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.     

Isolation of methicillin-resistant Staphylococcus pseudintermedius from breeding dogs

The overuse of antimicrobials can select resistant bacteria strains; staphylococci have the ability to become resistant to all beta-lactam antimicrobials and are a significant concern in human medicine and a growing issue for veterinary medicine. Because antimicrobials are sometimes incorrectly used in breeding kennels, the objective of the work was to assess the occurrence of methicillin-resistant coagulase-positive staphylococci in breeding dogs. The research was carried out in 13 kennels that were allotted to three categories according to the intensity of antimicrobial use. Vaginal and milk swabs were taken from 87 healthy bitches around parturition and also from multiple organs of 27 of their pups that died within the first 2 weeks. Standard bacteriological examinations were carried out and coagulase-positive staphylococci were identified. All the coagulase-positive staphylococci resulted to be Staphylococcus pseudintermedius. Susceptibility to oxacillin and the presence of the mecA gene were tested. Nine out of 89 strains (six isolated from the bitches' milk and three from dead puppies, all belonging to kennels characterized by an excessive use of antimicrobials) were multidrug-resistant, methicillin-resistant and mecA positive.Our results confirm that excessive use of antimicrobials entails the risk of selecting resistant staphylococci strains. Our data also indicate that the bacterial flora of healthy dogs belonging to specific populations may act as a reservoir of resistance genes.     

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.     

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.     

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.     

Production of lysozyme by staphylococci and its correlation with three other extracellular substances

Jay, James M. (Wayne State University, Detroit, Mich.). Production of lysozyme by staphylococci and its correlation with three other extracellular substances. J. Bacteriol. 91:1804-1810. 1966.-Lysozyme production was determined on plates containing 1 mg/ml of Lysozyme Substrate in Heart Infusion Agar with incubation at 37 C for 48 hr. Its production was compared with that of alpha-hemolysin and sheep hemolysin and egg-yolk precipitation, by use of both coagulase-positive and coagulase-negative strains of staphylococci. Of 126 coagulase-positive strains tested, 120 or 95.2% produced lysozyme, 117 or 92.9% produced alpha-hemolysin, 108 or 85.7% precipitated egg yolk, and 102 or 81% produced sheep hemolysin. Of the 49 coagulase-negative strains (which included 22 pathogens), only 4 or 8.1% produced lysozyme, 14 or 28.6% produced alpha-hemolysin, 13 or 26.5% produced sheep hemolysins, and 5 or 10.2% precipitated egg yolk. Only two of the six coagulase-positive strains which failed to produce lysozyme showed any consistent patterns in relation to the four characteristics determined. The four coagulase-negative strains which produced lysozyme were inconsistent for the other characteristics measured. It is suggested that lysozyme production is more a property of coagulase-positive staphylococci, and therefore a better ancillary test of pathogenicity, than either production of alpha-hemolysin or egg-yolk precipitation, because the incidence of lysozyme producers is higher among this group than among those producing the other substances and because fewer coagulase-negative staphylococci produced lysozyme than hemolysins or egg-yolk precipitation. Of 16 other species of bacteria and yeasts tested, all were found negative except Bacillus subtilis. Lysozyme production by staphylococci in heavily contaminated foods was not inhibited on plates containing sodium azide, whereas media containing 7.5% salt and sorbic acid were unsuitable. The possible relationship of lysozyme production to staphylococcal pathogenicity is discussed.     

Virulent and avirulent encapsulated variants of Staphyococcus aureus

There are at least two serologically distinct capsular types of coagulase-positive Staphylococcus aureus. Until now, unequivocal evidence for encapsulation of the Smith diffuse variant was lacking. However, the data presented in this paper provide definitive details of encapsulation of the Smith strain. A marked difference in ld(50) values for the two serologically distinct capsular types of S. aureus was demonstrated. The paradoxical behavior of these two strains suggested that the host was resistant to one and was susceptible to the other. A survey of the carriage incidence in mice for staphylococci and staphylococcal capsular antibodies disclosed the presence of staphylococci and capsular antibodies in these animals. The capsular antibodies detected were reactive against only one of the capsular types of S. aureus. None of the sera from the mice surveyed possessed capsular antibodies against the Smith diffuse variant, but the average incidence for the capsular antibodies against the wound mucoid type was 46%. We postulated that the susceptibility of the mice to the Smith diffuse variant was caused by the absence of protective, type-specific capsular antibodies. Conversely, the resistance of the mice to the wound mucoid staphylococci may have been a result of the presence of type-specific capsular antibodies.     

