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Comparing different isolates of Pasteurella haemolytica from beef calves using their in vitro antimicrobial sensitivity patterns
Vetennary M~crobmlogy, 20 (1989) 73-78 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
73
C o m p a r i n g D i f f e r e n t Isolates of PasteureUa haemolytica from B e e f Calves u s i n g their In Vitro Antimicrobial Sensitivity Patterns M.K SHOO*
Department of Veterinary Medicine, Unwerszty of Glasgow Vetermar3, School, Bearsden Road, Glasgow G61 1QH (Gt Brttam) (Accepted for publication 21 November 1988)
ABSTRACT Shoo, M.K., 1989. Comparing different isolates of PasteureUa haemolytzca from beef calves using their in vitro antimicrobial sensitivity patterns. Vet Mtcroblol, 20.73-78. In vitro studies, using disc diffusion and agar dilution techniques, were carried out to compare susceptibilities to selected antimicrobial agents of 30 isolates of Pasteurella haemolytwa from healthy calves and 30 isolates from calves with transit fever. There was no difference m susceptibility patterns between isolates from healthy calves and ~solates from diseased calves or between isolates of serots~e A1 and isolates of serotype A2. Penicillin resistance was assocmted with productlon of fl-lactamase.
INTRODUCTION
The percentages of PasteureUa haemolytica A1 susceptible to various drugs were found to be higher for isolates from the lungs of calves with transit fever than those from the upper respiratory tracts of the same calves (Allan et al., 1985 ). Data were not available on whether P. haemolytica isolates from clinically healthy calves differed significantly from those from calves with transit fever. It has also been reported that P. haemolytica biotypes A and T differ significantly in their susceptibilities to antimicrobial agents and several workers have suggested that these differences might be useful in biotyping (Smith, 1961; Biberstein and Gills, 1962; Wessman and Hilker, 1968; Mwangota et al., 1978; Biberstein and Kirkham, 1979). Information regarding m i n i m u m inhibitory concentrations (MICs) of antimicrobial agents to PasteureUa spp. isolated from cattle is limited to a few *Present address: Department of Veterinary Medicine and Public Health, Sokoine University of Agriculture, P.O. Box 3021, Morogoro, Tanzania.
0378-1135/89/$03.50
© 1989 Elsevier Science Pubhshers B.V.
74
selected isolates (Chang and Carter, 1976; Silver et al., 1979; Wray and Morrison, 1983 ). The major objective of the present study was to use disc diffusion and agar dilution tests to compare susceptibilities to selected antimicrobial agents of: ( 1 ) P. haemolytica isolates from healthy calves with calves with transit fever: ( 2 ) P. haemolytica isolates of serotypes A1 and A2. A secondary objective was to determine whether penicillin-resistant P. haemolytica produced fl-lactamase. MATERIALS AND METHODS
Pasteurella haemolytica isolates A total of 60 isolates from various beef farms in Scotland were tested. Thirty were obtained from nasopharyngeal swabs taken from clinically healthy calves and were of serotypes A1 (5), A2 (24) and A6 ( 1 ). The remainder were isolated from nasopharyngeal swabs of calves with transit fever or from the respiratory tract tissues at necropsy; they were serotypes A1 (27), A2 (2) and Tm (1). Seven of the transit fever cases were known to have been treated with one or more antimicrobial agents.
Determination of diameters of zones of inhibition of Pasteurella haemolytica by various drugs Oxoid multodiscs (Oxoid, London S.E.1, Gt. Britain), each consisting of individual discs of 30 zg chloramphenicol, 10 ~g ampicillin, 30 ~g oxytetracycline, 5 zg penicillin, 2 llg lincomycin, 10/~g streptomycin, 25/zg sulphamethaxazole-trimethoprim and a drug-free control disc, were used. Mueller-Hinton agar (Difco Laboratories, Detroit, MI) supplemented with sheep blood to a concentration of 5% was employed as culture medium. Preparation of inoculum, inoculation of plates, measurement of diameters of zones of inhibition of P. haemolytica around the various discs and interpretation of results were as described by Barry (1976).
