OTHERS S
-
I
4
s
kh
+4 kh (1': kh
4-4 kb
'..' k I--) k} I-)
Fig. 1. Genomic DNA restriction endonuclease fingerprints (REF) of the predominant REF type (indicated by parentheses) of 6 Staphylococcus species. Lanes: 1, 8. aureus (SA1); 2, 8. chromoqenes (SC1); 3. 8. hyicus (SHi1); 4 8. hominis (SHO1): 5. S. .sinulans (SS1); 6, S. xylosus (SX1); S, Hind III digested A DNA.
by densitometric scanning of REF were confirmed by visual comparison of REF photographs. REStTLTS
The predominant DNA REF for each Staphylococcus species is shown (Fig. 1). Comparison of DNA REF's were made between strains of a species and not between species. Reproducibility between gels was excellent provided DNA concentration and electrophoresis methods were standardized. Hind III digested A-DNA was included to further ensure standardization between gels. Graphical representation of staphylococcal DNA REF after laser densitometric scanning showed that the fragment length of the discriminating region was between 2-3 and 9 4 kb. Laser densitometric scans of the discriminating region show similarities (Fig. 2 top) and differences (Fig. 2 bottom) between strains of S. chromogenes. Staphylococcus aureus isolates (n = 7) cultured from mammary secretions of 6 cows belonged to 2 distinct REF patterns. Three distinct antibiogram patterns were observed and 5 of 7 isolates belonged to one pattern (Table 1). Paired isolates from cow L895 had the same REF type; however, the antibiogram differed.
DINA fingerprinting of staphylococci 7-11 MY A
xCkb
116
.
\
.
.
1.
+
Epidemiol. Infect. (1992), 109, 59-68 Printed in Great Britain
Restriction endonuclease fingerprinting of genomic DNA of Staphylococcus species of bovine origin K. R. MATTHEWS, B. M. JAYARAO AND S. P. OLIVER* Department of Animal Science, Institute of Agriculture, University of Tennessee, Knoxville, Tennessee 37901-1071, USA
(Accepted 3 January 1992) SUMMARY
Fifty-one staphylococcal isolates from mammary secretions of cows with subclinical mastitis were examined by antibiograms and DNA restriction endonuclease fingerprinting (REF). DNA REF differentiated closely related strains of each species isolated from mammary secretions of different mammary glands of the same cow and from the same mammary gland at different periods of the lactation cycle. In addition, REF analysis provided evidence concerning persistence of infection in the same or different mammary gland over different periods of the lactation cycle, and occurrence of infection with similar and dissimilar strains of each Staphylococcus species. Antibiograms were of limited value in differentiating closely related strains. The ease by which REF analysis can be performed together with the reproducibility and clarity of REF patterns suggest that this technique is useful for differentiating closely related and unrelated strains of Staphylococcus species isolated from bovine mammary secretions. INTRODUCTION
Staphylococci are isolated frequently from bovine milk. Staphylococcus aureus is probably the most frequent infectious agent in chronic mastitis in the bovine [1]. Coagulase-negative staphylococci (species other than S. aureus, S. intermedius and some strains of S. hyicus) are considered minor mastitis pathogens generally associated with a mild inflammatory response of the bovine mammary gland. Milk somatic cell counts from mammary glands infected with coagulase-negative staphylococci are generally two- to three-fold higher than from uninfected mammary glands. However, clinical mastitis can occur in mammary glands infected with coagulase-negative staphylococci [2]. During a 5-year study, Smith and colleagues [3] reported that coagulase-negative staphylococci were isolated from 9% of milk samples collected from mammary glands with clinical mastitis. From an epidemiological standpoint, it is important to determine the origin of organisms involved in the aetiology of a disease. In epidemiological studies of S. aureus, phage typing, serotyping, biotyping and plasmid pattern analysis have been used [1, 4-6]. Some of these typing schemes have been used successfully to * Address correspondence to: S. l'. Oliver. Department of Animal Science, 105 MeCord Hall, L'niversity, of Teiniessee. Knoxville. TN 37901-1071, USA.