Lysozyme Production as an Aid for Identification of Potentially Pathogenic Strains of Staphylococci

Lysozyme production is a frequent property of potentially pathogenic staphylococci. In the present study, 1,186 strains of human origin, 85 strains of animal origin, and 156 strains of Staphylococcus albus (epidermidis) were tested. Of 1,114 coagulase-positive strains of human and animal origin, 1,098 were lysozyme-positive (98.5%). On the other hand, of 157 coagulase-negative strains which, based on further investigations, belong to the potentially pathogenic staphylococci, all were lysozyme-positive. All of the 156 strains (100%) belonging to the species S. albus (epidermidis) were lysozyme-negative. We conclude that lysozyme production is a better index of potentially pathogenic staphylococci than the measurement of free coagulase, especially in cases of strains of animal origin. It is possible that lysozyme production allows a differentiation between pathogenic and nonpathogenic coagulase-negative staphylococci.     

Susceptibility of Coagulase-negative Staphylococci to Lysostaphin and Other Antibiotics

In general, coagulase-negative staphylococci were found to be relatively less susceptible to the lytic action of lysostaphin than coagulase-positive staphylococci. To achieve, arbitrarily, a lysis greater than 75%, it was necessary to use an increased concentration of enzyme or a longer incubation period than that usually required with coagulase-positive strains. For the most part, the cultures studied were sensitive to oxacillin, cloxacillin, dicloxacillin, nafcillin, ancillin, cephalothin, cephaloridine, fusidic acid, lincomycin, novobiocin, and neomycin [median minimal inhibitory concentrations (MIC) of 1.56 mug/ml or less]. Some degree of resistance (median MIC values of 12.5 mug/ml or greater) to benzylpenicillin, ampicillin, methicillin, tetracycline, chloretetracycline, erythromycin, ristocetin, and lysostaphin was found. Ten methicillin-resistant, coagulase-negative staphylococal strains were found to be cross-resistant to all nine of the penicillins tested, but much less resistant to the two cephalosporin analogues. In several instances, some of these strains seemed to be more sensitive to benzylpenicillin and to certain of the semisynthetic penicillins than to methicillin. Of the 18 antibiotics tested with the viable plate count method, the methicillin-resistant strains were found to be the most sensitive to lincomycin and novobiocin.     

Antibiotic sensitivity of staphylococci isolated from the surface of the skin of the breasts in pregnant women

Nine hundred and fourty coagulase-positive and coagulase-negative strains of staphylococci isolated from the skin surface of the mammary glands of 94 pregnant women were tested by the disc agar diffusion method for their sensitivity to five antibiotics. The highest number of the isolates were sensitive to erythromycin and lincomycin (87.7 and 89.7 per cent, respectively). The highest number of moderately resistant strains were detected with respect to methicillin. 19 out of 42 cultures of Staph. aureus were resistant to benzylpenicillin and 24 cultures were resistant to tetracycline. Among staphylococci 130 strains or 13.8 per cent were polyresistant.     

Drug resistant coagulase-positive staphylococci strains in food

From the 1572 food samples, examined in Microbiology Department of Frozen Food Industry Research Laboratory in Lód?, 79 (5.0%) coagulase-positive staphylococci strains were isolated. All the strains were sensitive to vancomycin, gentamycin and chloramphenicol. Only individual staphylococci strains were resistant to erythromycin (1.3%), lincomycin (2.5%) and ciprofloxacin (2.5%). 20.3% strains, isolated mainly from raw meat, were resistant to doxycycline and 6.3% to oxacillin. 38.0% of coagulase-positive staphylococci strains had positive results of cefinase test. One strain isolated from minced meat was resistant to methicillin and at the same time it was producing beta-lactamases.     

Antimicrobial sensivity and ability to slime production of koagulaze-negative staphylococci

The aim of this study was to assess the ability of slime production ofcoagulase-negative staphylococci (CONS) and evaluate the susceptibility of bacteria to antibiotics. Strains were isolated from clinical specimens obtained from hospitalized patients. The most frequently isolated species were S. epidermidis (51%), S. hominis (18%), S. haemolyticus (13%). The result of this study shows that 61% of S.epidermidis produce slime on CRA (Congo red agar), whereas none of the tested S. haemolyticus strains has this ability. All examined strains were susceptible to vancomycin, linezolid and quinupristin/ dalfopristin. The majority of strains were susceptible to minocycline, fusid acid, nitrofurantoin and rifampicin. Sixty six percent of isolates were determined as methicillin-resistant coagulase-negative staphylococci.     

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.     

Inhibition of Fibrinogen Reaction by Polysaccharide of Encapsulated Staphylococcus aureus

Encapsulated and nonencapsulated strains of Staphylococcus aureus which lack coagulase or clumping factor (bound coagulase), or both, were examined for the antigen associated with the fibrinogen-cell clumping reaction. Extracts of the cells were tested for the ability to react with fibrinogen or to inhibit fibrinogen precipitation. Antisera prepared against encapsulated (coagulase-positive, clumping factor-negative) variants, as well as against nonencapsulated wild-type (coagulase-positive, clumping factor-positive) S. aureus strains, contained high titers of clumping-inhibiting antibody. When coagulase-negative, clumping factor-negative mutants were the immunizing agents, antisera contained no demonstrable clumping-inhibiting antibody. Phenol extracts of all coagulase-positive strains tested precipitated fibrinogen, regardless of the ability of cells to clump in the presence of fibrinogen. Polysaccharide extracts of encapsulated, clumping factor-negative strains inhibited this fibrinogen-precipitating activity, whereas similar extracts of nonencapsulated staphylococci did not inhibit the fibrinogen reaction. From these results, it appeared that the coagulase-positive, encapsulated staphylococci which do not clump in fibrinogen solution possess clumping factor, but that their capsular polysaccharide inhibits clumping activity. These findings suggested a closer association of clumping factor and coagulase than is now recognized.     

