Fibronectin-binding capacity of staphylococci

The fibronectin-binding capacity of clinical isolates of the genus Staphylococcus has been studied in the flake-formation test on glass and by inoculation into agar with human fibronectin added. Most of 58 S. aureus strains have been found capable of binding fibronectin. None of coagulase-negative staphylococci has given flake formation with fibronectin. The possibilities of the quantitative evaluation of the fibronectin-binding by the above-mentioned methods have been shown. In the analysis of the monomers of bovine fibronectin its capacity for inducing the growth of compact colonies in semiliquid agar has been shown.     

Pseudocompact-Type Growth and Conversion of Growth Types of Strains of Staphylococcus aureus In Vitro and In Vivo

Some strains of Staphylococcus aureus grew as compact colonies in Brain Heart Infusion-serum-soft agar but as diffuse colonies in a modified Staphylococcus 110-serum-soft agar. These strains were designated "pseudocompact." Strains showing compact-type colonial morphology in both media were designated "compact," whereas strains showing diffuse-type growth in both media were designated "diffuse." It was observed that the most recently isolated strains of S. aureus were of the pseudocompact type, whereas most stock culture strains tested were of the compact type. Using cultures recently isolated from clinical material, it was shown that pseudocompact strains convert to compact-type growth after prolonged incubation. Interconversion of compact, diffuse, and pseudocompact growth forms could be induced in vitro by appropriate cultural conditions, and conversion of growth type was also observed in vivo. Femoral abscesses produced in mice by four different compact-type strains showed conversion to diffuse or pseudocompact-type growth during the course of the infection.     

The growth of compact and diffuse variants of Staphylococcus aureus in bovine mastitic and normal whey

Strains of Staphylococcus aureus producing either diffuse or compact colonies in serum-soft agar were grown in bovine normal and mastitic whey. Bacterial growth was followed by automated turbidometry. Compact strains multiplied faster and to higher final numbers in mastitic whey than diffuse strains, whereas diffuse strains grew to higher numbers in normal whey. Nutrients (hemolysed bovine blood, bovine serum, proteose-peptone) were added to normal whey to enhance bacterial growth as in mastitic whey. The growth-promoting effect of these nutrients was dose-dependent for compact strains but not for diffuse strains. The difference in the growth characteristics of diffuse and compact strains in bovine whey explains some aspects of the pathogenesis of bovine mastitis.     

Mechanism of compact-colony formation by strains of Staphylococcus aureus in serum soft agar.

Compact-colony forming active substance (CCFAS), the material responsible for the compact colonies of Staphylococcus aureus observed in serum soft agar, was found to be an alkaline-stable, associated polysaccharide containing galactose, N-acetylglucosamine, ribitol, phosphorus and a small quantity of alanine. This substance, when extracted from strains unable to produce protein A clumping factor, was able to absorb the serum-reacting factor whereas a teichoic acid preparation of one strain could not. The formation of CCFAS was unaffected by the age of the cells, whereas when staphylococci were cultured at alkaline pH, young cells produced more clumping factor than old ones. Both fibrinogen and its degradation products were capable of inducing compact colonies in a strain of S. aureus. The ability of human sera to interact in compact-colony formation was independent of the immunoglobin content. Thus neither protein A, clumping factor, nor teichoic acid participate in the CCFAS reaction.     

Comparative study of the binding of human blood plasma fibronectin with clinical strains of staphylococci

The interaction of 62 S. aureus clinical strains and, respectively, 20 and 17 isolated S. epidermidis and S. saprophyticus strains with human blood plasma fibronectin (FN) has been studied. The specific interaction of FN with bacteria has been evaluated simultaneously by the binding of 125I with FN (method 1), the FN-mediated agglutination of staphylococci (method 2) and the character of colonies formed in 0.15% agar medium containing FN (method 3). The data obtained in this investigation indicated that all S. aureus strains under study react with FN to a different extent. When evaluating the binding of FN with bacteria, the most pronounced correlation was observed between methods 1 and 3. None of the methods used in this investigation has revealed interaction between FN and S. epidermidis and S. saprophyticus strains under study. The authors suggest that a preliminary inference on the capacity of the isolated clinical strains of staphylococci for reaction with FN may be made from the character of colonies formed in 0.15% agar medium containing FN.     

Comparison of three methods detection of slime production by Staphylococcus aureus and Staphylococcus epidermidis

In this article, slime production of Staphylococcus aureus and Staphylococcus epidermidis strains from infective skin lesions was evaluated by three different methods: Congo red agar method (CRA), Christensen tube method (CT) and spectrophotometric method (SC). All strains by CT method interpreted as negative (dark-claret or red colonies of the surface). 12 (37.5%) strains of S. aureus, 16 (50.0%) strains of S. epidermidis produced slime as shown by CT method, 6 (18.7%) strains of S. aureus, 8 (25,0%) strains of S. epidermidis by SC method. They also found a correlation of slime production by CT and SC method (p > 0.05).     

