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Reliability of thermonuclease production for the identification of human and animal Staphylococcus aureus
Veterinary Microbiology, 9 (1984) 271--278 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
271
RELIABILITY OF THERMONUCLEASE PRODUCTION FOR THE IDENTIFICATION OF HUMAN AND ANIMAL STAPHYLOCOCCUS A U R E US
J.D. ADEKEYE
Department of Veterinary Pathology and Microbiology, Ahmadu Bello University, Zaria (Nigeria) (Accepted 9 December 1983)
ABSTRACT Adekeye, J.D., 1984. Reliability of thermonuclease production for the identification of human and animal Staphylococcus aureus. Vet. Microbiol., 9: 271--278. A total of 314 clinical and non-clinical isolates of the genus Staphylococcus was tested for coagulase production and glucose and mannitol fermentation. The isolates were tested for thermonuclease production and agglutination by sera 17H and 61218, which were specific for human and canine S. aureus biotypes, respectively. All produced coagulase and fermented glucose. A majority fermented mannitol anaerobically except for the canine isolates. A majority of human isolates produced thermonuclease (64.3%) and most were agglutinated by serum 17H. There was good correlation between thermonuclease production and agglutination by serum 17H of human and bovine clinical isolates (86.6 and 80%, respectively). This was also true of clinical canine isolates agglutinated by serum 61218, of which 75% were thermonuclease-positive. Over half of canine isolates (52.8%) were thermonuclease-positive and most were agglutinated by serum 61218. Bovine and caprine isolates were 34.1 and 25% thermonuclease-positive, respectively, while ovine isolates were only 14.2% thermonuclease-positive. Isolates from these ruminant sources were also poorly agglutinated by either serum. It was concluded that a greater number of clinical human and canine biotypes of S. aureus produced thermonuclease than their nonclinical isolates, and that a majority of other animal isolates were negative for thermonuclease. Therefore, the thermonuclease test may not be very useful for confirming the animal origin of S. aureus isolates.
INTRODUCTION F o l l o w i n g t h e d e m o n s t r a t i o n b y C u n n i n g h a m e t al. ( 1 9 5 6 ) , o f t h e p r o d u c t i o n o f t h e r m o s t a b l e d e o x y r i b o n u c l e a s e as a u n i q u e p r o p e r t y o f S t a p h y lococcus aureus, m a n y w o r k e r s ( C h e s b r o a n d A u b o r n , 1 9 6 7 ; S p e r b e r a n d Tatini, 1975; Yrios, 1976; Zarzour and Belle, 1977) have evaluated the relationship between the production of thermostable nuclease, enterotoxin production and coagulase formation. They concluded that thermonuclease p r o d u c t i o n c a n b e u s e d as a c o n f i r m a t o r y t e s t f o r t h e i d e n t i f i c a t i o n o f t o x i g e n i c a n d p a t h o g e n i c S. aureus. H o w e v e r , m o s t o f t h e S. aureus i s o l a t e s t h e y
0378-1135/84/$03.00
© 1984 Elsevier Science Publishers B.V.
272 tested were obtained either from h u m a n clinical materials or from foods possibly contaminated by S. a u r e u s from people. The same test has not been extensively used to verify whether or not such a conclusion can also be drawn in respect of S. a u r e u s isolates from clinical and non-clinical animal sources. To date, the most widely used criterion for distinguishing toxigenic and pathogenic S. a u r e u s from closely related saprophytic organism is the coagulase test. Although the result of the test can be affected by factors such as type of plasma used, the nature of the anticoagulant and possible loss of the characteristic on rare occasions by S. a u r e u s , m a n y laboratories rely solely on coagulase testing. Furthermore, in attempts to identify S. a u r e u s of different biotypes, Oeding (1960) and Pillet et al. (1968) obtained a typing serum designated 61218 which was considered specific for canine isolates of S. aureus. Similarly, Live (1975) produced an agglutinating serum designated 17H which is specific for thermostable agglutination of S. a u r e u s of h u m a n biotype. Since the antigenic properties of S. a u r e u s are stable (Griin, 1958; Hofstad and Oeding, 1960), the use of these sera may help to identify h u m a n and canine biotypes of S. a u r e u s from different sources. The purpose of this work was to evaluate the reliability of thermonuclease in confirming h u m a n and animal, clinical and non-clinical S. a u r e u s detected by coagulase and agglutination tests. MATERIALS AND METHODS B a c t e r i a l isolates