Evaluation of the usefulness of selected commercial systems for identification of Staphylococcus species

The aim of the study was the assessment of the usefulness of commercially available systems for rapid identification of staphylococci. API STAPH (bioMerieux, France), ID 32 STAPH (bioMerieux, France), GPL (HTL, Poland) and Staph-Zym (Rosco, Denmark). The identifications were carried out according to producer's instruction. The material for the study comprised 76 staphylococcal strains, coagulase-positive and coagulase-negative. The strains were isolated from throat, nasal, wound, bone slivers, pus and blood of inpatients and from throat and nasal swabs of outpatients. Besides that, for the study staphylococcal strains were obtained from the American Collection of Typical Cultures (ATCC) and from the Polish Collection of Microorganisms (PCM). All tested strains were identified on the basis of classic biochemical tests. In the light of obtained results it is concluded that the commercial system most suitable for identification of staphylococci was ID 32 STAPH (bioMerieux), which has a wide spectrum of species identifiable with it and the highest percent (95%) of correctly identified species. The least suitable system was the GPL 15 (HTL, Poland).     

Limitations of Fibrinogen-Polymyxin Medium in Detecting Coagulase-Positive Staphylococci in Raw Milk

A fibrinogen-polymyxin medium and Staphylococcus Medium 110 were used in the isolation of coagulase-positive staphylococci in raw milk. Results indicated that both media allow the growth of some rods and of many coagulase-negative cocci. A significantly greater number of coagulase-positive staphylococci were identified by the tube test than were revealed by halo formation on fibrinogen-polymyxin medium.     

A survey of microorganisms for thermonuclease production

A total of 1204 cultures comprising 16 genera were surveyed for production of thermonuclease (TNase) in milk. Cultures other than Staphylococcus capable of TNase production were restricted to two genera, Streptococcus and Bacillus. Nineteen percent of 338 group D streptococci comprising four species (85% of which were Streptococcus faecalis) and 17% of 60 streptococci belonging to other groups produced TNase. Nine percent of 130 Bacillus cultures comprising six species produced the enzyme. On the other hand, 99% of coagulase-positive staphylococci produced TNase and only 18% of the coagulase-negative staphylococci produced the enzyme. The amount of TNase produced by streptococci and bacilli was significantly lower than that produced by coagulase-positive staphylococci. The pH profile of the streptococci and Bacillus TNases was similar to that of the staphylococcal TNase; each enzyme exhibited a minor peak at pH 7.0 and a broad major peak ranging from pH 8.5 to 10. The nuclease produced by coagulase-positive Staphylococcus was more heat stable than the nucleases produced by Streptococcus and Bacillus; there was little loss in activity of the staphylococcal enzyme after 60 min at 100 degrees C, whereas 50% of the activity of the streptococcal and Bacillus nucleases was destroyed in 40-60 min and 60-80 min, respectively.     

Deoxyribonucleic acid homologies among staphylococci: Coagulase-positive reference strains

DNA hybridization results confirm the proposed separation of coagulase-positive staphylococci into two distinct species. Strains ofStaphylococcus aureus representing the various biotypes and different phage typing groups of the human biotype gave high values of reassociation with DNA fromS. aureus reference strain RN 450, at both optimal and restrictive reassociation temperatures. Similar results were obtained between strains ofS. intermedius and its reference strain K 3. Interspecific reassociation between the two coagulase-positive species was low, and each reference strain showed low DNA sequence homology with 10 coagulase-negative species.S. staphylolyticus, strain PS 73, and putative pleiotropic mutants ofS. aureus were shown to be unrelated toS. aureus.     

Antagonistic action of corynebacteria and bacilli of a skin ecotype on staphylococci

582 strain of corynebacteria and 235 cultures of bacilli isolated from the skin surface of mammary glands of 120 nursing women were studied for their antagonistic action on staphylococci. It is established that bacilli, especially Bacillus subtilis, B. firmus and B. alvei exert more pronounced inhibitory effect on staphylococci. Representatives of the family Corynebacteriaceae possess a weak antagonistic action both on coagulase-positive and on coagulase-negative species. The ability of coryneform bacteria to retain the colonization resistance is supposed to be due to other factors. Antagonistic properties of B. subtilis are shown expedient to be used for elimination of hospital strains of Staphylococcus aureus from the skin of mammary glands.