Some Microbiological Aspects of Staphylococcal Mastitis

(1) Mannitol fermentation is a reasonably reliable method for the detection of coagulase positive staphylococci in milk. This reliability can be improved if mannitol fermentation is carried out under anaerobic conditions.(2) Among hemolytic strains of staphylococci isolated from milk, beta hemolytic staphylococci predominate. Bovine and sheep blood agar plates give similar hemolytic patterns, but the hemolysis is more pronounced on sheep blood agar.(3) Gelatin liquefaction cannot be relied upon for the selection of coagulase positive staphylococci in milk.(4) Urease production is a feature of the majority of coagulase positive staphylococci isolated from milk.(5) Tellurite Glycine agar medium is not satisfactory for the selection of coagulase positive staphylococci in milk.     

Polymyxin-Coagulase-Mannitol-Agar: I. A Selective Isolation Medium for Coagulase-Positive Staphylococci1

A selective, differential plating medium was developed for the isolation and identification of coagulase-positive and mannitol-fermenting staphylococci. Coagulase produced by growing Staphylococcus aureus caused an opaque zone of fibrin to form around each colony. Several strains of S. aureus produced a visible coagulase reaction by 8 hr, and all strains gave a positive reaction before 12 hr. Mannitol fermentation was usually observed between 12 and 36 hr. Rabbit serum was filtered through Sephadex G-100 to obtain plasmin- and plasminogen-free coagulase-reacting factor (CRF). False-negative reactions, caused by staphylokinase and staphylococcal Müller factor action on plasminogen, were eliminated when this CRF was used. False-positive reactions by lipolytic, coagulase-negative staphylococci were reduced, since gel filtration removed the serum lipoprotein which served as a primary source of opacity. The addition of 75 mug of polymyxin B per ml selectively retarded the growth of S. epidermidis and minimized false-positive reactions caused by citrate-utilizing gram-negative rods. The preparation, characteristics, and use of the medium are presented.     

Coagulase and Deoxyribonuclease Activities of Staphylococci Isolated From Clinical Sources

A total of 504 clinical isolates of the family Micrococcaceae were tested for coagulase, deoxyribonuclease, clumping factor, and phosphatase to determine whether there is a correlation between the results of these tests and the pathogenicity of staphylococci. In the tests for coagulase production, it was found that either human or rabbit plasma could be used with broth cultures, whereas rabbit but not human plasma was satisfactory when microorganisms removed from solid culture medium were used. Deoxyribonuclease production correlated better than the fermentation of mannitol with coagulase production. The use of methyl green, Toluidine Blue O, or acridine orange offered no advantage over the use of HCl for detecting the production of deoxyribonuclease. Neither the presence of clumping factor nor the production of phosphatase correlated well with coagulase production. Strains of staphylococci that did not produce coagulase and deoxyribonuclease were isolated as frequently as, and from a greater variety of clinical sources than, strains which produced these substances. It is concluded that the production of coagulase and deoxyribonuclease are properties of staphylococci which are not necessarily indicative of potential pathogenicity of the organisms for man.     

THE OXIDASE ACTIVITY OF STAPHYLOCOCCI

SUMMARY: By the use of Kovacs'(1956) test, oxidase activity was demonstrated in 23 of 66 strains of coagulase negative staphylococci (or micrococci) but in none of 82 strains of coagulase positive staphylococci. Less sensitive methods showed fewer reactions or failed to demonstrate them at all. Oxidase activity could not be correlated with other biochemical features.     

Sensitivity to Lysostaphin as a Criterion for the Identification of Staphylococci from Animal Origin

A total of 195 Gram positive, catalase positive cocci, isolated from ovine mastitis, abscesses in slaughtered animals and parasitic pulmonary lesions in lambs were tested for glucose fermentation, anaerobic growth in thioglycollate medium, coagulase production and susceptibility to the lytic action of lysostaphin. On the basis of lysostaphin sensitivity, 192 strains were classified as staphylococci. The number of cultures able to produce acid anaerobically from glucose, or giving a positive result in the test of Evans and Kloos was lower. A good correlation among these three tests was not observed. Ninety-seven of the strains tested gave a positive coagulase reaction. Sensitivity to lysostaphin could not be used as a criterion for the differentiation of coagulase positive and coagulase negative strains. The turbidimetric method employed for the assessment of lysostaphin sensitivity is discussed.     

