A Comparison of Methods and the Validity of Deoxyribonuclease Tests for the Characterization of Staphylococci Isolated from Animals

Strains isolated from pigeons belonging to the coagulase-positive species Staphylococcus intermedius , coagulase-negative Staph. hyicus subsp. chromogenes strains from cattle and pigs, and Staph. aureus strains from poultry, gave weakly positive reactions in DNase plate culture tests and heat-resistant DNase tests. Staph. aureus and Staph. intermedius strains from other sources and coagulase-negative and coagulase-positive Staph. hyicus subsp. hyicus strains reacted strongly in these tests. A standardized plate culture test procedure is proposed and the use of DNase tests in the identification of staphylococci isolated from animals is discussed.     

Application of current methods for isolation and identification of staphylococci in raw bovine milk

Samples of raw milk were examined for counts of somatic cells, total viable bacteria, staphylococci (Schleifer & Kramer's medium) and Staphylococcus aureus (Baird-Parker medium, Baird-Parker medium with pig plasma and Baird-Parker medium with additional antibiotics). For the isolation of staphylococci from raw milk, Schleifer & Kramer's medium was found to be very selective and in general performed satisfactorily. From the results obtained with the three remaining media the continued use of Baird-Parker medium for isolation of Staph. aureus from raw milk is recommended with the proviso that colonies selected for identification should include those that clear and do not clear the egg yolk and are not limited to colonies with diameters greater than 1 mm. Staphylococci isolated from raw milk were identified by key tests using a multipoint inoculation procedure. A selected number were also examined by the API STAPH system in conjunction with the API LAB computer programme for identification of staphylococci. Of the staphylococci examined, 90.0% were identified using the multipoint procedure. For strains identified as Staph. aureus, Staph. hyicus subsp. hyicus, Staph. epidermidis, Staph. simulans, Staph. xylosus or members of the Staph. hominis/Staph. warneri/Staph haemolyticus group, the API system provided confirmatory evidence. With strains identified by the multipoint procedure as Staph. hyicus subsp. chromogenes, Staph. sciuri subsp. sciuri and Staph. sciuri subsp. lentus the API system did not always provide concurring results. Several strains which could not be identified by the multipoint procedure could be identified by the API system. Staph. aureus, Staph. hyicus subsp. hyicus and Staph. hyicus subsp. chromogenes strains isolated from milk were examined for production of enterotoxin A-E. Only 3.9% of Staph. aureus strains examined produced detectable enterotoxin (type C). None of the Staph. hyicus subsp. hyicus or Staph. hyicus subsp. chromogenes strains produced any of the known enterotoxins.     

Polar lipid and isoprenoid quinone composition in the classification of Staphylococcus

Representatives of 13 species of Staphylococcus were examined using a small-scale procedure for the sequential extraction of isoprenoid quinones and polar lipids. Menaquinones were the only isoprenoid quinones found in the 77 test strains which were divided into three groups based upon the predominant isoprenologue detected: (i) S. hyicus subsp. hyicus, S. sciuri subsp. lentus and S. sciuri subsp. sciuri contained unsaturated menaquinones with six isoprene units; (ii) S. capitis, S. cohnii, S. epidermidis, S. haemolyticus, S. hominis, S. hyicus subsp. chromogenes, S. intermedius, S. saprophyticus, S. simulans, S. warneri and S. xylosus contained unsaturated menaquinones with seven isoprene units and (iii) S. aureus contained unsaturated menaquinones with eight isoprene units and varying amounts of the corresponding lower isoprenologue. All of the organisms contained very similar polar lipid patterns consisting of diphosphatidylglycerol, phosphatidylglycerol, beta-gentiobiosyl diacylglycerol and a number of glycolipids and phospholipids. One of the glycolipids was chromatographically indistinguishable from beta-gentiotriosyl diacylglycerol. Lysylphosphatidylglycerol was a major component in S. aureus and S. intermedius but was usually present in minor amounts in the coagulase-negative strains. The polar lipid data underline the homogeneity of the genus Staphylococcus and distinguish staphylococci from aerobic, Gram-positive cocci and from the phylogenetically related aerobic, endospore-forming bacteria. Menaquinone composition can also be used to separate staphylococci from other aerobic, Gram-positive cocci.     

