SHORT COMMUNICATIONS Research in Veterinary Science 1991, 51, 339-340
Comparative in vitro activity of three cephalosporins against Staphylococcus aureus isolated from bovine mastitis C. A. M. LOPES, G. MORENO, Department o f Microbiology and Immunology, Institute o f Biosciences, Estadual Paulista University, UNESP, Botucatu-SP 18610, Brazil
This report compares the in vitro activity of three cephalosporins (cephalothin, cefoxitin and ceftriaxone) against 57 S t a p h y l o c o c c u s aureus strains isolated from cows with clinical mastitis on the basis of the minimal inhibitory (MIC) and minimal bactericidal concentrations (MUC). The majority of the S aureus strains showed resistance to cefoxitin and ceftriaxone and sensitivity to cephalothin. The highest MICs and MnCs were found for cefoxitin and ceftriaxone. Antimicrobial tolerance (MBC/MIC>~32:l) was observed in relation to cephalothin and ceftriaxone. The data suggest that these cephalosporins may not be effective for the treatment of staphylococcal bovine mastitis. The precise definition of their antimicrobial efficacies requires more detailed in vitro and in vivo studies. A L T H O U G H several papers haves focused attention on the in vitro susceptibilities of Staphylococcus aureus isolated from bovine mastitis in recent years (Araki et al 1985, Jones and Heath 1985, Craven et al 1986, Francis and Carroll 1986, Monsallier and Thomasson 1986, Wilson et a11986, Perry et al 1987, Mackie et al 1988), little information is available concerning the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of cephalosporins employed for the treatment of bovine mastitis. Since antibiotic therapy plays an important role in the treatment of staphylococcal mastitis (Craven et al 1986), the authors examined in vitro activity of cephalothin, cefoxitin and ceftriaxone against S aureus by the broth dilution method. Fifty-seven S aureus strains freshly isolated from cases of clinical bovine mastitis on dairy farms in Brazil during February to September 1989 were investigated by standard bacteriological methods (Koneman et al 1988). All isolates could be classed as bovine strains on the basis of phage typing using the standard method of Blair and Williams (1961) as modified by Parker (1972), with the basic sets of phages for typing human strains (Subcommittee on Phage Typing of Staphylococci 1975) and bovine strains (Working
Group on Phage Typing of Bovine Staphylococci 1971) and biotyping according to the methods of Hajek and Marsalek (1971). Antibiotics were obtained in powder form: cephalothin (Eli Lilly), cefoxitin (Merck Sharp & Dohme) and ceftriaxone (Roche A/A). A standard stock solution of each antibiotic was prepared according to the instructions of the manufacturer and used immediately. The MIC and MBC of each antibiotic was determined by a modification of the broth dilution method of the World Health Organization International Collaborative Study (Ericsson and Sherris 1971). Serial twofold dilutions from 128 to 0.06 gg m1-1 were made in 5 ml of Mueller Hinton broth (Difco Laboratories). All inocula (50 ~1) were quantitated to log 5" 7 colony forming units (CFU) per millilitre of a log-phase-growth subculture from the original plates. The rack of inoculated tubes was gently shaken to mix the contents and tubes were incubated at 35°C overnight. MIC endpoints were read as the lowest antibiotic concentration that resulted in no growth after the incubation period. Tubes showing no visible growth were then individually shaken by hand to resuspend the contents and subcultured by streaking 100/zl with a semiautomatic pipette across the diameter of a 90 mm 5 per cent sheep blood agar plate (Tryptic Soy Agar Base; Difco Laboratories). After 20 minutes, when the liquid was completely absorbed into the medium, the inoculum was spread over the entire surface of the plates with a sterile bent glass rod. Following this procedure to minimise the inhibition of the inoculum by antibiotic carry-over, CFU were counted after 18 to 24 hours of incubation at 35°C and MBC was read as the lowest antibiotic concentration killing at least 99" 9 per cent of the initial inoculum. Antimicrobial tolerance is defined as an MBC/MIC ratio /> 32:1 after 24 hours of incubation at 35°C (Sabath et al 1977). The results of the MIC and MBC determinations are summarised in Table 1. The majority of the S aureus strains were resistant to cefoxitin and ceftriaxone (MICin excess of 4