A total of 314 S. a u r e u s isolates obtained from clinical and non-clinical specimens from humans and others from animals was examined. Clinical isolates from h u m a n beings were from specimens for routine bacteriological culture submitted to the University Teaching hospitals at Zaria and Ibadan. Non-clinical isolates were from nose swabs of h u m a n volunteers who were apparently healthy. Clinical isolates from animals were from specimens submitted for bacteriological culture at the A h m a d u Bello University Veterinary Hospital. The non-clinical isolates were obtained from either nasal swabs from animals brought to the Veterinary Clinic for routine vaccinations, or obtained during ambulatory services. All the isolates were obtained from specimens cultured on blood agar plates incubated for 18--24 h at 37°C, and identified primarily as Gram-positive, catalase positive cocci. C o a g u l a s e test
Isolates from blood agar plates were subcultured in nutrient broth and incubated overnight at 27°C. Five drops (approximately 0.125 ml) of each overnight-culture were added to test-tubes (16 X 100 mm) containing 0.5 ml of one-fifth concentrations of rabbit (DIFCO) plasma, The tubes were incubated at 37°C and examined for coagulase formation at 4, 6, 12 and 24
273 h. A solid coagulum which did n o t fragment when the tubes were tilted was a positive test. Carbohydrate fermentation Fermentation of glucose and mannitol under anaerobic conditions was determined according to the recommendations of the Subcommittee on Taxo n o m y of Staphylococci and Micrococci (1965). Preparation and absorption o f antisera These were performed as described by Live (1975)using S. aureus strains 17, Cowan 1 and 61218. Cowan 1 is S. aureus Cowan serologic type strain 1. This is one of the 13 international-type strains of S. aureus in group sera (a, b, c, e) (Oeding, 1960), and was kindly supplied by I. Live. Slide agglutination tests These were carried out as described by Live (1975) using antisera 17H and 61218. Thermostable nuclease test Production of thermostable nuclease by the isolates was determined by the methods of Lachica et al. (1971) and Sperber and Tatini (1975). The toluidine blue-0-deoxyribonucleic acid agar used was prepared as described by Zarzour and Belle (1978). Fifteen ml of the agar was poured into a petri dish (100 X 15 mm). Twenty wells were cut in the agar with the tip of a 2 ml plastic syringe. Each well was filled with an overnight brain--heart infusion broth-culture of an isolate which had been steamed for 15 min at 100°C. Each plate was then placed in 150 X 15 mm petri dishes, the bottoms of which were covered with filter papers moistened with water. The plates were incubated at 37°C and observed at 1, 4 and 24 h. Organisms with bright pink zones surrounding the test-wells were regarded as thermostable deoxyribonuclease positive. RESULTS All the 314 Gram-positive, catalase-positive cocci were coagulase-positive and fermented glucose. Most of them, except canine isolates, fermented mannitol anaerobically. Table I shows the reactions of the isolates when agglutinated with sera 17H and 61218, compared with their thermonuclease production. Isolates from h u m a n beings had the highest frequency of thermonuclease production (64.3%), followed by dog isolates (52.8%). Bovine and caprine isolates were less then 50% thermonuclease-positive, while ovine isolates were only 14.2% thermonuclease-positive.