THE ISOLATION OF COAGULASE POSITIVE STAPHYLOCOCCI FROM ROUTINE COMPOSITE MILK SAMPLES

SUMMARY: Composite morning milk samples were taken once weekly for 9 weeks from 24 dairy farms. In all 208 samples were tested, and coagulase positive staphylococci ( aureus, albus and citreus ) were isolated from 127 (61%). The week by week recovery of these staphylococci from individual farm samples varied between 11% and 88% Penicillin resistant coagulase positive strains were recovered from 20 samples (10%).     

Interference of Neisseria gonorrhoeae growth by aerobic bacterial representatives of the urogenital flora

Aerobic bacterial isolates obtained from endocervical, vaginal and urethral swabbings were tested for interference of neisseria gonorrhoeae growth on solid medium. Simultaneous antagonism was studied using the lawn spotting method, and delayed antagonism by the basal spot/lawn method. From 58 swabbings we recuperated a total of 181 isolates, 71 of those were found interfering with at least one out of four gonococcal strains (G-1, G-2, G-3 and G-4). Similar percentages of interfering isolates were obtained from each of the isolation sites. The identification of the interfering isolates has revealed that similar numbers of coagulase negative staphylococci and identical numbers of group D streptococci were found for each of those sites. The majority of the interfering isolates and also of the inhibitory coagulase negative staphylococci showed only simultaneous antagonism. To complete the interference spectrum, we have tested all the active urogenital isolates against four other gonococcal strains (G-7, G-9, G-10 and G-11). This spectrum showed clearly that interference is not an all or none phenomenon. While the gonococcal interference spectrum of most of the Gram positive cocci and the Acinetobacter sp. strains is broad, that of all the other isolates is relatively narrow. Gonococcal strains G-7 and G-9 were the most susceptible to inhibition by the interfering urogenital isolates while strain G-3 was the most resistant one.     

Simple and direct detection of Staphylococcus aureus in milk by a tube coagulase test

A tube coagulase test (TCT) is described as a simple and non-expensive system for detection of Staphylococcus aureus directly in milk. The procedure is characterized by mixing milk samples with rabbit citrate plasma followed by incubation at 37 °C for clot formation. The tube coagulase test demonstrated 91·5% accuracy, 88·5% sensitivity and 100% specificity for the direct recognition of Staph. aureus in milk samples from quarters with subclinical mastitis, when compared with plating of milk on blood agar. The TCT has the potential to detect other coagulase positive staphylococci in milk. It is concluded that TCT may be of use to veterinary practitioners with limited laboratory facilities, or to dairy farmers as a simple diagnostic test on site.     

Staphylococcus schleiferi subsp. coagulans subsp. nov., isolated from the external auditory meatus of dogs with external ear otitis

A new subspecies, Staphylococcus schleiferi subsp. coagulans, was isolated from the external auditory meatus of dogs suffering from external ear otitis and is described on the basis of studies of 21 strains. Phenotypic studies showed that these strains are more closely related to Staphylococcus intermedius than to other staphylococci, but DNA hybridization studies indicated that they are closely related to Staphylococcus schleiferi N850274T. On the basis of biochemical distinctiveness (positive test tube coagulase test and different carbohydrate reactions) and the etiological importance (frequent isolation from otitis specimens from dogs) of these strains, we propose to classify them as a subspecies of S. schleiferi. The strains of this new subspecies are coagulase tube test, beta-hemolysin, and heat-stable nuclease positive but clumping factor negative. A simple scheme for the differentiation of S. schleiferi subsp. coagulans from the other coagulase-positive staphylococci is presented. The type strain is GA211 (= JCM 7470).     

