Serological characteristics of coagulase-positive staphylococci isolated from man and animals

A simplified method allowed Staphylococcus aureus, Staph. intermedius and coagulase-positive Staph. hyicus subsp. hyicus isolated from humans, dogs, monkey, sheep, poultry, rabbits, giant rats ( Cricetomys gambianus ) and other animals to be serotyped. The nine coagulase-positive staphylococcal strains of human origin possessed thermolabile and thermostable agglutinogens. Two strains of Staph. intermedius of human and canine origins examined had agglutinogen K₁K₂. The three Staph. aureus strains isolated from African giant rats ( Cricetomys gambianus ) had agglutinogens a₅ and P common to them. The Staph. aureus strain isolated from a monkey belonged to serotype b₁, c₁, o and the caprine strain of Staph. hyicus subsp. hyicus was serotype a₅, c₁.     

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.     

The inhibition of Staphylococcus hyicus on a selective medium for staphylococci

Growth of Staphylococcus hyicus subsp. hyicus and Staph. hyicus subsp. chromogenes strains was found to be severely inhibited when broth cultures of these organisms were streaked on Schleifer and Kramer's staphylococcal (SK) medium. Of the selective agents contained in SK medium, potassium thiocyanate was found to be inhibitory towards both subspecies of Staph. hyicus and sodium azide had an additional inhibitory effect on Staph. hyicus subsp. chromogenes. Of six different media supplements examined, sheep blood and Tween 80 were found to improve the growth of both Staph. hyicus subspecies when added to SK medium. These findings were confirmed in subsequent work where the supplemented SK media were used to isolate potential enterotoxin-producing organisms from simulated raw milk (Staph. aureus, Staph. hyicus subsp. hyicus, Staph. hyicus subsp. chromogenes, Staph. intermedius). SK medium supplemented with sheep blood proved more effective in allowing satisfactory recovery of Staph. aureus, Staph. hyicus subsp. hyicus and Staph. intermedius. However, neither supplement enabled satisfactory recovery of Staph. hyicus subsp. chromogenes to be achieved.     

The inhibition of Staphylococcus hyicus on a selective medium for staphylococci

Growth of Staphylococcus hyicus subsp. hyicus and Staph. hyicus subsp. chromogenes strains was found to be severely inhibited when broth cultures of these organisms were streaked on Schleifer and Kramer's staphylococcal (SK) medium. Of the selective agents contained in SK medium, potassium thiocyanate was found to be inhibitory towards both subspecies of Staph. hyicus and sodium azide had an additional inhibitory effect on Staph. hyicus subsp. chromogenes. Of six different media supplements examined, sheep blood and Tween 80 were found to improve the growth of both Staph. hyicus subspecies when added to SK medium. These findings were confirmed in subsequent work where the supplemented SK media were used to isolate potential enterotoxin-producing organisms from simulated raw milk (Staph. aureus, Staph. hyicus subsp. hyicus, Staph. hyicus subsp. chromogenes, Staph. intermedius ). SK medium supplemented with sheep blood proved more effective in allowing satisfactory recovery of Staph. aureus, Staph. hyicus subsp. hyicus and Staph. intermedius. However, neither supplement enabled satisfactory recovery of Staph. hyicus subsp. chromogenes to be achieved.     

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.     

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.     

Characteristics of staphylococci isolated from man, poultry and some other animals

Of 281 strains of staphylococci isolated from man and animals 36 (12.8%) were coagulase-positive and 245 (87.2%) were coagulase-negative. Staphylococcus aureus and Staph. intermedius were the commonest coagulase-positive staphylococci isolated from the hosts examined. Of the 20 strains that remained unclassifiable, 14 were isolated from sheep and goats.     

Characteristics of staphylococci isolated from man, poultry and some other animals

Of 281 strains of staphylococci isolated from man and animals 36 (12–8%) were coagulase-positive and 245 (87–2%) were coagulase-negative. Staphylococcus aureus and Staph. intermedius were the commonest coagulase-positive staphylococci isolated from the hosts examined. Of the 20 strains that remained unclassifiable, 14 were isolated from sheep and goats.     

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).     

