Staphylococci express a receptor for human transferrin: identification of a 42-kilodalton cell wall transferrin-binding protein

Staphylococcus aureus and the coagulase-negative staphylococci are commonly responsible for peritonitis in renal patients undergoing continuous ambulatory peritoneal dialysis. To simulate growth conditions in vivo, staphylococci isolated from peritoneal infections were cultured in used human peritoneal dialysate (HPD). Immunoblotting experiments using cell wall preparations from these staphylococci revealed the presence of the host iron-binding glycoprotein transferrin bound to S. aureus, S. epidermidis, S. capitis, S. haemolyticus, and S. hominis but not to S. warneri or S. saprophyticus. Similar results were obtained by incubating broth-grown staphylococci with human transferrin, although, in contrast to S. aureus, the coagulase-negative staphylococci bound more transferrin after growth in iron-restricted broth. To determine whether the staphylococci express a saturable specific receptor for human transferrin, the interaction of human 125I-transferrin with the staphylococci was examined. Both S. aureus and S. epidermidis bound the radiolabelled iron-saturated ligand in a time- and concentration-dependent manner. From competition binding assays, the affinity (Kd) and number of receptors were estimated for S. epidermidis (Kd, 0.27 microM; 4,200 receptors per cell) and S. aureus (Kd, 0.28 microM; 4,200 receptors per cell). S. epidermidis but not S. aureus receptor activity was partially iron regulated. Human apotransferrin and iron-saturated transferrin and rabbit and rat transferrins competed equally well for the staphylococcal receptor. Bovine and porcine transferrins and ovotransferrin as well as human and bovine lactoferrins were much less effective at competing with human transferrin. Treatment of whole staphylococci with protease abolished transferrin binding, indicating the involvement of cell surface protein. Western blots (immunoblots) of cell wall preparations probed with human transferrin revealed the presence of a 42-kDa transferrin-binding protein common to both S. aureus and S. epidermidis. On Western strip blots, the binding of human transferrin to this protein was blocked by labelled human transferrin but not by albumin, immunoglobulin G, or bovine transferrin or ovotransferrin. To assess the conservation of the 42-kDa transferrin-binding protein, cell wall proteins of S. epidermidis, S. haemolyticus, S. capitis, S. hominis, S. warneri, and S. saprophyticus were Western blotted and probed with human transferrin. Only S. warneri and S. saprophyticus lacked the 42-kDa wall protein, consistent with their inability to bind transferrin. These data show that the staphylococci express a specific receptor for human transferrin based at least in part on a common 42-kDa cell wall protein.     

Clumping of Staphylococcus aureus by human fibronectin

Clumping of different staphylococci by fibronectin and other purified plasma proteins has been investigated. Purified fibronectin was capable of clumping Staphylococcus aureus strains in concentrations identical with concentrations of fibronectin in human plasma. S. epidermidis and S saprophyticus were not clumped by fibronectin. The binding of fibronectin to S. aureus was not mediated by protein-A, as a strain lacking protein-A clumped in the presence of fibronectin, and the presence of IgG could not inhibit the clumping of S. aureus strains. The fibronectin-binding component on the staphylococcal cell wall seems to be unrelated to the fibrinogen-binding clumping factor.     

Comparative antimicrobial activity of RP 59,500 (quinupristin-dalfopristin), the first semisynthetic injectable streptgramin, against gram-positive cocci and other recent clinical pathogens