Determination of minimum inhibitory concentrattons of selected drugs to Pasteurella haemolytica The agar dilution technique as described by Barry (1976) was used. The drugs used were ampicillin, oxytetracycline hydrochloride, lincomycin, sulphadiazine and trimethoprim, all of which were supplied in powdered form (Sigma Chemical Company, Poole, Dorset BH 17 7NH, Gt. Britain), together with a sulphadiazine-trimethoprim combination which was prepared by mixing the stock solutions of sulphadiazine and trimethoprim at a 20: 1 ratio before diluting in agar (Krogstad and Moellering, 1980).
75
Testing for fl-lactamase production by Pasteurella haemolytica Two isolates shown to be penicillin-resistant and four that were penicillinsensitive were tested for production of fl-lactamase by use of Oxoid fl-lactamase identification sticks. RESULTS
Relattonship between Pasteurella haemolytica from healthy calves and those from calves with transit fever Most of the isolates from clinically healthy calves and calves with transit fever were susceptible to most of the drugs used in the disc diffusion test (Table 1). The only significant difference between the two groups was that isolates from clinically healthy calves were more susceptible to streptomycin than those from calves with transit fever ( P < 0.05). The distributions of MICs of the various drugs for isolates from calves with transit fever are shown in Fig. 1. Those from clinically healthy calves were not significantly different from those obtained from calves with transit fever (P<0.05).
Relationship between Pasteurella haemolytica serotypes AI and As as judged by their in vitro sensitivities There were 32 isolates of P. haemolytica A1 and 26 of A2. The means of diameters of the zones of inhibition were larger for P. haemolytica A2 than for A1 for all of the antimicrobial agents apart from sulphamethoxazole-trimethoprim for which the reverse was the case. Such differences, however, were only significant ( P < 0.05 ) for streptomycin and oxytetracycline. The means of the MICs were lower for P. haemolytica A2 than for A~ for all TABLE 1 Percentage susceptibilities of Pasteurella haemolyt~ca to various antimicrobial drugs as determined by dmc diffusion test Sources ( N o )
Chmcally healthy calves
% susceptibility Amp.
Chlor.
Lmc
Oct.
Pen.
Str
Sm-Tm
100
100
0
100
90
30
100
93
100
0
90
90
17
100
(30) Cases of transit fever (30)
Amp --ampicillin; Chlor.=chloramphemcol; Line.=lincomycm; Oct.=oxytetracycline; Pen.= penicillin; Str. = streptomycin; S m - T m = sulphamethaxazole-trimethoprim.
76
TRANSIT FEVER
TRANSIT FEVER OXYTETRACYCLINE
TRIMETHOPRIM
2G
% 59
o MINIMUM INHIBITORY
o
MINIMUM INHIBITORY
CONCENTRATION ( p g , ' m l )
CONCENTRATION
(iJg/ml)
SULPHADIAZINE-TRIMETHOPRIM
AMPICILLIN I-._1
o
o
IJ-
IC
O
cc U..I m
Z
z=
o
2
o
n~$
MINIMUM
INHIBITORY
CONCENTRATION
(pg/ml)
o o
o
....
MINIMUM INHIBITORY
~
g
~
CONCENTRATION
~ ,~ ~v ~ (ug/ml)
LINCOMYCIN
SULPHADIAZINE
2O
o
111 I-_1
O
r.,0
1.1_ 10 O rrw m
z
0
~
~ o o o o MINIMUM
INHIBITORY
~v
oo
o
CONCENTRATION
(pg/ml)
MINIMUMINHIBITORYCONCENTAATION(pg/ml)
Fig. 1. Distribution of m i n i m u m inhabltory concentrations (MICs) of various antlmieroblal drugs for Pasteurella haemolytlca from calves with transit fever.