60
K. R. MATTHEWS AND OTHERS
type coagulase-negative staphylococci of human origin [7, 8] and to a limited extent isolates of bovine origin [4, 9, 10]. However, most of these methods resulted in loW typability for staphylococci of bovine origin. DNA restriction endonuclease fingerprinting (REF) has been developed for many pathogens including Bacteroides species [11], campylobacter [12], and moraxella [13]. RecentlV, Jayarao and co-workers [14] reported that DNA REF was an effective method for differentiating closely related and unrelated strains of Streptococcus uberis isolated from bovine mammary secretions. Tlhis is in agreement with previous reports [15, 16]. Restriction endonuclease fingerprinting, which measures DNA restriction fragment length polymorphism. offers perhaps the most specific current method of typing bacteria. This paper describes DNA REF analysis of staphylococci isolated from mammary secretions of cows with subclinical mastitis and assesses its potential as a repeatable and practical technique to distinguish between strains of a species. M1ATERIALS AND IIETHOI)S
Bacteria Staphylococci (n = 51) were isolated from mammary secretions of cows with subclinical mastitis at the University of Tennessee dairy research herd. Lewisburg, TN. Staphylococci were identified by Gram reaction. catalase and coagulase tests and speciated by the API Staph-Trac system (Analytab Inc., Plainview, NY, USA) and conventional biochemical methods as described [17]. Seven strains obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) were used as control organisms: S. aureus ATCC 10832. 8. aureus ATCC 13709, S. chromogenes ATCC 43764, S. hominis ATCC 27844, S. simulans ATCC 27848, S. xylosus ATCC 29971., and S. xylosus ATCC 35663. Before examination, strains were stored at -80 °C in sterile skim milk.
Antibiotic susceptibility patterns of staphylococci to antibiotics was evaluated as described by Susceptibility Bauer and co-workers [18] using antibiotic impregnated disks (BBL Microbiology Systems. Cockeysville, MD, USA). All isolates were tested for susceptibility to: ampicillin (10,ug), bacitracin (10 U), cephalothin (30,ug), erythromycin (15 fig), gentamicin (10,ag). kanamycin (30,ug), methicillin (5ufg), novobiocin (30 ,ig), penicillin (10 U), streptomycin (10 fig) and tetracycline (30 fig). Zones of inhibition around disks were recorded as susceptible or resistant. Isolated colonies within a zone of growth inhibition were considered resistant. Preparation of genomic I)NA A modification of the procedure for isolating DNA described by Burnie and Lee [19] was employed. A dense suspension of each isolate was made by transferring bacterial growth from blood agar plates into 1 ml of 10 mm-Tris-hydroxymethylaminomethane (TE), pH 8, in Eppendorf tubes (Brinkmann Instruments Inc., Westbury, NY, USA). Cells were washed once in 1 ml TE, and 500,fl of fresh TE containing 100 fIl of 1 mg/ml lysostaphin (ICN Biochemicals, Cleveland, OH,
DNA fingerprinting of staphylococci
61
USA) were added to each pellet. The mixture was vortexed gently and incubated at 37 'C in a block heater for 1 h. A solution (500 ltl) containing 0 05 m-EDTA (pH 85) and 2 mg/ml sodium dodecyl sulphate was added, vortex mixed and incubated at 68 °C for 30 min. The solution was centrifuged for 5 min and the supernatant pipetted into 1 ml cold ethanol. This was vortex mixed, left at -20 °C for 30 min, clarified by centrifugation (5 min) and the supernatant discarded. The precipitate was washed with 1 ml cold 70% ethanol and the ethanol decanted prior to vacuum drying (10 min). The precipitate was resuspended in 100 Ial TE containing 12 ,al RNase (Ribonuclease-A from Bovine Pancrease Type 1-AS, Sigma, St Louis, MO, USA) which had been treated previously by boiling for 10 min in a TE solution (10 mg/ml). The solution was incubated 30 min at 37 'C. A 3 M sodium