TABLE 1 : In vitro susceptibilities of 5 7 Staphylococcus aureus strains isolated from bovine mastitis Antibiotic Cephalothin Cefoxitin Ceftriaxone
50% of strains MIC MBC O. 5 32 4
90% of strains MIC MBC
2 64 16
1 64 8
MIC Minimal inhibitory concentration MBC Minimal bactericidal concentration
339
8 128 64
Range MIC
MBC
O- 06-2 2-128 0.12-16
O- 05-64 4 - > 128 0"12-128
340
C. A . M . L o p e s , G. M o r e n o TABLE 2- MBC/MIC ratios of 57 Staphylococcus aureus strains isolated from bovine mastitis Antibiotic Cephalothin Cefoxitin* Ceftriaxone
1: 1 21 (36-8) 18 (33' 3) 18 (31.6)
Number (%) of strains with the indicated MBC/MIC ratio 2:1 4:1 8: I 16:1 3(5.3) 33 (61 • 1) 18 (31 .6)
12(21) 0 3 (5-3)
6(10.5) 3 (5- 5) 6 (10.5)
6(10.5) 0 0
/> 32:1 9(15.8) 0 2 (21)
• Three strains not tested
/zg m l - 1) and sensitive to cephalothin. The highest MIC and MBC values were found for cefoxitin. Table 2 gives the MBC/MIC ratios for the tested strains. An MBC/MIC ratio of 4 or greater was observed for all drugs, but most frequently for cephalothin and ceftriaxone. The authors found a considerable deg~'ee of bacterial tolerance to cephalothin (15.8 per cent) and ceftriaxone (21 per cent). According to Sabath et al (1977), the growth of a tolerant strain is inhibited at low drug concentrations, like a non-tolerant strain, but killing occurs at much higher concentrations than for a non-tolerant strain. This concept may have practical implications and clinical significance for the choice of an antibiotic for therapeutic purposes, although it has not been currently investigated in veterinary science. However, Craven et al (1983) observed cloxacillin tolerance in vitro in isolates of S aureus from bovine mastitis, but suggested that it was of doubtful relevance to the response of therapy in vivo. Tolerance to/3-1actam antibiotics was not found by Craven et al (1986). In order to assess the efficacy of new cephalosporins for the treatment of bovine mastitis, some investigators (Mackie et al 1988, Monsallier and Thomasson 1986, Wilson et al 1986) have employed the disc diffusion method and found good antimicrobial activity of these drugs against most S aureus isolates tested. Since these cephalosporins can show remarkable differences in their antimicrobial activities and little is known about their interactions in the bovine udder, it was concluded that further bacteriological studies, as well as more clinical investigations, are needed to understand their in vivo efficacies. Until that time and for practical purposes, the use of cephalothin, cefoxitin and ceftriaxone should be viewed with caution in the treatment of staphylococcal bovine mastitis.
Acknowledgements We thank Professor Ana Maria Uthida Tanaka and Professor Maria Aparecida de Araujo of the department of parasitology, microbiology and immunology, Faculty of Medicine, Ribeirao Preto, Sao Paulo University, for phage typing our S aureus strains. We also thank Antonia Horacio and Antonio Corr~a for excellent technical assistance.
References ARAKI, S., KASHIWAZAKI, M. & KUME, T. (1985) Antimicrobial activity of amoxiciUin and other penicillins against clinical isolates from bovine udders. Japanese Journal of Veterinary Science 47, 323-324 BLAIR, J. E. & WILLIAMS, R. E. O. (1961) Phage typing of staphylococci. Bulletin of the World Health Organization 24, 771-784
CRAVEN, N., ANDERSON, J. C. & JONES, T. O. (1986) Antimicrobial drug susceptibilities of Staphylococcus aureus isolates from bovine mastitis. Veterinary Record 118, 290-291 CRAVEN, N., ANDERSON, J. C. & WILSON, C. D. (1983) Penicillin (cloxacillin-tolerant) Staphylococcus aureus from bovine mastitis: identification and lack of correlation between tolerance in vitro and response to therapy in vivo. Research in Veterinary Science 34, 266-271 ERICSSON, H. M. & SHERRIS, J. C. (1971) Antibiotic sensitivity testing: report of an international collaborative study. Acta Pathologica et Microb~ologica Scandinavica, Sec B 217 (supplement),