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The number of isolates which were agglutinated by serum 17H was generally n o t very high regardless of source, but h u m a n isolates were most frequently agglutinated (57% of the clinical isolates and 47% of the non-clinical isolates). Agglutination of animal isolates of S. a u r e u s by the same serum was poor (35% of canine isolates down to 0% of caprine clinical isolates). Of all the clinical isolates from different sources, only those from h u m a n (57%) and bovine (23.3%) sources were agglutinated by serum 17H. Non-clinical isolates from h u m a n and animal sources were agglutinated by the serum at varying degrees. Again, human isolates were most frequently agglutinated (46.8%) and ovine isolates were the least (11.8%). The clinical isolates which were agglutinated by serum 17H were much better producers of thermonuclease than their non-clinical counterparts. There was a very high agreement between agglutinability by serum 17H and thermonuclease production by the clinical isolates from h u m a n and cattle (86.6 and 80%, respectively}. Clinical and non-clinical isolates of S. a u r e u s from all the sources examined were poorly agglutinated by serum 61218 except clinical isolates from dogs, of which 100% were agglutinated. Of these isolates, 75% were thermonuclease-positive, but other isolates also agglutinated by serum 61218 were poor producers of thermonuclease except those from humans, of which 50% were positive. Non-agglutinability by either serum was very high with isolates from sheep, goat and cattle; 82--100%, 75--87% and 54--58%, respectively. The majority of isolates (75%) from other sources (mainly horses) were also not agglutinated. Very few non-agglutinable animal isolates produced thermonuclease apart from the canine isolates. DISCUSSION
Coagulase production by a member of the genus S t a p h y l o c o c c u s is usually accepted as proof that it is S. a u r e u s . Anaerobic utilization of glucose and mannitol are other properties of S. aureus. In this work, all the isolates investigated had those characteristics, except for a n u m b e r of the canine isolates which were negative for mannitol fermentation. Furthermore, because of the specificity of sera 17H and 61218 (Live, 1975), and the stability of the antigenic properties of S. a u r e u s (Grun, 1958; Oeding, 1960; Hofstad, 1964}, isolates agglutinated by serum 17H were regarded as h u m a n biotypes and those by serum 61218 were regarded as canine biotypes, irrespective of their sources. This was further supported in this work by the fact that most isolates agglutinated by serum 17H were of h u m a n origin and those agglutinated by serum 61218 were of canine origin. Few isolates from other sources were agglutinated and any that were agglutinated were regarded as human and canine biotypes t h a t had contaminated other animals. Generally the percentage of isolates agglutinated by either serum was not very high with either h u m a n or canine isolates. This was because their hosts probably carried large numbers of S. a u r e u s of other biotypes, contracted from contaminated environments jointly shared by animals and people -- a situation very com-
276 m o n in Nigeria (Adekeye, 1981). The scarcity of human biotypes of S. from clinical specimens, apart from humans and a few from cattle, supports the general belief expressed by some authors that different biotypes produce disease mainly in their homologous hosts (Elek, 1959; Fluharty et al., 1966). Direct correlation of anaerobic utilization of glucose and coagulase production with thermonuclease production was 64.3% for h u m a n isolates. This was less than the findings of other workers like Zarzour et al. (1977) and R a y m a n et al. (1975), and was probably due to differences in the criterion used to judge coagulase production. Those workers recorded a higher correlation because they worked with organisms that produced either 4+ or 3+ clots. In the work reported here any coagulum, irrespective of the degree of clotting, that did not disintegrate when the tubes were tilted, was regarded as coagulase positive. It is possible that those isolates with lower than 3+ clots did not produce thermonuclease. R a y m a n et al. (1975) found that isolates with 2+ or lower coagulase reactions were invariably negative for thermonuclease production. The much lower correlations between thermonuclease production and coagulase formation by isolates from cattle, goats and sheep may have a similar reason, but is more probably due to the fact that most of these isolates were n o t actually thermonuclease-positive. This was further supported by the fact t h a t most isolates from these animals were not agglutinated by sera 17H and 61218, indicating that they were neither h u m a n nor canine biotypes and thus were probably biotypes of the host animal species (H~jek and Marsalek, 1971). The majority of these nonagglutinated isolates did not produce thermonuclease. Therefore, most isolates of S. a u r e u s from cattle, sheep and goats can be regarded as being negative for thermonuclease production. The production of thermonuclease by a greater percentage of clinical than non-clinical h u m a n isolates suggests that thermonuclease production may be a property of clinical isolates rather than those from the environment. This is further supported