Antimicrobial susceptibility of staphylococci isolated from otitis externa in dogs

Samples were obtained from 65 unmedicated adult dogs, processed for isolation of Staphylococcus species and tested for susceptibility to penicillin G, gentamicin, oxacillin, tetracycline, trimethoprim-sulphamethoxazole, streptomycin, ampicillin and rifampin. Forty-four isolates were obtained, which represents 67.7% of samples. Coagulase-negative species were most commonly found, and the most frequently isolated staphylococcus species were Staph. epidermidis and Staph. aureus. Other species, such as Staph. simulans, Staph. haemolyticus, Staph. saprophyticus and Staph. intermedius were also isolated. Resistance to antibiotics was frequently observed, with 90.9% of the isolates showing resistance to at least one drug. The most active antimicrobial agents against staphylococci isolated from otitis externa of dogs were rifampin and oxacillin. Multidrug resistance was a common finding, and one strain of Staph. haemolyticus species, was resistant to all tested antimicrobial agents. Resistance to three or more different drugs was a common finding, observed in 16 strains (36.4%) of both coagulase-positive and coagulase-negative staphylococci. This study highlights the emergence of cases of otitis externa determined by coagulase-negative staphylococcus strains and once more emphasizes the need for bacterial culture with species identification and susceptibility testing of swab specimens from the ear canal in order to choose appropriate antimicrobial agents.     

Classification and biological characteristics of coagulase-positive Staphylococci isolated from animals

A total of 165 coagulase-positive staphylococcal strains of different origin (142 S. aureus strains and 23 S. intermedius strains) were subjected to biological typing in accordance with the schemes of Hajek-Marsalek and Meyer-Witte. The former of these schemes permitted to identify 68% and the latter 18% of S. aureus strains. The cultures isolated from swine and chickens had the most uniform composition: 85-86% of the strains belonged to biotype B. 44% of the strains isolated from cows and sheep belonged to biotypes C (ecovars bovis and ovis) and A (ecovar hominis); the rest of the strains could not be identified. 96% of the strains isolated from minks were made up of S. intermedius, more than a half of them belonging to biotype E (ecovar canis). In 80% of S. aureus strains and 48% S. intermedius cultures protein A was detected. Only 9% of S. aureus strains of animal origin were found capable of producing enterotoxins (A-D). The expediency of working out a unified scheme for the biotyping of coagulase-positive staphylococci is discussed.     

Staphylococci associated with the rumen of young and wild ruminants

Staphylococcus warneri, Staph, xylosus, Staph. saprophyticus, Staph. epidermidis, Staph. sciuri subsp. lentus, Staph. sciuri subsp. sciuri and Staph. cohnii subsp. urealyticum were the most frequently occurring staphylococci in the rumen content and wall of young and wild ruminants. Staphylococcus warneri formed a high percentage mainly among 2–9-week-old ruminants. Staphylococcus hominis was found only in mouflons. Staphylococcus gallinarum was detected only in calves. Staphylococcus aureus was the predominant representative of coagulase-positive staphylococci in the rumen of wild ruminants. Most of the strains examined could not be identified as known species.     

Phage typing of the coagulase-positive staphylococci isolated from various species of animals

More than 200 coagulase-positive strains of animal origin have been studied by means of Staphylococcus aureus typing phages, belonging to two international sets and intended for typing staphylococci isolated from large cattle and humans, and experimental "chicken" phage A 1591. Among S. aureus strains the cultures isolated from swine, cows, chickens, and belonging to biotypes B1, C1, B2, respectively, have been mostly (in 78.5-90.0% of cases) determined by phage typing. The strains belonging to one biotype have proved to be sensitive predominantly to the same phages. In this connection further differentiation of staphylococci within individual biotypes by means of the phages used in these experiments seems to be impracticable. S. intermedius strains have been found to be completely resistant to the above phages, which confirms that S. intermedius is rightly considered to be an independent species of coagulase-positive staphylococci.     

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.     

Streptomycin and tetracycline resistance plasmids in Staphylococcus hyicus and other staphylococci.