1-90 FRANCIS, P. G. & CARROLL, P. J. (1986) Antibiotic resistance patterns of Staphylococcus aureus strains from clinical bovine mastitis. Veterinary Record 118, 361-363 HAJEK, V. & MARSALEK, E. (197'1) The differentiation of pathogenic staphylococci and a suggestion for their taxonomic classification. Zentralblatt fiir Bakteriologie, Parasitenkunde, lnfektionskrankheiten und Hygiene. I Abt Orig Reihe A 217, 176-182 JONES, T. O. & HEATH, P. J. (1985)/3-Lactamase production in Staphylococcus aureus isolated from bovine mastitis milk. Veterinary Record 117, 340 KONEMAN, E. W., ALLEN, S. D., DOWELL Jr, V. R., JANA, W~ M., SOMMERS, H. M. & WINN Jr, W. C. (1988) Diagnostic Microbiology. 3rd edn. Philadelphia, J. B. Lippincott. p 890 MACKIE, D. P., LOGAN, E. F., POLLOCK, D. A. & RODGERS, S. P. (1988) Antibiotic sensitivity of bovine staphylococcal and coliform mastitis isolates over four years. Veterinary Record 123, 515-517 MONSALLIER, G. & THOMASSON, C. (1986) Traitement des mammites bovines en lactation par une infusion unique de cefoperazone: Essais clinique. Revue du Mddecin Vdtdrinaire 137, 15-22 PARKER, H. T. (1972) Methods in Microbiology. Volume 7B. New York, Academic Press. p 345 PERRY, B. D., CARTER, M. E., HILL, F. W. G. & MILNE, A. C. (1987) Mastitis and milk production in cattle in a communal land of Zimbabwe. British Veterinary Journal 143, 44-50 SABATH, L. D., LAVARDIERE, M., WHEELER, N., BLAZEVIC, D. & WILKINSON, B. J. (1977) A new type of penicillin resistance of Staphylococcus aureus. Lancet i, 443-447 SUBCOMMITTEE ON PHAGE TYPING OF STAPHYLOCOCCI (1975) Report (1970-1974) o f the subcommittee on phage typing o f staphylococci to the international committee on nomenclature of bacteria. International Journal of Systematic Bacteriology 25, 241-242 WILSON, C. D., AGGER, N., GILBERT, G. A., THOMASSON, C. A. & TOLLING, S. T. (1986) Field trials with cefoperazone in the treatment of bovine clinical mastitis. Veterinary Record 118, 17-19 WORKING GROUP ON PHAGE TYPING OF BOVINE STAPHYLOCOCCI (1971) Minutes of meeting, 5 August 1970. International Journal of Systematic Bacteriology 21, 171-176
Received March 11, 1991 Accepted July 10, 1991
Comparative in vitro activity of three cephalosporins against Staphylococcus aureus isolated from bovine mastitis C. A. M. LOPES, G. MORENO, Department o f Microbiology and Immunology, Institute o f Biosciences, Estadual Paulista University, UNESP, Botucatu-SP 18610, Brazil
This report compares the in vitro activity of three cephalosporins (cephalothin, cefoxitin and ceftriaxone) against 57 S t a p h y l o c o c c u s aureus strains isolated from cows with clinical mastitis on the basis of the minimal inhibitory (MIC) and minimal bactericidal concentrations (MUC). The majority of the S aureus strains showed resistance to cefoxitin and ceftriaxone and sensitivity to cephalothin. The highest MICs and MnCs were found for cefoxitin and ceftriaxone. Antimicrobial tolerance (MBC/MIC>~32:l) was observed in relation to cephalothin and ceftriaxone. The data suggest that these cephalosporins may not be effective for the treatment of staphylococcal bovine mastitis. The precise definition of their antimicrobial efficacies requires more detailed in vitro and in vivo studies. A L T H O U G H several papers haves focused attention on the in vitro susceptibilities of Staphylococcus aureus isolated from bovine mastitis in recent years (Araki et al 1985, Jones and Heath 1985, Craven et al 1986, Francis and Carroll 1986, Monsallier and Thomasson 1986, Wilson et a11986, Perry et al 1987, Mackie et al 1988), little information is available concerning the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of cephalosporins employed for the treatment of bovine mastitis. Since antibiotic therapy plays an important role in the treatment of staphylococcal mastitis (Craven et al 1986), the authors examined in vitro activity of cephalothin, cefoxitin and ceftriaxone against S aureus by the broth dilution method. Fifty-seven S aureus strains freshly isolated from cases of clinical bovine mastitis on dairy farms in Brazil during February to September 1989 were investigated by standard bacteriological methods (Koneman et al 1988). All isolates could be classed as bovine strains on the basis of phage typing using the standard method of Blair and Williams (1961) as modified by Parker (1972), with the basic sets of phages for typing human strains (Subcommittee on Phage Typing of Staphylococci 1975) and bovine strains (Working