Relation of Mucoid Growth of Staphylococcus aureus to Clumping Factor Reaction, Morphology in Serum-Soft Agar, and Virulence

The growth characteristics of several strains of Staphylococcus aureus in Brain Heart Infusion and in a modified Staphylococcus Medium No. 110 were compared. In the latter medium all of the strains studied showed an increased mucoid character. Some of the strains studied showed a greater potential to synthesize excess slime layer material than others. The highly mucoid strains grew as diffuse-type colonies in modified Staphylococcus Medium No. 110 serum-soft agar and reacted as though they were negative in the test for clumping factor. These strains were also found to be more virulent when used to challenge normal mice intraperitoneally.     

A modified pork plasma agar for the enumeration of Staphylococcus aureus in foods

The coagulase reaction of Staphylococcus aureus on the PPSA (pork plasma for S. aureus) agar of Devoyod et al. was found to be fibrinogen-deficient. By including bovine fibrinogen (BFG) in the medium, the fibrin halos around S. aureus colonies became more distinct, preparations of pork plasma previously unacceptable for inclusion in the original PPSA agar were performing well, and the amount of pork plasma required in PPSA agar could be reduced by nearly 90%. In the modified medium, designated PPF (pork plasma fibrinogen) agar, the agar base (Baird-Parker agar without egg yolk) was unchanged. After surface plating, the base was covered with 8 mL of a modified overpour agar: 2.5% pork plasma, 0.38% BFG, and 0.0015% soy trypsin inhibitor in 0.7% Bacto agar. Most S. aureus strains could be enumerated after 24 h of incubation at 35 degrees C; the others required 44 h. Without soy trypsin inhibitor, a number of strains showed considerable fibrinolysis between 24 and 44 h of growth; this activity was neutralized by the inhibitor. The S. aureus counts of 27 food samples on PPF agar were essentially the same as the confirmed S. aureus counts obtained by the Baird-Parker method.     

Tandem coagulase/thermonuclease agar method for the detection of Staphylococcus aureus.

In optimizing previously reported coagulase agar media to obtain a rapid, reliable, and inexpensive coagulase test agar, variations in plasmas, pH, buffer system, fibrinogen, and fibrinolytic inhibitor were investigated. The agar with the following composition was determined best for the demonstration of coagulase production by Staphylococcus aureus: 25 ml of 15% bovine fibrinogen (fraction I, type I, citrated, Sigma Chemical Co.), 25 ml of rehydrated rabbit plasma (coagulase plasma ethylenediaminetetraacetic acid, Difco), 10.0 mg of soybean trypsin inhibitor (Schwarz/Mann), and 450 ml of brain heart infusion agar (Difco). In additional studies involving 7 different temperatures and 11 heating times, the thermal destruction of microbial nucleases on plate count agar and coagulase test agar was investigated. Heating the plates for 2.5 h at 65 degrees C destroyed all heatlabile nucleases, but not thermonucleases of S. aureus. A tandem agar plate method for the identification of S. aureus was developed. Coagulase and thermonuclease activity of 50 colonies can be detected on a single agar plate. Suspect S. aureus colonies isolated on various selective media are transferred to coagulase test agar, the plates are incubated at 37 degrees C for 18 h, and the coagulase reaction is recorded. The plates are then heated at 65 degrees C for 2.5 h, overlaid with toluidine blue-metachromatic diffusion agar, and reincubated at 37 degrees C for 3 h, and the thermonuclease reaction is recorded. Studies based on 88 enterotoxigenic S. aureus strains and 133 and 48 suspect S. aureus strains isolated from fresh salami mixtures on mannitol salt and tellurite-polymyxin-egg yolk agars, respectively, demonstrated 100% agreement between the tandem agar plate method and standard coagulase and thermonuclease tests. Overall, the tandem agar plate method is a rapid and convenient approach contributing to the identification of S. aureus from foods.     

Factors affecting growth of Staphylococcus aureus L forms on semidefined medium

Banville, Robert R. (The Catholic University of America, Washington, D.C.). Factors affecting growth of Staphylococcus aureus L forms on semidefined medium. J. Bacteriol. 87:1192-1197. 1964.-A semidefined agar medium was found suitable for production and cultivation of the L form of Staphylococcus aureus. In semidefined liquid medium, growth of the L form took place in the form of a sediment containing large masses of cells, but heavy and diffuse growth occurred in the same medium with 0.05% agar. The optimal pH for L-colony formation on solid medium was 6.5. More L colonies developed on 0.75% agar than at higher agar concentrations. L colonies developed in greater numbers on pour plates than on streak plates, and in some cases more L colonies appeared under anaerobic incubation. L-colony formation appeared to be inhibited by sodium citrate. The vitamin requirements of the L forms studied were similar to those of the classical form.     