77
of the drugs apart from sulphadiazine for which the means were larger. The differences, however, were only significant (P < 0.05) for oxytetracycline.
fl-lactamase activity of selected Pasteurella haemolytica isolates Of the six isolates tested, only the two which were resistant to penicillin were fl-lactamase positive and this enzymatic activity did not require pre-induction. Growth near a disc containing penicillin did not induce fl-lactamase activity in the four negative isolates. DISCUSSION
In a previous study, isolates of P. haemolytica A1 from the lungs of calves with transit fever were more susceptible to various drugs than isolates from the upper respiratory tract of the same calves (Allan et al., 1985) indicating a possible difference in virulence. When strains from healthy calves were compared with those from transit fever in the present study, however, there were no major differences in their sensitivities to antimicrobial compounds. Therefore, if these isolates differed in virulence, it was not related to their in vitro sensitivities to the antimicrobial agents tested. Similarly, P. haemolytica AI and A2 did not differ significantly in terms of their in vitro sensitivities to most of the drugs used; however, serotype A2 strains were more sensitive to oxytetracycline and streptomycin. Although P. haemolytica biotypes A and T differed significantly in their in vitro sensitivities to many antimicrobial agents (Smith, 1961; Biberstein and Gills, 1962; Wessman and Hilker, 1968; Mwangota et al., 1978; Biberstein and Kirkham, 1979), differences within the biotype A group were minor in the present study. Accordingly, it is unlikely that antibiotic sensitivity will aid in serotyping the biotype A strains. Insufficient strains of biotype T were examined to make any judgement. Beta-lactamase activity was demonstrated only in the penicillin-resistant isolates. It is very likely that genetic information coding for the production of this enzyme is plasmid controlled and could have been acquired from other bacteria. Plasmids have been demonstrated in P. haemolytica and their possible acquisition from enteric bacteria has been suggested (Zimmerman and Hirsh, 1980). ACKNOWLEDGEMENTS
The author wishes to thank Dr. A. Wiseman, Prof. I.E. Selman and Dr. E.M. Allan for their encouragements. Twenty of the isolates used in this study were provided by Dr. E.M. Allan to whom the author is very grateful. Thanks are also given to Her Majesty's Government, Foreign and Commonwealth Scholarships for the study grant.
78 REFERENCES Allan, E M., Gibbs, H A., Shoo, M.K, Dalgleish, R., Selman, I.E. and Wlseman, A., 1985 Antimicrobial senslttvltms ofPasteurella haemo~'tzca A1 from beef calves. Vet. Rec, 177- 506-507 Barry, A L , 1976. The antimicroblc susceptibility test. In. Principle and Practices. Lea and Febiger, Philadelphia, PA, pp. 3-11, 61-103, 164-219 Biberstein, E.L. and Gills, M.G., 1962. The relation of the antigenic types to the A and T types of Pasteurella haemolyttca. J. Comp. Pathol., 32: 316-320. Bibersteln, E.L. and Kirkham, C , 1979. Antimicroblal susceptibd~ty patterns of the A and T types of Pasteurella haemolytwa. Res. Vet Sci., 26: 324-328. Chang, W.H and Carter, G.R., 1976. Muhlple drug resistance m PasteureUa multoctda and Pasteurella haemolyttca from cattle and swine. J. Am Vet. Med Assoc, 169' 710-712. Krogstad, D.J and Moellermg, R.C., 1980 Combinations of antibiotics, mechanisms of interaction against bacteria. In: V Lorian (Editor), Antibiotics in Laboratory Medicine. Williams and Wflkins, Baltimore, MD, pp 298-341 Mwangota, A.U., Muhammed, S.I. and Thomson, R.G., 1978. Serological types of Pasteurella haemolytzca m Kenya. Cornell Vet., 68: 84-93. Silver, R.P., Lemmg, B., Garon, C.F. and Hjerpe, C.A., 1979. R-plasmid m Pasteurella multoctda. Plasmld, 2 493-497 Smith, G.R., 1961. The characteristics of two types of Pasteurella haemolytwa associated with different pathologmal conditions m sheep. J. Pathol. Bacterlol., 81. 431-440. Wessman, G.E and Hilker, G., 1968. Characterization of Pasteurella haemolytlca isolated from respiratory tract of cattle. Can. J. Comp Med, 32: 498-504. Wray, C and Morrison, J R A., 1983. Antibiotic resistant Pasteurella haemolytlca. Vet. Rec, 113' 143 Zimmerman, M L. and Hirsh, D.C, 1980. Demonstration of an R-plasmld m a stram of PasteureUa haemolytzca isolated from feedlot cattle. Am. J Vet Res., 41:166-169
73
C o m p a r i n g D i f f e r e n t Isolates of PasteureUa haemolytica from B e e f Calves u s i n g their In Vitro Antimicrobial Sensitivity Patterns M.K SHOO*
Department of Veterinary Medicine, Unwerszty of Glasgow Vetermar3, School, Bearsden Road, Glasgow G61 1QH (Gt Brttam) (Accepted for publication 21 November 1988)
ABSTRACT Shoo, M.K., 1989. Comparing different isolates of PasteureUa haemolytzca from beef calves using their in vitro antimicrobial sensitivity patterns. Vet Mtcroblol, 20.73-78. In vitro studies, using disc diffusion and agar dilution techniques, were carried out to compare susceptibilities to selected antimicrobial agents of 30 isolates of Pasteurella haemolytwa from healthy calves and 30 isolates from calves with transit fever. There was no difference m susceptibility patterns between isolates from healthy calves and ~solates from diseased calves or between isolates of serots~e A1 and isolates of serotype A2. Penicillin resistance was assocmted with productlon of fl-lactamase.