Group on Phage Typing of Bovine Staphylococci 1971) and biotyping according to the methods of Hajek and Marsalek (1971). Antibiotics were obtained in powder form: cephalothin (Eli Lilly), cefoxitin (Merck Sharp & Dohme) and ceftriaxone (Roche A/A). A standard stock solution of each antibiotic was prepared according to the instructions of the manufacturer and used immediately. The MIC and MBC of each antibiotic was determined by a modification of the broth dilution method of the World Health Organization International Collaborative Study (Ericsson and Sherris 1971). Serial twofold dilutions from 128 to 0.06 gg m1-1 were made in 5 ml of Mueller Hinton broth (Difco Laboratories). All inocula (50 ~1) were quantitated to log 5" 7 colony forming units (CFU) per millilitre of a log-phase-growth subculture from the original plates. The rack of inoculated tubes was gently shaken to mix the contents and tubes were incubated at 35°C overnight. MIC endpoints were read as the lowest antibiotic concentration that resulted in no growth after the incubation period. Tubes showing no visible growth were then individually shaken by hand to resuspend the contents and subcultured by streaking 100/zl with a semiautomatic pipette across the diameter of a 90 mm 5 per cent sheep blood agar plate (Tryptic Soy Agar Base; Difco Laboratories). After 20 minutes, when the liquid was completely absorbed into the medium, the inoculum was spread over the entire surface of the plates with a sterile bent glass rod. Following this procedure to minimise the inhibition of the inoculum by antibiotic carry-over, CFU were counted after 18 to 24 hours of incubation at 35°C and MBC was read as the lowest antibiotic concentration killing at least 99" 9 per cent of the initial inoculum. Antimicrobial tolerance is defined as an MBC/MIC ratio /> 32:1 after 24 hours of incubation at 35°C (Sabath et al 1977). The results of the MIC and MBC determinations are summarised in Table 1. The majority of the S aureus strains were resistant to cefoxitin and ceftriaxone (MICin excess of 4
TABLE 1 : In vitro susceptibilities of 5 7 Staphylococcus aureus strains isolated from bovine mastitis Antibiotic Cephalothin Cefoxitin Ceftriaxone
50% of strains MIC MBC O. 5 32 4
90% of strains MIC MBC
2 64 16
1 64 8
MIC Minimal inhibitory concentration MBC Minimal bactericidal concentration
339
8 128 64
Range MIC
MBC
O- 06-2 2-128 0.12-16
O- 05-64 4 - > 128 0"12-128
340
C. A . M . L o p e s , G. M o r e n o TABLE 2- MBC/MIC ratios of 57 Staphylococcus aureus strains isolated from bovine mastitis Antibiotic Cephalothin Cefoxitin* Ceftriaxone
1: 1 21 (36-8) 18 (33' 3) 18 (31.6)
Number (%) of strains with the indicated MBC/MIC ratio 2:1 4:1 8: I 16:1 3(5.3) 33 (61 • 1) 18 (31 .6)
12(21) 0 3 (5-3)
6(10.5) 3 (5- 5) 6 (10.5)
6(10.5) 0 0
/> 32:1 9(15.8) 0 2 (21)
• Three strains not tested
/zg m l - 1) and sensitive to cephalothin. The highest MIC and MBC values were found for cefoxitin. Table 2 gives the MBC/MIC ratios for the tested strains. An MBC/MIC ratio of 4 or greater was observed for all drugs, but most frequently for cephalothin and ceftriaxone. The authors found a considerable deg~'ee of bacterial tolerance to cephalothin (15.8 per cent) and ceftriaxone (21 per cent). According to Sabath et al (1977), the growth of a tolerant strain is inhibited at low drug concentrations, like a non-tolerant strain, but killing occurs at much higher concentrations than for a non-tolerant strain. This concept may have practical implications and clinical significance for the choice of an antibiotic for therapeutic purposes, although it has not been currently investigated in veterinary science. However, Craven et al (1983) observed cloxacillin tolerance in vitro in isolates of S aureus from bovine mastitis, but suggested that it was of doubtful relevance to the response of therapy in vivo. Tolerance to/3-1actam antibiotics was not found by Craven et al (1986). In order to assess the efficacy of new cephalosporins for the treatment of bovine mastitis, some investigators (Mackie et al 1988, Monsallier and Thomasson 1986, Wilson et al 1986) have employed the disc diffusion method and found good antimicrobial activity of these drugs against most S aureus isolates tested. Since these cephalosporins can show remarkable differences in their antimicrobial activities and little is known about their interactions in the bovine udder, it was concluded that further bacteriological studies, as well as more clinical investigations, are needed to understand their in vivo efficacies. Until that time and for practical purposes, the use of cephalothin, cefoxitin and ceftriaxone should be viewed with caution in the treatment of staphylococcal bovine mastitis.