Characterization and identification of coagulase-negative, heat-stable deoxyribonuclease-positive staphylococci

Various characteristics of 13 coagulase-negative, weakly heat-stable deoxyribonuclease-positive staphylococci from human, veterinary and food sources were determined in an effort to identify them. Nine of the isolates were identified as coagulase-negative Staphylococcus aureus (2), Staphylococcus xylosus (2), Staphylococcus simulans (3), Staphylococcus capitis (1) and Staphylococcus sciuri subsp. lentus (1); the other four isolates, from food and veterinary sources, could not be identified as currently accepted or proposed species. Teichoic acid and peptidoglycan compositions were used as key taxonomic characteristics. The determination of heat-stable deoxyribonuclease activity can be useful to detect coagulase-negative S. aureus strains. However, this activity also appears to be present in strains of other staphylococcal species.     

Antagonistic activities of coagulase-positive staphylococci

Antagonistic activities were investigated by Frédérioq's plate method in 1,014 coagulase-positive staphylococcal strains of different species and origins. Staphylococcin effect was demonstrated in 12 (2%) of the 559 Staphylococcus aureus strains, in 51 (18%) of the 283 S. intermedius strains, and in 1 (3%) of the 36 S. hyicus subsp. chromogenes strains. Lysostaphin was identified in the 15 (5%) S. intermedius strains. In addition, heat-stable bacteriostatic agent was detected in 45 (33%) and heat-labile bacteriolytic agent in 7 (5%) of the 136 S. hyicus subsp. hyicus strains. An attempt was made at antagonistic activity typing in all of the active staphylococcal strains.     

Enterotoxin production by atypical Staphylococcus aureus from poultry*

Phenotypically typical Staphylococcus aureus was isolated frequently from the necrotic bone and liver of poultry suffering from femoral head necrosis. Occasionally strains were isolated that differed from typical Staph. aureus in one or more of the major diagnostic tests, i.e. coagulase production, anaerobic fermentation of mannitol and production of a heat-stable deoxyribonuclease. Such atypical strains were also isolated from nasal swabs of healthy birds. Tests for enterotoxin production demonstrated that some atypical strains from both sick and healthy birds are capable of producing staphylococcal enterotoxins.     

Additional differentiating characters of the two subspecies of Staphylococcus hyicus

Forty-five strains of Staphylococcus hyicus subsp. hyicus and 36 strains of S. hyicus subsp. chromogenes were examined for bacteriolytic activity with the same assay system previously used in taxonomic studies on staphylococci. The two subspecies differed from each other chiefly in that for optimal lytic activity S. hyicus subsp. hyicus strains required a higher salt concentration in the test medium than S. hyicus subsp. chromogenes strains. The lack of lytic activity on B15TP1 medium was a major difference between S. hyicus and S. aureus, and the lack of activity on TP2P medium was a major difference between S. hyicus and S. intermedius. Penicillin-binding proteins (PBPs) were studied in 40 S. hyicus strains. The S. hyicus subsp. hyicus strains had only one PBP (mol. wt 79 000) while the S. hyicus subsp. chromogenes strains had three distinct PBPs (mol. wts 84 000, 82 000 and 79 000).     

Enterotoxigenicity of Staphylococcus strains isolated from Spanish dry-cured hams.

The ability of 135 Staphylococcus strains isolated from Spanish dry-cured hams to produce enterotoxins in culture was investigated by the reversed passive latex agglutination method. A high percentage of enterotoxigenic Staphylococcus aureus strains (85.9%) was recorded, and 54.3% of these produced enterotoxin A. One of the two Staphylococcus epidermidis strains produced enterotoxin C. The reversed passive latex agglutination method yielded satisfactory results.     