Bacterial Interference with L-Forms

During the course of studying the effect of normal nasal flora on the growth of L-forms, a clear zone of inhibition was observed around colonies of many coagulase-negative staphylococci. Subsequent investigation demonstrated that Staphylococcus aureus and some S. albus strains produce a substance which is capable of markedly inhibiting the growth of stable staphylococcal and streptococcal L-forms. This interfering substance is separable from the staphylococcal organism and is diffusible through 1.5% agar, but not through a dialysis membrane. It is heat-stable.     

Different effects of fibronectin on the phagocytosis of Staphylococcus aureus and coagulase-negative staphylococci by murine peritoneal macrophages

Fibronectin is known to be an important factor in colonization by Staphylococcus aureus of host tissues as well as other extracellular matrix proteins such as collagen and laminin. We investigated the effect of fibronectin on the phagocytosis of the S. aureus Cowan I strain by macrophages and of coagulase-negative staphylococci (CNS) strains for comparison. Fibronectin-reduced serum in place of normal serum lowered the phagocytic activity of the macrophages on the Cowan I strain. Purified fibronectin enhanced the phagocytic activity of the strain in a dose-dependent manner. On the other hand, fibronectin did not show any opsonic effect on the ingestion of CNS strains, though the binding of fibronectin occurred equally well in CNS strains and the Cowan I strain. Fibronectin-binding protein (FnBP), the specific fibronectin receptor on the surface on S. aureus, was detected in both the Cowan I strain and CNS strains. Polymerase chain reaction confirmed that not only the Cowan I strain, but also CNS strains possessed the FnBP gene. These results indicate that fibronectin shows an opsonic effect on the S. aureus, Cowan I strain but not on CNS strains, and suggest that the binding of fibronectin to FnBP is not sufficient for efficient phagocytosis of the staphylococci strains by macrophages.     

Evaluation of methicillin-resistance in Staphylococcus aureus by the agar disk diffusion method and PCR

Staphylococcus aureus is a widespread human pathogen. One the most striking characteritics of this bacterium is resistance to methicillin and all beta-lactam antibiotics. The agar disk diffusion method is the most widely used in vitro susceptibility test, but recently molecular methods, e.g. Polymerase Chain Reaction, have been also introduced. We compared the detection of methicillin resistant coagulase positive Staphylococcus aureus isolated from clinical materials in Silesian microbiological laboratories by diffusion method and PCR through the detection of nuc and mec A genes. Our results show that PCR used for the detection of mec A gene increases the detection of methicillin-resistant Staphylococcus aureus strains by 10% as compared to the agar disk diffusion method. Among Staphylococcus aureus strains, detected as methicillin-resistant, 17% of organisms showed no presence of mec A gene.     

Enumeration of Vital and Thermally Stressed Staphylococcus aureus in Foods Using Baird-Parker Pig Plasma Agar (BPP)

Baird-Parker (BP), modified BP medium (egg yolk replaced with pig plasma, BPP) and modified Vogel and Johnson agar (phosphatidyl choline, deoxyribonucleic acid, with catalase added, PCVJ) were equally efficient for enumerating both non-stressed and thermally-stressed populations of Staphylococcus aureus from pure cultures and naturally contaminated foods. Vogel and Johnson agar was inferior. All colonies exhibiting coagulase activity on BPP were subsequently confirmed as Staph. aureus ; this was not the case with presumptive colonies from BP and PCVJ. Based on selectivity, definitive diagnostic characterization of colonies and increased sensitivity (more sample plated), BPP should be considered as the reference method for the routine enumeration of Staph. aureus in foods.     

Colony spreading in Staphylococcus aureus

Wild-type Staphylococcus aureus rapidly expands on the surface of soft agar plates. The rates of expansion and the shapes of the resultant giant colonies were distinct for different strains of laboratory stocks and clinical isolates. The colony spreading abilities did not correlate with the biofilm-forming abilities in these strains. Insertional disruption of the dltABCD operon, which functions at the step of D-alanine addition to teichoic acids, and of the tagO gene, which is responsible for the synthesis of wall teichoic acids, decreased the colony spreading ability. The results indicate that wall teichoic acids and D-alanylation of teichoic acids are required for colony spreading.     

Detection of fibronectin-binding protein genes in staphylococcal strains from peri-prosthesis infections.