Bacterial strains colonizing subcutaneous catheters of personal insulin pumps

Continuous subcutaneous insulin infusion (CSII) is a commonly used, safe intensive insulin therapy method effective in maintaining normoglycaemia. The disadvantage of CSII are skin infections of the catheter injection site. The aim of the study was to gain insight on the colonization of subcutaneous insulin pump catheters by skin flora and to investigate the correlation between Staphylococcus aureus carrier state (presence in the nose), its presence on the skin and catheter. 141 catheters obtained from 94 children with T1DM and CSII were examined using the semi quantitative culture technique of Maki. The result was positive in 34 examinations (24.1%) in 30 children (31.9%). Most often coagulase negative staphylococci were isolated (30), mainly Staphylococcus epidermidis, 1/3 of the staphylococci were methicillin resistant. S. aureus was detected in 7 examinations in 6 children. S. aureus carrier state was proved in 31.9% of all examined patients, more often in children with a positive catheter culture (41.4%), there were no MRSA. No correlation between S. aureus carrier state and catheter colonization was shown. Statistically significant correlations between: coagulase negative staphylococci presence, including the methicillin resistant strains, on the skin and on the catheter surface (p< 0.0001); glycosylated hemoglobin (HbA1c) and bacteria catheter colonization (p = 0.0335) were observed. Subcutaneous catheter colonization by microorganisms often occurs in CSII. Microorganisms found on the skin are the most frequent cause of the subcutaneous catheter infection.     

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.     

Circadian rhythms of antibacterial resistance, coagulase and antilysozyme activity of Staphylococcus aureus

AIM: To determine daily dynamics of antibacterial resistance as well as antilysozyme and coagulase activity of S. aureus strains. MATERIALS AND METHODS: On an example of clinical strains of S.aureus isolated from patients with surgical infections daily dynamics of biological characteristics of staphylococci was studied. After 12 hours of incubation strains were tested for coagulase activity by standard method (test tube method), antilysozyme activity by photometric method, and antibacterial resistance by method of serial dilutions in agar. Tests were repeated each 3-hours during a day. RESULTS: Variation of levels of studied biological characteristics of staphylococci during a day was revealed. Structures of coagulase and antilysozyme circadian rhythms had some differences in different S. aureus strains. Alongside with it, similarity in temporal expression of such biological characteristics of staphylococci as antibacterial resistance and antilysozyme activity was noted. CONCLUSION: Obtained data open prospect to use biorhythmological approach in study of biological characteristics of microorganisms during evaluation of their mechanisms of adaptation to changing environmental conditions. Chronobiological approach allows to reveal periods of maximal expression of S. aureus characteristics that could be used for increasing of effectiveness of antibacterial treatment by the choice of optimal time for administration of antibiotic.     

The Tellurite Reactions of Coagulase Negative Staphylococci and Micrococci

S ummary . Methods for determining the tellurite reactions of coagulase negative staphylococci and micrococci have been applied to strains causing urinary tract infections in human patients. The tellurite positive strains were assigned to Micrococcus subgroup 3 and Staphylococcus subgroup VI of the Baird-Parker (1963) classification.     

The toxicity of potassium tellurite to Staphylococcus aureus in rabbit plasma fibrinogen agar

The potassium tellurite concentration, 0.01% w/v, in Baird-Parker agar has been recommended for plasma coagulase media such as pig or rabbit fibrinogen agar. Comparative tests have shown that with some strains of Staphylococcus aureus this level of potassium tellurite is too inhibitory and it should be reduced four-fold to 0.0025% w/v to maximize the isolation rate. It is postulated that egg yolk in Baird-Parker agar has a protective effect on staphylococci against the inhibitory action of tellurite.     

Identification of Staphylococci Isolated from Clinical Material

A total of 350 staphylococci isolated from various clinical sources were examined for bound and free coagulase, fermentation of mannitol, and deoxyribonuclease. The economical coagulase-mannitol-agar method of Esber and Faulcomer was found to be suitable for the detection of free coagulase and mannitol fermentation. A significant number of coagulase- and mannitol-negative staphylococci proved to be deoxyribonuclease-positive.     

The toxicity of potassium tellurite to Staphylococcus aureus in rabbit plasma fibrinogen agar

The potassium tellurite concentration, 0.01% w/v, in Baird-Parker agar has been recommended for plasma coagulase media such as pig or rabbit fibrinogen agar. Comparative tests have shown that with some strains of Staphylococcus aureus this level of potassium tellurite is too inhibitory and it should be reduced four-fold to 0.0025% w/v to maximize the isolation rate. It is postulated that egg yolk in Baird-Parker agar has a protective effect on staphylococci against the inhibitory action of tellurite.     