Plasmids associated with resistance to streptomycin, to streptomycin plus chloramphenicol or to tetracycline in Staphylococcus hyicus isolated from the skin of pigs have been compared, by restriction endonuclease digest patterns, with similar staphylococci from human sources and with published DNA base sequences. Several plasmids from Staph. hyicus have proved to have a very similar structure to those described from Staph. aureus but others appeared very dissimilar. This confirms the opinion that staphylococci from animal skin share a pool of plasmids with those from human skin but may also possess some of quite different structure.     

Isolation of strains of a new ecological variant of Staphylococcus aureus from asses

47 coagulase-positive staphylococcal strains were isolated from the nasal smears of 175 healthy donkeys. In accordance with the schemes of Akatov--Devriese and Mayer-Witte--Akatov, 10.6% of the cultures were classified with the coagulase-positive species S. hyicus and 89.3%, with the species S. aureus. Out of S. aureus strains, 11.9% were found to have the characteristics of ecovar hominis, while 16.2% of the cultures could not be classified with definite ecovars. Most of the strains (71.4%) were found to differ from the known ecovars of S. aureus in their biological properties. For this reason, the above strains were classified with the new ecovar asinae. The authors propose to make the existing S. aureus identification scheme (the scheme of Mayer-Witte--Akatov) more complete by adding the tests for hyaluronidase and phosphatase.     

Streptomycin and tetracycline resistance plasmids in Staphylococcus hyicus and other staphylococci

Plasmids associated with resistance to streptomycin, to streptomycin plus chloramphenicol or to tetracycline in Staphylococcus hyicus isolated from the skin of pigs have been compared, by restriction endonuclease digest patterns, with similar staphylococci from human sources and with published DNA base sequences. Several plasmids from Staph. hyicus have proved to have a very similar structure to those described from Staph. aureus but others appeared very dissimilar. This confirms the opinion that staphylococci from animal skin share a pool of plasmids with those from human skin but may also possess some of quite different structure.     

Deoxyribonucleic acid homologies among strains of Bacteroides melaninogenicus and related species

Saccharolytic, black-pigmented Bacteroides strains, which at present belong to the species Bacteroides melaninogenicus were classified on the basis of deoxyribonucleic acid (DNA) base ratios and DNA hybridization studies. These strains were divided into several DNA homology groups, which showed no or low mutual DNA homology. A DNA homology group with a percentage guanine plus cytosine (G + C) of 42–43% was formed by three strains of Bact. melaninogenicus subsp. melaninogenicus ; the type strain of this subspecies, strain ATCC 25845, had about 60% DNA homology with this group. Strain ATCC 15930, which has been assigned to this subspecies, had a percentage G + C of 47% and showed no DNA homology with the former group. All strains of Bact. melaninogenicus subsp. intermedius had a percentage G + C of 39–45%. A DNA homology group was formed by eight strains of this subspecies. The type strain of Bact. melaninogenicus subsp. intermedius , ATCC 25611, showed relatively low DNA homology with this main DNA homology group. A strain of Bact. melaninogenicus subsp. intermedius serotype C1 showed no DNA homology with the other strains tested. Furthermore two strains labelled 'Bact. melaninogenicus subsp. levii' were found to form a distinct DNA homology group. On the basis of the DNA homology results, the strains, which at present are classified in the species Bact. melaninogenicus , were clearly distinguished from strains of Bact. asaccharolyticus and Bact. gingivalis , and also from strains of related non-pigmented Bacteroides species.     

Plasmids in Staphylococcus hyicus

Fifty-one strains of Staphylococcus hyicus and six of Staph, chromogenes were collected from porcine and bovine sources in England and Belgium. Antibiotic resistance and plasmid profiles were established. Plasmids associated with resistance to tetracycline, erythromycin and streptomycin were identified in many strains; these plasmids were about the same size as those mediating similar resistances in Staph. aureus of human origin. Many small cryptic plasmids were also seen.     

Plasmids in Staphylococcus hyicus

Fifty-one strains of Staphylococcus hyicus and six of Staph. chromogenes were collected from porcine and bovine sources in England and Belgium. Antibiotic resistance and plasmid profiles were established. Plasmids associated with resistance to tetracycline, erythromycin and streptomycin were identified in many strains; these plasmids were about the same size as those mediating similar resistances in Staph. aureus of human origin. Many small cryptic plasmids were also seen.