RP 59,500 (Quinupristin-Dalfopristin) is the first semisynthetic injectable streptogramin antimicrobial agent, which is a combination of quinupristin and dalfopristin in a 30:70 ratio. The components of RP 59,500 act synergically to provide bactericidal activity through action at different sites on bacterial ribosomes. In the present study, the antimicrobial activity of RP 59,500 was compared with those of four macrolides (erythromycin, clarithromycin, azithromycin, roxithromycin). Susceptibility testing was carried out by microdilution method on 303 strains of 10 species, especially antibiotic-resistant Gram-positive cocci. RP 59,500 was active against a wide range of Gram-positive cocci including methicillin-resistant Staphylococci and penicillin-resistant Streptococcus pneumoniae. The MICs90 of RP 59,500 against methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis were both 0.25 microgram/ml, although those of four macrolides were higher than 32 micrograms/ml. The MICs90 of RP 59,500 against penicillin-sensitive, -intermediate and -resistant S. pneumoniae were all 0.5 microgram/ml, although those of four macrolides against penicillin-resistant S. pneumoniae were higher than 32 micrograms/ml. RP 59,500 also exhibited equivalent activities to the four macrolides against strains of Streptococcus pyogenes. Streptococcus agalactiae and Moraxella catarrhalis. RP 59,500 exhibited the highest activities against Enterococcus faecalis, Enterococcus faecium and Enterococcus avium strains which are intrinsically resistant to most antimicrobial agents. No cross-resistance was observed between RP 59,500 and the four macrolides, which will merit attention in future clinical trials of the agent. The effect of human serum on the MIC of RP 59,500 was studied with strains of S. aureus, S. epidermidis and E. faecalis. The presence of 20% (V/V) serum had little or no effect on the MIC, although 50% (V/V) serum increased MICs by 4-8 folds. Laboratory-induced resistance to RP 59,500 occurred in a stepwise fashion in broth cultures of S. aureus, S. epidermidis and E. facalis strains and the induction rate was slow and no more than four fold increases were observed. Population analysis was performed on RP 59,500 and the reference macrolides against S. aureus ATCC 25,923 strain. Although low frequencies (less than 0.01%) of resistant sub-population were detected with EM, CAM, AZM and RXM, no RP 59,500-resistant sub-population was detected in this study.     

Extracellular and bacterial factors influencing staphylococcal phagocytosis and killing by human polymorphonuclear leukocytes

Extracellular and bacterial factors that influence the phagocytosis and killing of staphylococci by human polymorphonuclear leukocytes have been studied. Staphylococcus epidermidis strains were, in general, more rapidly phagocytized than were S. aureus strains. However, two strains of S. epidermidis had a very slow rate of ingestion. Although the rate of phagocytosis of S. aureus Wood 46 was greater than that of S. aureus 502A, the Wood 46 strain was more difficult to kill. Serum was essential for phagocytosis of both S. aureus and S. epidermidis. The opsonic titer of pooled serum was similar for S. aureus and S. epidermidis. In normal pooled serum, heat-labile factors were more important for effective phagocytosis than they were in immune serum. Although a saturation point for ingestion was reached, the percentage of ingested bacteria that remained alive within the leukocyte remained relatively fixed. Heat-killed and live staphylococci were igested in a similar fashion. The rate of phagocytosis was greatly reduced at 41 degrees C.     

Differentiation of staphylococci from sheep and goat milk samples

A total of 447 micrococcaceae strains isolated from 88 ewe and 359 goat milk samples from cases of chronic mastitis were differentiated by means of the ATB 32 STAPH-test. Of these strains 389 (= 87%) could be identified. Fourteen strains were sensitive in the bacitracin-resistance-test and therefore classified as Micrococcus spp. In ewe milk following Staphylococcus spp. were found: S. epidermidis, S. aureus, S. lentus, S. xylosus, S. warneri, S. equorum, S. haemolyticus, S. simulans, S. hominis and S. saprophyticus. Staphylococcus spp. identified in goat milk samples were: S. epidermidis, S. aureus, S. caprae, S. lentus, S. simulans, S. capitis, S. lugdunensis, S. xylosus, S. chromogenes, S. hominis, S. arlettae, S. warneri, S. sciuri, and S. saprophyticus. Highest cell counts in the milk of both animals species, and the highest incidence of clinical udder alterations were caused by S. aureus. Increases in milk cell counts as well as pathological udder findings were observed in coagulase-negative staphylococcal infections for novobiocin-sensitive Staphylococcus spp. (S. epidermidis, S. warneri, S. simulans, S. lugdunensis, and S. chromogenes) and several S. lentus strains.     