in this work by the fact that a greater percentage of clinical canine isolates produced thermonuclease than those from the nasal passages of dogs. In the assessment of the thermonuclease test to identify pathogenic and toxigenic S. a u r e u s , various closely related suggestions have been made by authors like Zarzour and Belle (1977) and R a y m a n et al. (1975). They propose that the thermonuclease test should be performed on isolates with a doubtful coagulase result. This is certainly a useful suggestion except that R a y m a n et al. (1975) found t h a t presumptive staphylococcal isolates from food yielding 2+ or lower coagulase reactions were negative for nuclease production. Coupled with the findings in this work that less than 50% of non-clinical h u m a n and canine biotypes of S. a u r e u s produced thermonuclease, and that isolates f~om cattle, sheep, goats and horses are poor producers of this enzyme, lack of its production, even by isolates with d o u b t f u l coagulase reactions, would n o t justify classifying t h e m as not
aureus
277 S. a u r e u s micrococci. This is further confirmed by the fact that Rayman et
al. (1975) found that 2+ or less coagulase-positive isolates were thermonuclease-negative, and Zarzour and Belle (1977) found all their 2+ coagulase positive. Thermonuclease production has also been reported by some authors, notably Tatini (1981), as a better indicator of enterotoxin than the coagulase test. In the light of the present findings, care must be taken to avoid false negative results when thermonuclease is used to indicate toxigenicity with S. a u r e u s , especially if there is the possibility that the organisms may be animal biotypes, some of which are known to be toxigenic (Adekeye, 1981). It is concluded from this study that clinical human and canine biotypes of S. a u r e u s are greater producers of thermonuclease than their non-clinical counterparts and that animal isolates, particularly from cattle, sheep and goats, are poor producers of the enzyme. While thermonuclease testing of doubtful coagulase positive isolates of S. aureus, as suggested by some authors, may be helpful in confirming the identity of the organisms, those which do not produce the enzyme may still be S. a u r e u s of animal origin. REFERENCES Adekeye, J.D., 1981. Epidemiological study of human and canine S t a p h y l o c o c c u s aureus in Nigeria by serological means. In: J. Jeljaszewicz (Editor), Staphylococci and Staphylococcal Infections. Zentralbl. Bakteriol. Suppl. 10, Gustav Fischer Verlag, Stuttgart, New York, pp. 991--995. Chesbro, W.R. and Auborn, K., 1967. Enzymatic detection of the growth of S t a p h y l o coccus aureus in foods. Appl. Microbiol., 15: 1150--1159. Cunningham, L., Catlin, B.W. and Privat de Garilhe, 1956. A deoxyribonuclease of Micrococcus pyogenes. J. Am. Schem. Soc., 78: 4642--4645. Elek, S.D., 1959. S t a p h y l o c o c c u s p y o g e n e s and its Relation to Disease. Livingstone, London. Fluharty, D.M., Spencer, G.R., Buttermore, R.M. and Cherrington, V.A., 1966. Effects of exposure to animals and antibiotics on the staphylococci infection of veterinary hospital personnel. Am. J. Vet. Res., 27: 1113--1122. Grun, L., 1958. lJber ein neues Staphylokokken-Antigen. Z. Hyg. Infektionskr., 145: 259--262. Hfijek, V. and Marsalek, E., 1971. The differentiation of pathogenic Staphylococci and suggestion for their taxonomic classification. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1: Orig. Reihe A, 217: 176--182. Hofstad, T., 1964. Studies on the antigenic structure of the 80/81 complex of S t a p h y lococcus aureus. Acta Pathol. Microbiol. Scand., 61: 558--570. Lachica, R.V.F., Genigeorgis, C. and Hoeprich, P.D., 1971. Metachromatic agar-diffusion methods for detecting staphyloccoccal nuclease activity. Appl. Microbiol., 21: 585-587. Live, I., 1975. Agglutinating serum for distinguishing S t a p h y l o c o c c u s aureus of human biotype. Infect. Immun. 12: 443--445. Oeding, P., 1960. Antigenic properties of S t a p h y l o c o c c u s aureus. Bacteriol. Rev., 24: 374--396. Pillet, J., Orta, B., Corrieras, F. and Petillon, C., 1968. Etude serologique de 61 souches de Staphylocoques isolates de l~sions cutan~es chez le chien. Mise en ~vidence d'un nouveau serotype. Ann. Inst. Pasteur, Paris 114: 658--688.
278 Rayman, M.K., Park, C.E., Philpott, J. and Todd, E.C.D., 1975. Reassessment of the coagulase and thermostable nucelase tests as means of identifying S t a p h y l o c o c c u s aureus. Appl. Microbiol., 29: 451--454. Sperber, W.H. and Tatini, S.R., 1975. Interpretation of the tube coagulase test for identification of S t a p h y l o c o c c u s aureus. Appl. Microbiol., 29: 502--505. Subcommittee on T a x o n o m y on Staphylococci and Micrococci. 1965. Recommendations of the subcommittee. Int. Bull. Bacteriol. Nomencl. Taxon, 15: 109--110. Tatini, S.R., 1981. Thermonuclease as an indicator of Staphylococcal enterotoxins in food. In: R.L. Ory (Editor), Antinutrients and Natural Toxicants in Foods. Food Nutrition Press, Westport, U.S.A., pp. 53--75. Yrios, J.W., 1976. Comparison of rabbit and pig plasma in the tube coagulase test. J. Clin. Microbiol., 5: 221--224. Zarzour, J.Y. and Belle, E.A., 1977. Evaluation of three test procedures for identification of S t a p h y l o c o c c u s aureus from clinical sources. J. Clin. Microbiol., 7: 133--135.