INTRODUCTION
The percentages of PasteureUa haemolytica A1 susceptible to various drugs were found to be higher for isolates from the lungs of calves with transit fever than those from the upper respiratory tracts of the same calves (Allan et al., 1985 ). Data were not available on whether P. haemolytica isolates from clinically healthy calves differed significantly from those from calves with transit fever. It has also been reported that P. haemolytica biotypes A and T differ significantly in their susceptibilities to antimicrobial agents and several workers have suggested that these differences might be useful in biotyping (Smith, 1961; Biberstein and Gills, 1962; Wessman and Hilker, 1968; Mwangota et al., 1978; Biberstein and Kirkham, 1979). Information regarding m i n i m u m inhibitory concentrations (MICs) of antimicrobial agents to PasteureUa spp. isolated from cattle is limited to a few *Present address: Department of Veterinary Medicine and Public Health, Sokoine University of Agriculture, P.O. Box 3021, Morogoro, Tanzania.
0378-1135/89/$03.50
© 1989 Elsevier Science Pubhshers B.V.
74
selected isolates (Chang and Carter, 1976; Silver et al., 1979; Wray and Morrison, 1983 ). The major objective of the present study was to use disc diffusion and agar dilution tests to compare susceptibilities to selected antimicrobial agents of: ( 1 ) P. haemolytica isolates from healthy calves with calves with transit fever: ( 2 ) P. haemolytica isolates of serotypes A1 and A2. A secondary objective was to determine whether penicillin-resistant P. haemolytica produced fl-lactamase. MATERIALS AND METHODS
Pasteurella haemolytica isolates A total of 60 isolates from various beef farms in Scotland were tested. Thirty were obtained from nasopharyngeal swabs taken from clinically healthy calves and were of serotypes A1 (5), A2 (24) and A6 ( 1 ). The remainder were isolated from nasopharyngeal swabs of calves with transit fever or from the respiratory tract tissues at necropsy; they were serotypes A1 (27), A2 (2) and Tm (1). Seven of the transit fever cases were known to have been treated with one or more antimicrobial agents.
Determination of diameters of zones of inhibition of Pasteurella haemolytica by various drugs Oxoid multodiscs (Oxoid, London S.E.1, Gt. Britain), each consisting of individual discs of 30 zg chloramphenicol, 10 ~g ampicillin, 30 ~g oxytetracycline, 5 zg penicillin, 2 llg lincomycin, 10/~g streptomycin, 25/zg sulphamethaxazole-trimethoprim and a drug-free control disc, were used. Mueller-Hinton agar (Difco Laboratories, Detroit, MI) supplemented with sheep blood to a concentration of 5% was employed as culture medium. Preparation of inoculum, inoculation of plates, measurement of diameters of zones of inhibition of P. haemolytica around the various discs and interpretation of results were as described by Barry (1976).