acetate (10 ,al, pH 5 5) solution and two volumes (200 ,ul) of cold absolute ethanol were added and kept at -20 'C overnight. The solution was centrifuged, the precipitate washed with 70 % ethanol. and vacuum dried and resuspended in 90 ,al TE. DNA in TE was dissolved bv incubation at 55 'C for 30-60 min and stored at -20 'C. Prior to restriction endonuclease digestion, DNA samples were electrophoresed overnight at 50 V (constant voltage) in gels containing 0 8 % agarose to screen for presence of plasmids and shearing of chromosomal DNA. Strains containing plasmids were eliminated from the study. New DNA samples were prepared if shearing was detected. Restriction endonuclease digestion of DNA Ten microlitres of Eco RI (10 x ) reaction buffer and 2 1A1 Eco RI enzyme (Gibco BRL, Gaithersburg, MD, USA) containing 100 U were added to 90 ,al of DNA in TE. Digestion of DNA was completed after incubation at 37 'C for 3-5 h. The mixture was heated to 70 'C for 10 min to stop digestion. DNA from a strain was prepared at least twice and subsequently digested to establish reproducibility of the method. Hind III digested A-DNA (Gibco BRL) (2 ,dl; 500 ,ug/803 ,ll) was used as the DNA marker. Three microlitres of bromophenol blue loading dye (50% glycerol. 1OO mm-EDTA. 1 % SDS, 01 0% bromophenol blue and 01 % xylene eyanol) were added to 12 pl of digested DNA sample just prior to loading wells. Gel electrophoresis and photography Digested DNA samples were electrophoresed overnight at 50 V (constant voltage) in a horizontal gel containing 0 8 % agarose with Trisborate-EDTA buffer described previously. After electrophoresis, the gel was stained with ethidium bromide (1 ,ug/ml) and visualized under u.v. light and photographed with Polaroid type 55 film. Evaluation of D;\NA REF The polaroid negative was scanned along each track using a computer integrated laser densitometer (Ultroscan XL. LKB Produkter AB. Bromma. Sweden). The Gelscan XL version 2.0 software package (Pharmacia, LKB Biotechnology. Uppsala, Sweden) was used to store scans for comparison and evaluation. Two REF's were evaluated at one time to determine similarities and dissimilarities. Each REF was designated as a distinct pattern if differences in number of fragments and size (in kilobase pairs) were observed. Results obtained
62
K. R. MATTHEWS AND OTHERS S
-
I
4
s
kh
+4 kh (1': kh
4-4 kb
'..' k I--) k} I-)
Fig. 1. Genomic DNA restriction endonuclease fingerprints (REF) of the predominant REF type (indicated by parentheses) of 6 Staphylococcus species. Lanes: 1, 8. aureus (SA1); 2, 8. chromoqenes (SC1); 3. 8. hyicus (SHi1); 4 8. hominis (SHO1): 5. S. .sinulans (SS1); 6, S. xylosus (SX1); S, Hind III digested A DNA.
by densitometric scanning of REF were confirmed by visual comparison of REF photographs. REStTLTS
The predominant DNA REF for each Staphylococcus species is shown (Fig. 1). Comparison of DNA REF's were made between strains of a species and not between species. Reproducibility between gels was excellent provided DNA concentration and electrophoresis methods were standardized. Hind III digested A-DNA was included to further ensure standardization between gels. Graphical representation of staphylococcal DNA REF after laser densitometric scanning showed that the fragment length of the discriminating region was between 2-3 and 9 4 kb. Laser densitometric scans of the discriminating region show similarities (Fig. 2 top) and differences (Fig. 2 bottom) between strains of S. chromogenes. Staphylococcus aureus isolates (n = 7) cultured from mammary secretions of 6 cows belonged to 2 distinct REF patterns. Three distinct antibiogram patterns were observed and 5 of 7 isolates belonged to one pattern (Table 1). Paired isolates from cow L895 had the same REF type; however, the antibiogram differed.
DINA fingerprinting of staphylococci 7-11 MY A
xCkb
116
.
\
.
.
1.
+