COAGULASE POSITIVE STAPHYLOCOCCI FROM BULK MILK SUPPLIES LOW IN SOLIDS-NOT-FAT

SUMMARY: Tests carried out on coagulase positive staphylococci isolated from bulk milk supplies showed a nearly perfect relationship between α-lysin production and pigment production at 42°. Ninety-nine per cent of the organisms were classed as pathogenic, while 75% were probably responsible for sub-clinical conditions in the udder.     

Delineation of the Site of Action of an Antistaphylococcal α-Globulin

S ummary . The isolation of an antibacterial α-globulin from the sera of humans as well as selected animal species has been reported. While antibacterial agent (ABA) reduced the respiration of intact cells by 55%, the anti-respiratory effect was increased to 67% and 85% for spheroplasts and L-forms, respectively. Studies indicated that neither cell wall nor peptidoglycan could absorb ABA quickly enough to inhibit its membrane damaging effects. Although the ribitol teichoic acid-free mutant Staphylococcus aureus H52A5 was not susceptible to ABA, the lack of ribitol teichoic acid may have altered structurally the cell wall so that ABA access to the cell membrane was precluded. The activity of ABA was neutralized by prior exposure of staphylococci to exogenous coagulase, presumably by masking unknown receptor sites for ABA on the cell surface. In our studies with cell wall deficient organisms, we could not demonstrate coagulase reversal of ABA activity.     

Public transport as a reservoir of methicillin‐resistant staphylococci

Aims: The aim of this study was to explore the occurrence of methicillin‐resistant staphylococci in a large urban public transport system. Methods and Results: Samples were taken from hand rails, which passengers hold onto when they are standing. In total, 1400 swabs taken from 55 vehicles (trolleybuses, trams and buses) were examined. As many as 30·1% samples were positive for the presence of methicillin‐resistant coagulase‐negative staphylococci (MRCoNS), but none for methicillin‐resistant Staphylococcus aureus (MRSA). MRCoNS were isolated from all 55 vehicles. Nearly 50% of MRCoNS isolates displayed resistance not only to beta‐lactams, but at least to two or more other classes of antimicrobials as well. Conclusions: This study demonstrated widespread occurrence of MRCoNS on hand rails in public transport vehicles. MRSA was not detected. Significance and Impact of the Study: The recovery of methicillin‐resistant staphylococci from public transport system implies a potential risk for transmission of these bacteria in an out‐hospital environment.     

Characterization of Staphylococci Isolated from Raw Milk

To evaluate the pathogenicity of staphylococci from bovine raw milk, the general characteristics of 775 strains isolated from 798 samples of milk were studied. The coagulase test was performed by use of rabbit plasma. Chromogenesis, mannitol fermentation, and gelatin liquefaction were investigated on Chapman's Medium 110, after 48 hr of incubation. Production of β-hemolysin, which has been considered indicative of pathogenic staphylococci of animal origin, was determined by streaking different strains on sheep blood-agar plates in the presence of a strain of Lancefield group B streptococci. Plates were incubated at 37 C for 24 hr, and strong hemolysis was produced in the zone of interaction of β-hemolysin and some substance liberated by streptococcus (CAMP test). Of 404 strains found to be coagulase-positive, 95.8% exhibited a deep-orange pigment, 76.5% produced β-hemolysin, 91.8% fermented mannitol, and 75% liquefield gelatin. Of 371 strains which gave a negative coagulase test, about 16% fermented mannitol and liquefied gelatin; none of these strains produced β-hemolysin. When results are grouped according to pigmentation and coagulase production, β-hemolysin seems to be developed by pathogenic strains of Staphylococcus aureus only. If suitability of these tests for investigation of pathogenicity is compared, production of β-hemolysin appears to be the most useful one, since no “false positive” results were found. The use of the CAMP test as a simple and rapid technique to determine production of β-hemolysin by pathogenic strains of animal staphylococci during routine bacteriological work is suggested.