Enterotoxigenicity of Staphylococcus strains isolated from Spanish dry-cured hams.

The ability of 135 Staphylococcus strains isolated from Spanish dry-cured hams to produce enterotoxins in culture was investigated by the reversed passive latex agglutination method. A high percentage of enterotoxigenic Staphylococcus aureus strains (85.9%) was recorded, and 54.3% of these produced enterotoxin A. One of the two Staphylococcus epidermidis strains produced enterotoxin C. The reversed passive latex agglutination method yielded satisfactory results.     

A quantitative study of enterotoxin production by sheep milk staphylococci

Of 124 staphylococcal strains isolated from sheep milk, 78 produced enterotoxin A, B, C, or D when evaluated by an enzyme-linked immunosorbent assay. Enterotoxins A and D, elaborated by 44 and 43 strains, respectively, showed the highest incidence. Enterotoxin production by coagulase-negative strains (one Staphylococcus cohnii, three S. epidermidis, five S. haemolyticus, and four S. xylosus) was detected. Linear and logarithmic-logarithmic regressions of optical density on enterotoxin concentration yielded the best-fitting equations for enterotoxin quantitation. A significantly higher incidence of enterotoxin producers and significantly higher levels of enterotoxins produced were recorded for coagulase-positive, thermostable nuclease-positive, hemolysis-positive, or mannitol-positive strains. Mannitol utilization was the best test for discriminating between enterotoxigenic and nonenterotoxigenic staphylococci.     

A quantitative study of enterotoxin production by sheep milk staphylococci.

Of 124 staphylococcal strains isolated from sheep milk, 78 produced enterotoxin A, B, C, or D when evaluated by an enzyme-linked immunosorbent assay. Enterotoxins A and D, elaborated by 44 and 43 strains, respectively, showed the highest incidence. Enterotoxin production by coagulase-negative strains (one Staphylococcus cohnii, three S. epidermidis, five S. haemolyticus, and four S. xylosus) was detected. Linear and logarithmic-logarithmic regressions of optical density on enterotoxin concentration yielded the best-fitting equations for enterotoxin quantitation. A significantly higher incidence of enterotoxin producers and significantly higher levels of enterotoxins produced were recorded for coagulase-positive, thermostable nuclease-positive, hemolysis-positive, or mannitol-positive strains. Mannitol utilization was the best test for discriminating between enterotoxigenic and nonenterotoxigenic staphylococci.     

Coagulase and Heat-resistant Nuclease Producing Staphylococcus epidermidis Strains from Animals

Strains isolated from the skin and the upper respiratory tract of chickens and from bovine udders showed characteristics typical for Staphylococcus epidermidis biotype 2, but produced a heat-resistant nuclease (DNase) and reacted in the tube coagulase test. The strains were physiologically, biochemically and serologically indistinguishable from Staphylococcus hyicus strains isolated from swine. However, in contrast to S. hyicus , they appeared to be devoid of pathogenicity when tested in colostrum deprived piglets. The poultry strains occur frequently as skin inhabitants.     

Chemical composition and structure of cell wall teichoic acids of staphylococci

The cell wall teichoic acid structures of 22 staphylococci including 13 type strains were determined. Most of the strains contain a poly(polyolphosphate) teichoic acid with glycerol and/or ribitol as polyol component. The polyolphosphate backbone is partially substituted with various combinations of sugars and/or amino sugars. Most of the substituents occur in a monomeric form but some strains also contain dimers of N-acetylglucosamine as substituents. Staphylococcus hyicus subsp. hyicus NCTC 10350 and S. sciuri DSM 20352 revealed rather complex cell wall teichoic acids. They consist of repeating sequences of phosphate-glycerol-phosphate-N-acetylglucosamine. The amino sugar component is present in this case as a monomer or an oligomer (n less than or equal to 3). Moreover, the glycerol residues are partially substituted with N-acetylglucosamine. The cell wall teichoic acid of S. auricularis is a poly(N-acetylglucosaminyl-phosphate) polymer similar to that found in S. caseolyticus ATCC29750. The cell wall teichoic acid structures for type strains of S. auricularis, S. capitis, S. cohnii, S. haemolyticus, S. hominis, S. hyicus subsp. hyicus, S. sciuri, S. xylosus and S. warneri were determined for the first time in detail. The structures of some of the previously described teichoic acids had to be revised (S. epidermidis, S. simulans, S. aureus phage type 187).     