271
RELIABILITY OF THERMONUCLEASE PRODUCTION FOR THE IDENTIFICATION OF HUMAN AND ANIMAL STAPHYLOCOCCUS A U R E US
J.D. ADEKEYE
Department of Veterinary Pathology and Microbiology, Ahmadu Bello University, Zaria (Nigeria) (Accepted 9 December 1983)
ABSTRACT Adekeye, J.D., 1984. Reliability of thermonuclease production for the identification of human and animal Staphylococcus aureus. Vet. Microbiol., 9: 271--278. A total of 314 clinical and non-clinical isolates of the genus Staphylococcus was tested for coagulase production and glucose and mannitol fermentation. The isolates were tested for thermonuclease production and agglutination by sera 17H and 61218, which were specific for human and canine S. aureus biotypes, respectively. All produced coagulase and fermented glucose. A majority fermented mannitol anaerobically except for the canine isolates. A majority of human isolates produced thermonuclease (64.3%) and most were agglutinated by serum 17H. There was good correlation between thermonuclease production and agglutination by serum 17H of human and bovine clinical isolates (86.6 and 80%, respectively). This was also true of clinical canine isolates agglutinated by serum 61218, of which 75% were thermonuclease-positive. Over half of canine isolates (52.8%) were thermonuclease-positive and most were agglutinated by serum 61218. Bovine and caprine isolates were 34.1 and 25% thermonuclease-positive, respectively, while ovine isolates were only 14.2% thermonuclease-positive. Isolates from these ruminant sources were also poorly agglutinated by either serum. It was concluded that a greater number of clinical human and canine biotypes of S. aureus produced thermonuclease than their nonclinical isolates, and that a majority of other animal isolates were negative for thermonuclease. Therefore, the thermonuclease test may not be very useful for confirming the animal origin of S. aureus isolates.
INTRODUCTION F o l l o w i n g t h e d e m o n s t r a t i o n b y C u n n i n g h a m e t al. ( 1 9 5 6 ) , o f t h e p r o d u c t i o n o f t h e r m o s t a b l e d e o x y r i b o n u c l e a s e as a u n i q u e p r o p e r t y o f S t a p h y lococcus aureus, m a n y w o r k e r s ( C h e s b r o a n d A u b o r n , 1 9 6 7 ; S p e r b e r a n d Tatini, 1975; Yrios, 1976; Zarzour and Belle, 1977) have evaluated the relationship between the production of thermostable nuclease, enterotoxin production and coagulase formation. They concluded that thermonuclease p r o d u c t i o n c a n b e u s e d as a c o n f i r m a t o r y t e s t f o r t h e i d e n t i f i c a t i o n o f t o x i g e n i c a n d p a t h o g e n i c S. aureus. H o w e v e r , m o s t o f t h e S. aureus i s o l a t e s t h e y
0378-1135/84/$03.00
© 1984 Elsevier Science Publishers B.V.
272 tested were obtained either from h u m a n clinical materials or from foods possibly contaminated by S. a u r e u s from people. The same test has not been extensively used to verify whether or not such a conclusion can also be drawn in respect of S. a u r e u s isolates from clinical and non-clinical animal sources. To date, the most widely used criterion for distinguishing toxigenic and pathogenic S. a u r e u s from closely related saprophytic organism is the coagulase test. Although the result of the test can be affected by factors such as type of plasma used, the nature of the anticoagulant and possible loss of the characteristic on rare occasions by S. a u r e u s , m a n y laboratories rely solely on coagulase testing. Furthermore, in attempts to identify S. a u r e u s of different biotypes, Oeding (1960) and Pillet et al. (1968) obtained a typing serum designated 61218 which was considered specific for canine isolates of S. aureus. Similarly, Live (1975) produced an agglutinating serum designated 17H which is specific for thermostable agglutination of S. a u r e u s of h u m a n biotype. Since the antigenic properties of S. a u r e u s are stable (Griin, 1958; Hofstad and Oeding, 1960), the use of these sera may help to identify h u m a n and canine biotypes of S. a u r e u s from different sources. The purpose of this work was to evaluate the reliability of thermonuclease in confirming h u m a n and animal, clinical and non-clinical S. a u r e u s detected by coagulase and agglutination tests. MATERIALS AND METHODS B a c t e r i a l isolates