Determination of minimum inhibitory concentrattons of selected drugs to Pasteurella haemolytica The agar dilution technique as described by Barry (1976) was used. The drugs used were ampicillin, oxytetracycline hydrochloride, lincomycin, sulphadiazine and trimethoprim, all of which were supplied in powdered form (Sigma Chemical Company, Poole, Dorset BH 17 7NH, Gt. Britain), together with a sulphadiazine-trimethoprim combination which was prepared by mixing the stock solutions of sulphadiazine and trimethoprim at a 20: 1 ratio before diluting in agar (Krogstad and Moellering, 1980).
75
Testing for fl-lactamase production by Pasteurella haemolytica Two isolates shown to be penicillin-resistant and four that were penicillinsensitive were tested for production of fl-lactamase by use of Oxoid fl-lactamase identification sticks. RESULTS
Relattonship between Pasteurella haemolytica from healthy calves and those from calves with transit fever Most of the isolates from clinically healthy calves and calves with transit fever were susceptible to most of the drugs used in the disc diffusion test (Table 1). The only significant difference between the two groups was that isolates from clinically healthy calves were more susceptible to streptomycin than those from calves with transit fever ( P < 0.05). The distributions of MICs of the various drugs for isolates from calves with transit fever are shown in Fig. 1. Those from clinically healthy calves were not significantly different from those obtained from calves with transit fever (P<0.05).
Relationship between Pasteurella haemolytica serotypes AI and As as judged by their in vitro sensitivities There were 32 isolates of P. haemolytica A1 and 26 of A2. The means of diameters of the zones of inhibition were larger for P. haemolytica A2 than for A1 for all of the antimicrobial agents apart from sulphamethoxazole-trimethoprim for which the reverse was the case. Such differences, however, were only significant ( P < 0.05 ) for streptomycin and oxytetracycline. The means of the MICs were lower for P. haemolytica A2 than for A~ for all TABLE 1 Percentage susceptibilities of Pasteurella haemolyt~ca to various antimicrobial drugs as determined by dmc diffusion test Sources ( N o )
Chmcally healthy calves
% susceptibility Amp.
Chlor.
Lmc
Oct.
Pen.
Str
Sm-Tm
100
100
0
100
90
30
100
93
100
0
90
90
17
100
(30) Cases of transit fever (30)
Amp --ampicillin; Chlor.=chloramphemcol; Line.=lincomycm; Oct.=oxytetracycline; Pen.= penicillin; Str. = streptomycin; S m - T m = sulphamethaxazole-trimethoprim.
76
TRANSIT FEVER
TRANSIT FEVER OXYTETRACYCLINE
TRIMETHOPRIM
2G
% 59
o MINIMUM INHIBITORY
o
MINIMUM INHIBITORY
CONCENTRATION ( p g , ' m l )
CONCENTRATION
(iJg/ml)
SULPHADIAZINE-TRIMETHOPRIM
AMPICILLIN I-._1
o
o
IJ-
IC
O
cc U..I m
Z
z=
o
2
o
n~$
MINIMUM
INHIBITORY
CONCENTRATION
(pg/ml)
o o
o
....
MINIMUM INHIBITORY
~
g
~
CONCENTRATION
~ ,~ ~v ~ (ug/ml)
LINCOMYCIN
SULPHADIAZINE
2O
o
111 I-_1
O
r.,0
1.1_ 10 O rrw m
z
0
~
~ o o o o MINIMUM
INHIBITORY
~v
oo
o
CONCENTRATION
(pg/ml)
MINIMUMINHIBITORYCONCENTAATION(pg/ml)
Fig. 1. Distribution of m i n i m u m inhabltory concentrations (MICs) of various antlmieroblal drugs for Pasteurella haemolytlca from calves with transit fever.