Identification of Micrococcaceae in Clinical Bacteriology

The cellular morphology, identifying physiological characteristics, and a key to the human genera of Micrococcaceae are presented with flow charts for identification of aerobic and anaerobic isolates. These flow charts can be amended as desired, depending upon the degree of accuracy desired. Micrococcaceae isolates in a 350-bed private general hospital during a 15-week period are tabulated to show relative numbers of the different genera and species, with their probable relationship to infection or contamination. Only 11 of the 220 Micrococcaceae isolates were not Staphylococcus; no Sarcina or Peptococcus were isolated. Of the Staphylococcus isolates, 61% were S. epidermidis. Almost 18% of the S. aureus isolates were coagulase-negative. Of the S. aureus isolates, 80% of the coagulase-positive isolates were infecting agents, as were 67% of the coagulase-negative S. aureus isolates, compared to only 48% of S. epidermidis isolates. Two of four Gaffkya isolates but only one of seven Micrococcus isolates were infecting agents. If coagulase production is used as the sole criterion for speciation of staphylococci, and Micrococcus is not differentiated from Staphylococcus, the term "coagulase-negative staphylococci" does not differentiate three distinct levels of pathogenicity. Coagulase-negative S. aureus is more virulent than S. epidermidis or Gaffkya, which are more virulent than Micrococcus or Sarcina.     

Evaluation of six commercial identification kits for the identification of Staphylococcus aureus isolated from bovine mastitis

AIMS: Comparison of six commercially available in human medicine well-established slide agglutination systems for the identification of Staphylococcus aureus. METHODS AND RESULTS: Slide agglutination tests were compared with the conventional tube coagulase test, biochemical identification and with the molecular identification by polymerase chain reaction (PCR) amplification of species-specific parts of the gene encoding the 23S RNA. Systems evaluated included Masta-Staph (Mast Diagnostics), Staphylase-Test (Oxoid), Staphytect-Plus (Oxoid), Staphyloslide Latex (Becton Dickinson), Slidex Staph Plus (bioMerieux) and Dry Spot Staphytect Plus (Oxoid). A total of 141 staphylococcal strains isolated from cases of bovine mastitis including 90 S. aureus, 14 Staphylococcus epidermidis, 10 Staphylococcus warneri, 13 Staphylococcus xylosus, 11 Staphylococcus haemolyticus and three other coagulase-negative staphylococci were tested with each method. Staphylococcus aureus strains were selected by macrorestriction analysis with pulsed field gel electrophoresis (PFGE). Only genetically unrelated strains were included in the study. The sensitivities and specificities of the test were as follows: Masta-Staph 86.7 and 90.1%, Staphylase-Test 78.4 and 85.1%, Staphytect-Plus 81.1 and 86.5%, Staphyloslide Latex 77.8 and 84.4%, Slidex Staph Plus 77.8 and 84.4%, Dry Spot Staphytect Plus 75.6 and 83.0%. CONCLUSIONS: The results of this evaluation suggest that the six slide agglutination methods tested can provide rapid identification of S. aureus also from bovine mastitis. The sensitivity and specificity seems to be less than those reported from human S. aureus isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first comparative reported investigations about the applicability of different commercially available slide agglutination tests for the detection of S. aureus from bovine mastitis using PFGE selected clinical isolates.     