A total of 314 S. a u r e u s isolates obtained from clinical and non-clinical specimens from humans and others from animals was examined. Clinical isolates from h u m a n beings were from specimens for routine bacteriological culture submitted to the University Teaching hospitals at Zaria and Ibadan. Non-clinical isolates were from nose swabs of h u m a n volunteers who were apparently healthy. Clinical isolates from animals were from specimens submitted for bacteriological culture at the A h m a d u Bello University Veterinary Hospital. The non-clinical isolates were obtained from either nasal swabs from animals brought to the Veterinary Clinic for routine vaccinations, or obtained during ambulatory services. All the isolates were obtained from specimens cultured on blood agar plates incubated for 18--24 h at 37°C, and identified primarily as Gram-positive, catalase positive cocci. C o a g u l a s e test
Isolates from blood agar plates were subcultured in nutrient broth and incubated overnight at 27°C. Five drops (approximately 0.125 ml) of each overnight-culture were added to test-tubes (16 X 100 mm) containing 0.5 ml of one-fifth concentrations of rabbit (DIFCO) plasma, The tubes were incubated at 37°C and examined for coagulase formation at 4, 6, 12 and 24
273 h. A solid coagulum which did n o t fragment when the tubes were tilted was a positive test. Carbohydrate fermentation Fermentation of glucose and mannitol under anaerobic conditions was determined according to the recommendations of the Subcommittee on Taxo n o m y of Staphylococci and Micrococci (1965). Preparation and absorption o f antisera These were performed as described by Live (1975)using S. aureus strains 17, Cowan 1 and 61218. Cowan 1 is S. aureus Cowan serologic type strain 1. This is one of the 13 international-type strains of S. aureus in group sera (a, b, c, e) (Oeding, 1960), and was kindly supplied by I. Live. Slide agglutination tests These were carried out as described by Live (1975) using antisera 17H and 61218. Thermostable nuclease test Production of thermostable nuclease by the isolates was determined by the methods of Lachica et al. (1971) and Sperber and Tatini (1975). The toluidine blue-0-deoxyribonucleic acid agar used was prepared as described by Zarzour and Belle (1978). Fifteen ml of the agar was poured into a petri dish (100 X 15 mm). Twenty wells were cut in the agar with the tip of a 2 ml plastic syringe. Each well was filled with an overnight brain--heart infusion broth-culture of an isolate which had been steamed for 15 min at 100°C. Each plate was then placed in 150 X 15 mm petri dishes, the bottoms of which were covered with filter papers moistened with water. The plates were incubated at 37°C and observed at 1, 4 and 24 h. Organisms with bright pink zones surrounding the test-wells were regarded as thermostable deoxyribonuclease positive. RESULTS All the 314 Gram-positive, catalase-positive cocci were coagulase-positive and fermented glucose. Most of them, except canine isolates, fermented mannitol anaerobically. Table I shows the reactions of the isolates when agglutinated with sera 17H and 61218, compared with their thermonuclease production. Isolates from h u m a n beings had the highest frequency of thermonuclease production (64.3%), followed by dog isolates (52.8%). Bovine and caprine isolates were less then 50% thermonuclease-positive, while ovine isolates were only 14.2% thermonuclease-positive.