Clinical applications of nuclear medicine in gastroenterology

This study describes the antibiotic resistance of 1961 staphylococcal strains that were isolated at the University Hospital of Vienna from July to December 1991. Staphylococcus aureus (SA) represented 43.2%; coagulase-negative (CNS) staphylococci 56.8%, three quarters of which were Staphylococcus epidermidis. Excepting netilmicin, the proportion of resistant strains to all antibiotics was higher with CNS than SA. Methicillin resistance (M(r)) was found in 11.8% of SA and 30.3% of CNS. Borderline oxacillin resistance (BOR) was noted in 7.4% of SA and 32.5% of CNS. It is important to note that severe or generalized infections due to M(r) staphylococci should be treated with glycopeptide antibiotics such as vancomycin or teicoplanin from the very beginning, whereas chemotherapy of those with BOR strains may also be carried out with beta lactamase-stable beta lactam antibiotics. Comparing the results of this study with those of the first half of 1991, the respective proportion of M(r) staphylococci was significantly lower than 23.6% for SA and 47.6% for CNS recorded then. As compared with the foregoing period, however, these strains demonstrated increased resistance frequencies to gentamicin (from 81.3 to 90%), amikacin (from 35.4 to 69%), netilmicin (35.4 to 55%), and ciprofloxacin (56.2 to 64%). This is taken as an indication for the epidemic spread of a clone of resistant strains.     

Molecular cloning of a 32-kilodalton lipoprotein component of a novel iron-regulated Staphylococcus epidermidis ABC transporter

Our previous studies identified two iron-regulated cytoplasmic membrane proteins of 32 and 36 kDa expressed by both Staphylococcus epidermidis and Staphylococcus aureus. In this study we show by Triton X-114 phase partitioning and tritiated palmitic acid labelling that these proteins are lipoproteins which are anchored into the cytoplasmic membrane by their lipid-modified N termini. In common with those of some other gram-positive bacteria, these highly immunogenic lipoproteins were released from the bacterial cell into the culture supernatants, with release being promoted by growth of the bacteria under iron-restricted conditions. Immunoelectron microscopy with a monospecific rabbit antiserum to the 32-kDa S. epidermidis lipoprotein showed that the majority of the antigen was distributed throughout the staphylococcal cell wall. Only minor quantities were detected in the cytoplasmic membrane, and exposure of the lipoprotein on the bacterial surface was minimal. A monoclonal antibody raised to the 32-kDa lipoprotein of S. aureus was used in immunoblotting studies to investigate the conservation of this antigen among a variety of staphylococci. The monoclonal antibody reacted with polypeptides of 32 kDa in S. epidermidis and S. aureus and of 40 kDa in Staphylococcus hominis. No reactivity was detected with Staphylococcus lugdunensis, Staphylococcus cohni, or Staphylococcus haemolyticus. The gene encoding the 32-kDa lipoprotein from S. epidermidis has been isolated from a Lambda Zap II genomic DNA library and found to be a component of an iron-regulated operon encoding a novel ABC-type transporter. The operon contains three genes, designated sitA, -B, and -C, encoding an ATPase, a cytoplasmic membrane protein, and the 32-kDa lipoprotein, respectively. SitC shows significant homology both with a number of bacterial adhesins, including FimA of Streptococcus parasanguis and ScaA of Streptococcus gordonii, and with lipoproteins of a recently described family of ABC transporters with proven or putative metal ion transport functions. Although the solute specificity of this novel transporter has not yet been determined, we speculate that it may be involved in either siderophore- or transferrin-mediated iron uptake in S. epidermidis.     

Identification of atypical strains of Staphylococcus epidermidis by use of molecular tools

Four atypical coagulase-negative staphylococcal (CNS) isolates from clinical sources were compared with Staphylococcus epidermidis strains by ribotyping. The ribotypes of the four strains shared close rDNA restriction profiles with those of the S. epidermidis strains used. The DNA sequence encoding 16S rRNA demonstrated 99.9% homology with S. epidermidis. S1 nuclease experiments showed that these atypical strains formed a homogeneous genomic group. DNA-DNA homologies between the S. epidermidis type strain CCM 2124 and the four CNS isolates ranged from 70 to 89%. The guanine-plus-cytosine content of the deoxyribonucleic acid of the four strains ranged from 31 to 32 mol%.     

Receptor-mediated recognition and uptake of iron from human transferrin by Staphylococcus aureus and Staphylococcus epidermidis