Enterotoxin production by staphylococci isolated from healthy goats

The ability of 342 staphylococcal isolates from different anatomical sites in healthy goats to produce staphylococcal enterotoxins (SE) was investigated. SE were produced by 74.3% of the 70 coagulase-positive strains and by 22% of the coagulase-negative strains studied. Most enterotoxigenic strains were isolated from the skin of udders and teats and from milk. SEC was the SE type most frequently produced, either alone (67.9%) or in combination with others. Five coagulase-negative species not previously reported as SE producers were identified (Staphylococcus chromogenes, S. warneri, S. sciuri, S. saprophyticus, and S. lentus). SEA, SEB, and SEC were detected in the milk of 17 of the 133 healthy goats studied. These results suggest that the goat is an important reservoir of enterotoxigenic staphylococci, most of which produce SEC.     

Enterotoxin production by staphylococci isolated from healthy goats.

The ability of 342 staphylococcal isolates from different anatomical sites in healthy goats to produce staphylococcal enterotoxins (SE) was investigated. SE were produced by 74.3% of the 70 coagulase-positive strains and by 22% of the coagulase-negative strains studied. Most enterotoxigenic strains were isolated from the skin of udders and teats and from milk. SEC was the SE type most frequently produced, either alone (67.9%) or in combination with others. Five coagulase-negative species not previously reported as SE producers were identified (Staphylococcus chromogenes, S. warneri, S. sciuri, S. saprophyticus, and S. lentus). SEA, SEB, and SEC were detected in the milk of 17 of the 133 healthy goats studied. These results suggest that the goat is an important reservoir of enterotoxigenic staphylococci, most of which produce SEC.     

Deoxyribonucleic Acid Base Composition and Biochemical Properties of Certain Coagulase-Negative Enterotoxigenic Cocci

Eight coagulase-negative, enterotoxigenic strains of cocci and one weakly coagulase-positive strain isolated from a number of different sources, including cases of food poisoning incidents, were evaluated for their relationship to Staphylococcus aureus on the basis of deoxyribonucleic acid (DNA) buoyant density and physiological studies. One strain of cocci produced enterotoxins A and C, two strains produced types B and C, four strains produced only type C, and one strain only type D. The enterotoxin produced by one strain of cocci was serologically untypable. None of the test organisms produced detectable amounts of enterotoxin in broth cultures. The test strains of cocci exhibited the following profile: all produced catalase; all grew anaerobically and fermented glucose; five were sensitive to lysostaphin; the percentage of guanine plus cytosine content of their DNA varied from 32.7 to 37.6; five produced acid from mannitol both aerobically and anaerobically; two formed δ-hemolysin; five produced phosphatase and acetoin; and all produced heat-stable nuclease. None of the organisms exhibited typical characteristics of S. aureus, S. epidermidis, or S. saprophyticus. On the basis of the present data and data reported elsewhere, these organisms should be considered as variants or mutants of S. aureus.     

Enterotoxigenicity of Staphylococcus aureus Cultures Isolated from Acute Cases of Bovine Mastitis

To determine whether staphylococci causing bovine mastitis are potential causes of human intoxications, 142 cultures identified as etiological agents of acute cases and 18 cultures causing chronic cases of staphylococcal mastitis were obtained from investigators in the United States and Canada, examined microscopically, and tested for carbohydrate utilization, terminal pH, catalase, coagulase, egg yolk hydrolysis, gelatin hydrolysis, cytochrome oxidase, urease production, nitrate reduction, micrococcal nuclease, phage type, and enterotoxin production. Three cultures were not confirmed as Staphylococcus aureus. Of the 157 S. aureus cultures, 23 produced staphylococcal enterotoxins. Although a direct relationship between staphylococcal mastitis and outbreaks of staphylococcal food poisoning was not proved, results indicated that staphylococcal infections of the bovine mammary gland represent a significant reservoir of enterotoxigenic strains of S. aureus.