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The number of isolates which were agglutinated by serum 17H was generally n o t very high regardless of source, but h u m a n isolates were most frequently agglutinated (57% of the clinical isolates and 47% of the non-clinical isolates). Agglutination of animal isolates of S. a u r e u s by the same serum was poor (35% of canine isolates down to 0% of caprine clinical isolates). Of all the clinical isolates from different sources, only those from h u m a n (57%) and bovine (23.3%) sources were agglutinated by serum 17H. Non-clinical isolates from h u m a n and animal sources were agglutinated by the serum at varying degrees. Again, human isolates were most frequently agglutinated (46.8%) and ovine isolates were the least (11.8%). The clinical isolates which were agglutinated by serum 17H were much better producers of thermonuclease than their non-clinical counterparts. There was a very high agreement between agglutinability by serum 17H and thermonuclease production by the clinical isolates from h u m a n and cattle (86.6 and 80%, respectively}. Clinical and non-clinical isolates of S. a u r e u s from all the sources examined were poorly agglutinated by serum 61218 except clinical isolates from dogs, of which 100% were agglutinated. Of these isolates, 75% were thermonuclease-positive, but other isolates also agglutinated by serum 61218 were poor producers of thermonuclease except those from humans, of which 50% were positive. Non-agglutinability by either serum was very high with isolates from sheep, goat and cattle; 82--100%, 75--87% and 54--58%, respectively. The majority of isolates (75%) from other sources (mainly horses) were also not agglutinated. Very few non-agglutinable animal isolates produced thermonuclease apart from the canine isolates. DISCUSSION
Coagulase production by a member of the genus S t a p h y l o c o c c u s is usually accepted as proof that it is S. a u r e u s . Anaerobic utilization of glucose and mannitol are other properties of S. aureus. In this work, all the isolates investigated had those characteristics, except for a n u m b e r of the canine isolates which were negative for mannitol fermentation. Furthermore, because of the specificity of sera 17H and 61218 (Live, 1975), and the stability of the antigenic properties of S. a u r e u s (Grun, 1958; Oeding, 1960; Hofstad, 1964}, isolates agglutinated by serum 17H were regarded as h u m a n biotypes and those by serum 61218 were regarded as canine biotypes, irrespective of their sources. This was further supported in this work by the fact that most isolates agglutinated by serum 17H were of h u m a n origin and those agglutinated by serum 61218 were of canine origin. Few isolates from other sources were agglutinated and any that were agglutinated were regarded as human and canine biotypes t h a t had contaminated other animals. Generally the percentage of isolates agglutinated by either serum was not very high with either h u m a n or canine isolates. This was because their hosts probably carried large numbers of S. a u r e u s of other biotypes, contracted from contaminated environments jointly shared by animals and people -- a situation very com-
276 m o n in Nigeria (Adekeye, 1981). The scarcity of human biotypes of S. from clinical specimens, apart from humans and a few from cattle, supports the general belief expressed by some authors that different biotypes produce disease mainly in their homologous hosts (Elek, 1959; Fluharty et al., 1966). Direct correlation of anaerobic utilization of glucose and coagulase production with thermonuclease production was 64.3% for h u m a n isolates. This was less than the findings of other workers like Zarzour et al. (1977) and R a y m a n et al. (1975), and was probably due to differences in the criterion used to judge coagulase production. Those workers recorded a higher correlation because they worked with organisms that produced either 4+ or 3+ clots. In the work reported here any coagulum, irrespective of the degree of clotting, that did not disintegrate when the tubes were tilted, was regarded as coagulase positive. It is possible that those isolates with lower than 3+ clots did not produce thermonuclease. R a y m a n et al. (1975) found that isolates with 2+ or lower coagulase reactions were invariably negative for thermonuclease production. The much lower correlations between thermonuclease production and coagulase formation by isolates from cattle, goats and sheep may have a similar reason, but is more probably due to the fact that most of these isolates were n o t actually thermonuclease-positive. This was further supported by the fact t h a t most isolates from these animals were not agglutinated by sera 17H and 61218, indicating that they were neither h u m a n nor canine biotypes and thus were probably biotypes of the host animal species (H~jek and Marsalek, 1971). The majority of these nonagglutinated isolates did not produce thermonuclease. Therefore, most isolates of S. a u r e u s from cattle, sheep and goats can be regarded as being negative for thermonuclease production. The production of thermonuclease by a greater percentage of clinical than non-clinical h u m a n isolates suggests that thermonuclease production may be a property of clinical isolates rather than those from the environment. This is further supported