Acknowledgements We thank Professor Ana Maria Uthida Tanaka and Professor Maria Aparecida de Araujo of the department of parasitology, microbiology and immunology, Faculty of Medicine, Ribeirao Preto, Sao Paulo University, for phage typing our S aureus strains. We also thank Antonia Horacio and Antonio Corr~a for excellent technical assistance.
References ARAKI, S., KASHIWAZAKI, M. & KUME, T. (1985) Antimicrobial activity of amoxiciUin and other penicillins against clinical isolates from bovine udders. Japanese Journal of Veterinary Science 47, 323-324 BLAIR, J. E. & WILLIAMS, R. E. O. (1961) Phage typing of staphylococci. Bulletin of the World Health Organization 24, 771-784
CRAVEN, N., ANDERSON, J. C. & JONES, T. O. (1986) Antimicrobial drug susceptibilities of Staphylococcus aureus isolates from bovine mastitis. Veterinary Record 118, 290-291 CRAVEN, N., ANDERSON, J. C. & WILSON, C. D. (1983) Penicillin (cloxacillin-tolerant) Staphylococcus aureus from bovine mastitis: identification and lack of correlation between tolerance in vitro and response to therapy in vivo. Research in Veterinary Science 34, 266-271 ERICSSON, H. M. & SHERRIS, J. C. (1971) Antibiotic sensitivity testing: report of an international collaborative study. Acta Pathologica et Microb~ologica Scandinavica, Sec B 217 (supplement),
1-90 FRANCIS, P. G. & CARROLL, P. J. (1986) Antibiotic resistance patterns of Staphylococcus aureus strains from clinical bovine mastitis. Veterinary Record 118, 361-363 HAJEK, V. & MARSALEK, E. (197'1) The differentiation of pathogenic staphylococci and a suggestion for their taxonomic classification. Zentralblatt fiir Bakteriologie, Parasitenkunde, lnfektionskrankheiten und Hygiene. I Abt Orig Reihe A 217, 176-182 JONES, T. O. & HEATH, P. J. (1985)/3-Lactamase production in Staphylococcus aureus isolated from bovine mastitis milk. Veterinary Record 117, 340 KONEMAN, E. W., ALLEN, S. D., DOWELL Jr, V. R., JANA, W~ M., SOMMERS, H. M. & WINN Jr, W. C. (1988) Diagnostic Microbiology. 3rd edn. Philadelphia, J. B. Lippincott. p 890 MACKIE, D. P., LOGAN, E. F., POLLOCK, D. A. & RODGERS, S. P. (1988) Antibiotic sensitivity of bovine staphylococcal and coliform mastitis isolates over four years. Veterinary Record 123, 515-517 MONSALLIER, G. & THOMASSON, C. (1986) Traitement des mammites bovines en lactation par une infusion unique de cefoperazone: Essais clinique. Revue du Mddecin Vdtdrinaire 137, 15-22 PARKER, H. T. (1972) Methods in Microbiology. Volume 7B. New York, Academic Press. p 345 PERRY, B. D., CARTER, M. E., HILL, F. W. G. & MILNE, A. C. (1987) Mastitis and milk production in cattle in a communal land of Zimbabwe. British Veterinary Journal 143, 44-50 SABATH, L. D., LAVARDIERE, M., WHEELER, N., BLAZEVIC, D. & WILKINSON, B. J. (1977) A new type of penicillin resistance of Staphylococcus aureus. Lancet i, 443-447 SUBCOMMITTEE ON PHAGE TYPING OF STAPHYLOCOCCI (1975) Report (1970-1974) o f the subcommittee on phage typing o f staphylococci to the international committee on nomenclature of bacteria. International Journal of Systematic Bacteriology 25, 241-242 WILSON, C. D., AGGER, N., GILBERT, G. A., THOMASSON, C. A. & TOLLING, S. T. (1986) Field trials with cefoperazone in the treatment of bovine clinical mastitis. Veterinary Record 118, 17-19 WORKING GROUP ON PHAGE TYPING OF BOVINE STAPHYLOCOCCI (1971) Minutes of meeting, 5 August 1970. International Journal of Systematic Bacteriology 21, 171-176
Received March 11, 1991 Accepted July 10, 1991