Several studies have been devoted to identify the adhesion mechanisms of Staphylococcus aureus and Staphylococcus epidermidis, which are the most frequent causes of prosthesis-associated infections. Recently, in particular for Staphylococcus aureus, considerable attention has been given to the host protein receptors as mediators for bacterial adherence. Fibronectin, in important matrix protein, seems to be a major ligand for bacterial adherence in the early stages of infection. To determine the importance of the fibronectin adhesin as virulence factor in Staphylococcus-induced prosthesis infection, a simple and reliable method using a polymerase chain reaction (PCR) was devised to identify fibronectin adhesin genes (fnbA and fnbB). Results obtained by this method were in accordance with those obtained by the in vitro phenotypic characterization of binding ability to fibronectin of Staphylococcus strains.     

The binding of microorganisms isolated from patients with complicated forms of acute cholecystitis with immobilized proteins in the bacterial sorption reaction

57 strains of different microbial species, isolated from patients having complicated forms of acute cholecystitis, were studied with the use of the reaction of bacteriosorption on immobilized proteins: fibrinogen, fibronectin, gamma globulin, ovalbumin, etc. The reaction of bacteriosorption was positive with all 4 Staphylococcus aureus strains: they were adsorbed on fibrinogen, fibronectin and gamma globulin. Besides, 4 out of 18 Staphylococcus epidermidis strains were selectively adsorbed on fibronectin and 1 of these strains was adsorbed on ovalbumin, while 2 out of 15 Escherichia coli strains were adsorbed on ovalbumin. S. epidermidis strains studied in this work differed from 15 strains of the same species, isolated from the urine of patients and studied earlier, by their adsorption properties.     

Escherichia coli strains of serogroup O139 isolated from oedema disease of swine bind fibronectin

Abstract 15 Escherichia coli strains of the serogroup O139, isolated from oedema disease of swine, were examined for their ability to interact with ¹²⁵I-labelled fibronectin. All strains were positive, and all except one showed higher fibronectin binding than Staphylococcus aureus strain Cowan 1 cells (to which fibronectin bound in the order of 15% of total protein added). 7 E. coli strains isolated from diarrhoea in young piglets were also tested, and 3 were positive. 2 of these strains showed higher binding than S. aureus Cowan 1 cells. E. coli strains expressing either K99 or K88 antigen were poor binders, comparable to cells of S. aureus strain Wood 46. There was no correlation between cell surface hydrophobicity, as determined by chromatography on Octyl-Sepharose, and the fibronectin-binding property.     

Comparison of methods for the detection of methicillin resistance in Staphylococcus aureus isolates from food products

AIMS: To compare several methods for detection of methicillin resistance in Staphylococcus aureus isolates from food. METHODS AND RESULTS: Two hundred S. aureus isolates from food of animal origin were screened for methicillin resistance by a PCR assay specific for the mecA gene, an oxacillin agar screen test and a cefoxitin disk diffusion test. Six out of 200 strains (3%) were found to be methicillin-resistant Staphylococcus aureus (MRSA) by PCR. The oxacillin agar screen test detected only one of the MRSA isolates (sensitivity of 16.7%) and mischaracterized three additional strains as MRSA (specificity of 98.45%). None of the MRSA strains was detected by the cefoxitin test (sensitivity of 0%), while 15 methicillin-susceptible S. aureus (MSSA) strains were misclassified as resistant (specificity of 92.3%). Fifteen MSSA strains displayed a beta-lactamase hyperproducer-like phenotype. The six MRSA (mecA-positive) strains resembled the characteristics of heteroresistant strains. CONCLUSIONS: As MRSA of animal origin may display atypical phenotypes, PCR appears to be more reliable for detection of methicillin resistance in animal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The study stresses the need for implementing the methods of screening S. aureus from food of animal origin for methicillin resistance.     

Expression of glycocalyx by coagulase-negative Staphylococcus species isolated from bovine milk

Two hundred and six strains of coagulase-negative Staphylococcus species were assessed for expression of glycocalyx on serum soft agar, india ink and adherence techniques. The organisms were maintained on trypticase soy agar plates at 4 degrees C for 30 d (120 strains) or stored at -80 degrees C in skim milk for 90 d (60 strains). Additionally, 26 milk samples from cows known to have excreted coagulase-negative staphylococci were used to inoculate serum soft agar directly. Nine of 26 direct culture samples and 43 of 180 strains maintained for an extended period had diffuse-type growth on serum soft agar. The proportion that exhibited an unstained halo by india ink was similar regardless of storage time. Slime production determined by in vitro adherence revealed a higher proportion of positive strains than had been predicted by serum soft agar or india ink techniques. More strains of Staphylococcus chromogenes, Staph. epidermidis, Staph. hominis, Staph. simulans and Staph. warneri expressed glycocalyx than other coagulase-negative Staphylococcus species. These results suggest that most coagulase-negative staphylococci produce slime rather than a capsule. However, evidence for classical encapsulation was demonstrated in several strains by india ink. The finding that Staphylococcus species other than Staph. aureus isolated from bovine milk are capable of glycocalyx production may be of importance in investigations on the relationship between staphylococci and host defence mechanisms.