in this work by the fact that a greater percentage of clinical canine isolates produced thermonuclease than those from the nasal passages of dogs. In the assessment of the thermonuclease test to identify pathogenic and toxigenic S. a u r e u s , various closely related suggestions have been made by authors like Zarzour and Belle (1977) and R a y m a n et al. (1975). They propose that the thermonuclease test should be performed on isolates with a doubtful coagulase result. This is certainly a useful suggestion except that R a y m a n et al. (1975) found t h a t presumptive staphylococcal isolates from food yielding 2+ or lower coagulase reactions were negative for nuclease production. Coupled with the findings in this work that less than 50% of non-clinical h u m a n and canine biotypes of S. a u r e u s produced thermonuclease, and that isolates f~om cattle, sheep, goats and horses are poor producers of this enzyme, lack of its production, even by isolates with d o u b t f u l coagulase reactions, would n o t justify classifying t h e m as not
aureus
277 S. a u r e u s micrococci. This is further confirmed by the fact that Rayman et
al. (1975) found that 2+ or less coagulase-positive isolates were thermonuclease-negative, and Zarzour and Belle (1977) found all their 2+ coagulase positive. Thermonuclease production has also been reported by some authors, notably Tatini (1981), as a better indicator of enterotoxin than the coagulase test. In the light of the present findings, care must be taken to avoid false negative results when thermonuclease is used to indicate toxigenicity with S. a u r e u s , especially if there is the possibility that the organisms may be animal biotypes, some of which are known to be toxigenic (Adekeye, 1981). It is concluded from this study that clinical human and canine biotypes of S. a u r e u s are greater producers of thermonuclease than their non-clinical counterparts and that animal isolates, particularly from cattle, sheep and goats, are poor producers of the enzyme. While thermonuclease testing of doubtful coagulase positive isolates of S. aureus, as suggested by some authors, may be helpful in confirming the identity of the organisms, those which do not produce the enzyme may still be S. a u r e u s of animal origin. REFERENCES Adekeye, J.D., 1981. Epidemiological study of human and canine S t a p h y l o c o c c u s aureus in Nigeria by serological means. In: J. Jeljaszewicz (Editor), Staphylococci and Staphylococcal Infections. Zentralbl. Bakteriol. Suppl. 10, Gustav Fischer Verlag, Stuttgart, New York, pp. 991--995. Chesbro, W.R. and Auborn, K., 1967. Enzymatic detection of the growth of S t a p h y l o coccus aureus in foods. Appl. Microbiol., 15: 1150--1159. Cunningham, L., Catlin, B.W. and Privat de Garilhe, 1956. A deoxyribonuclease of Micrococcus pyogenes. J. Am. Schem. Soc., 78: 4642--4645. Elek, S.D., 1959. S t a p h y l o c o c c u s p y o g e n e s and its Relation to Disease. Livingstone, London. Fluharty, D.M., Spencer, G.R., Buttermore, R.M. and Cherrington, V.A., 1966. Effects of exposure to animals and antibiotics on the staphylococci infection of veterinary hospital personnel. Am. J. Vet. Res., 27: 1113--1122. Grun, L., 1958. lJber ein neues Staphylokokken-Antigen. Z. Hyg. Infektionskr., 145: 259--262. Hfijek, V. and Marsalek, E., 1971. The differentiation of pathogenic Staphylococci and suggestion for their taxonomic classification. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1: Orig. Reihe A, 217: 176--182. Hofstad, T., 1964. Studies on the antigenic structure of the 80/81 complex of S t a p h y lococcus aureus. Acta Pathol. Microbiol. Scand., 61: 558--570. Lachica, R.V.F., Genigeorgis, C. and Hoeprich, P.D., 1971. Metachromatic agar-diffusion methods for detecting staphyloccoccal nuclease activity. Appl. Microbiol., 21: 585-587. Live, I., 1975. Agglutinating serum for distinguishing S t a p h y l o c o c c u s aureus of human biotype. Infect. Immun. 12: 443--445. Oeding, P., 1960. Antigenic properties of S t a p h y l o c o c c u s aureus. Bacteriol. Rev., 24: 374--396. Pillet, J., Orta, B., Corrieras, F. and Petillon, C., 1968. Etude serologique de 61 souches de Staphylocoques isolates de l~sions cutan~es chez le chien. Mise en ~vidence d'un nouveau serotype. Ann. Inst. Pasteur, Paris 114: 658--688.
278 Rayman, M.K., Park, C.E., Philpott, J. and Todd, E.C.D., 1975. Reassessment of the coagulase and thermostable nucelase tests as means of identifying S t a p h y l o c o c c u s aureus. Appl. Microbiol., 29: 451--454. Sperber, W.H. and Tatini, S.R., 1975. Interpretation of the tube coagulase test for identification of S t a p h y l o c o c c u s aureus. Appl. Microbiol., 29: 502--505. Subcommittee on T a x o n o m y on Staphylococci and Micrococci. 1965. Recommendations of the subcommittee. Int. Bull. Bacteriol. Nomencl. Taxon, 15: 109--110. Tatini, S.R., 1981. Thermonuclease as an indicator of Staphylococcal enterotoxins in food. In: R.L. Ory (Editor), Antinutrients and Natural Toxicants in Foods. Food Nutrition Press, Westport, U.S.A., pp. 53--75. Yrios, J.W., 1976. Comparison of rabbit and pig plasma in the tube coagulase test. J. Clin. Microbiol., 5: 221--224. Zarzour, J.Y. and Belle, E.A., 1977. Evaluation of three test procedures for identification of S t a p h y l o c o c c u s aureus from clinical sources. J. Clin. Microbiol., 7: 133--135.