77
of the drugs apart from sulphadiazine for which the means were larger. The differences, however, were only significant (P < 0.05) for oxytetracycline.
fl-lactamase activity of selected Pasteurella haemolytica isolates Of the six isolates tested, only the two which were resistant to penicillin were fl-lactamase positive and this enzymatic activity did not require pre-induction. Growth near a disc containing penicillin did not induce fl-lactamase activity in the four negative isolates. DISCUSSION
In a previous study, isolates of P. haemolytica A1 from the lungs of calves with transit fever were more susceptible to various drugs than isolates from the upper respiratory tract of the same calves (Allan et al., 1985) indicating a possible difference in virulence. When strains from healthy calves were compared with those from transit fever in the present study, however, there were no major differences in their sensitivities to antimicrobial compounds. Therefore, if these isolates differed in virulence, it was not related to their in vitro sensitivities to the antimicrobial agents tested. Similarly, P. haemolytica AI and A2 did not differ significantly in terms of their in vitro sensitivities to most of the drugs used; however, serotype A2 strains were more sensitive to oxytetracycline and streptomycin. Although P. haemolytica biotypes A and T differed significantly in their in vitro sensitivities to many antimicrobial agents (Smith, 1961; Biberstein and Gills, 1962; Wessman and Hilker, 1968; Mwangota et al., 1978; Biberstein and Kirkham, 1979), differences within the biotype A group were minor in the present study. Accordingly, it is unlikely that antibiotic sensitivity will aid in serotyping the biotype A strains. Insufficient strains of biotype T were examined to make any judgement. Beta-lactamase activity was demonstrated only in the penicillin-resistant isolates. It is very likely that genetic information coding for the production of this enzyme is plasmid controlled and could have been acquired from other bacteria. Plasmids have been demonstrated in P. haemolytica and their possible acquisition from enteric bacteria has been suggested (Zimmerman and Hirsh, 1980). ACKNOWLEDGEMENTS
The author wishes to thank Dr. A. Wiseman, Prof. I.E. Selman and Dr. E.M. Allan for their encouragements. Twenty of the isolates used in this study were provided by Dr. E.M. Allan to whom the author is very grateful. Thanks are also given to Her Majesty's Government, Foreign and Commonwealth Scholarships for the study grant.
78 REFERENCES Allan, E M., Gibbs, H A., Shoo, M.K, Dalgleish, R., Selman, I.E. and Wlseman, A., 1985 Antimicrobial senslttvltms ofPasteurella haemo~'tzca A1 from beef calves. Vet. Rec, 177- 506-507 Barry, A L , 1976. The antimicroblc susceptibility test. In. Principle and Practices. Lea and Febiger, Philadelphia, PA, pp. 3-11, 61-103, 164-219 Biberstein, E.L. and Gills, M.G., 1962. The relation of the antigenic types to the A and T types of Pasteurella haemolyttca. J. Comp. Pathol., 32: 316-320. Bibersteln, E.L. and Kirkham, C , 1979. Antimicroblal susceptibd~ty patterns of the A and T types of Pasteurella haemolytwa. Res. Vet Sci., 26: 324-328. Chang, W.H and Carter, G.R., 1976. Muhlple drug resistance m PasteureUa multoctda and Pasteurella haemolyttca from cattle and swine. J. Am Vet. Med Assoc, 169' 710-712. Krogstad, D.J and Moellermg, R.C., 1980 Combinations of antibiotics, mechanisms of interaction against bacteria. In: V Lorian (Editor), Antibiotics in Laboratory Medicine. Williams and Wflkins, Baltimore, MD, pp 298-341 Mwangota, A.U., Muhammed, S.I. and Thomson, R.G., 1978. Serological types of Pasteurella haemolytzca m Kenya. Cornell Vet., 68: 84-93. Silver, R.P., Lemmg, B., Garon, C.F. and Hjerpe, C.A., 1979. R-plasmid m Pasteurella multoctda. Plasmld, 2 493-497 Smith, G.R., 1961. The characteristics of two types of Pasteurella haemolytwa associated with different pathologmal conditions m sheep. J. Pathol. Bacterlol., 81. 431-440. Wessman, G.E and Hilker, G., 1968. Characterization of Pasteurella haemolytlca isolated from respiratory tract of cattle. Can. J. Comp Med, 32: 498-504. Wray, C and Morrison, J R A., 1983. Antibiotic resistant Pasteurella haemolytlca. Vet. Rec, 113' 143 Zimmerman, M L. and Hirsh, D.C, 1980. Demonstration of an R-plasmld m a stram of PasteureUa haemolytzca isolated from feedlot cattle. Am. J Vet Res., 41:166-169