Staphylococcus aureus and Staphylococcus epidermidis both recognize and bind the human iron-transporting glycoprotein, transferrin, via a 42-kDa cell surface protein receptor. In an iron-deficient medium, staphylococcal growth can be promoted by the addition of human diferric transferrin but not human apotransferrin. To determine whether the staphylococcal transferrin receptor is involved in the removal of iron from transferrin, we employed 6 M urea-polyacrylamide gel electrophoresis, which separates human transferrin into four forms (diferric, monoferric N-lobe, and monoferric C-lobe transferrin and apotransferrin). S. aureus and S. epidermidis but not Staphylococcus saprophyticus (which lacks the transferrin receptor) converted diferric human transferrin into its apotransferrin form within 30 min. During conversion, iron was removed sequentially from the N lobe and then from the C lobe. Metabolic poisons such as sodium azide and nigericin inhibited the release of iron from human transferrin, indicating that it is an energy-requiring process. To demonstrate that this process is receptor rather than siderophore mediated, we incubated (i) washed staphylococcal cells and (ii) the staphylococcal siderophore, staphyloferrin A, with porcine transferrin, a transferrin species which does not bind to the staphylococcal receptor. While staphyloferrin A removed iron from both human and porcine transferrins, neither S. aureus nor S. epidermidis cells could promote the release of iron from porcine transferrin. In competition binding assays, both native and recombinant N-lobe fragments of human transferrin as well as a naturally occurring human transferrin variant with a mutation in the C-lobe blocked binding of 125I-labelled transferrin. Furthermore, the staphylococci removed iron efficiently from the iron-loaded N-lobe fragment of human transferrin. These data demonstrate that the staphylococci efficiently remove iron from transferrin via a receptor-mediated process and provide evidence to suggest that there is a primary receptor recognition site on the N-lobe of human transferrin.     

Identification of mecA-related oxacillin resistance in staphylococci by the E test and the broth microdilution method

A set of 165 strains of different staphylococcal species, 67 Staphylococcus aureus, 71 novobiocin-sensitive coagulase-negative staphylococci (CNS) and 27 novobiocin-resistant CNS was used. The oxacillin and methicillin MICs were recorded after 24 and 42 h of incubation at 35 degrees C and at 30 degrees C. Significantly higher MICs were recorded at 30 degrees C compared with 35 degrees C. While a poor discrimination between mecA-positive and mecA-negative strains was obtained with methicillin, the oxacillin MICs enabled identification of resistant strains under certain conditions. The distribution of MICs differed between the three groups of species. Separation of uninduced mecA-positive (> or = 4.0 mg oxacillin/L) and mecA-negative (< or = 2.0 mg oxacillin/L) strains of S. aureus was only achieved with the E test and after 42 h of incubation. Oxacillin-induction yielded higher MICs for mecA-positive strains of S. aureus, and a separation from mecA-negative strains was achieved with the E test after 24 h and with the broth microdilution method after 42 h. Separation of mecA-positive and mecA-negative strains of novobiocin-sensitive CNS required agar supplemented with 5% blood, incubation of MIC trays and E test for 42 h, and species-specific oxacillin MIC breakpoints (S < or = 0.5 mg/L and R > or = 1.0 mg/L). The mecA-positive and mecA-negative strains of novobiocin-resistant CNS were clearly separated after 24 h of incubation by either method.     

A simple and inexpensive method for the identification of Staphylococcus epidermidis and Staphylococcus hominis

Eight hundred and ninety-two strains of Staphylococcus species were identified by means of desferrioxamine susceptibility and fermentation results of three carbohydrates, with the API Staph system (bioMérieux, France) as reference method. No identification could be obtained for 34 strains with API Staph. Of the remaining 858 strains, identical identification was obtained with 842 (98.1%). All 707 strains identified as Staphylococcus epidermidis or Staphylococcus hominis by the API Staph system were found to be desferrioxamine susceptible, and all but 5 (3.3%) of 151 strains identified as other staphylococcal species were found to be resistant, yielding an identification correlation of 99.4% for desferrioxamine. The five additional strains which were susceptible to desferrioxamine were identified as Staphylococcus capitis (2 strains), Staphylococcus lugdunensis (2 strains), and Staphylococcus warneri (1 strain) by API Staph, and as Staphylococcus epidermidis (1 strain), Staphylococcus hominis (3 strains), and one other staphylococcal species by the experimental system.     

Characteristics of the staphylococci isolated from the udder of cows with mastitis

A total of 175 strains of Staphylococcus aureus and 67 strains of Staphylococcus epidermidis were studied, isolated from 486 samples of milk secretion taken aceptically from the individual quarters of the udder of cows affected with subclinical and purulent (clinical) mastitis. The staphylococci were referred to as the causative agent of mastitis in case they were the only microflora in the seedings of the investigated material. Tests were applied as given in Fig. 1 to characterize the strains. It was found that mastitis in cows could be due to both plasma coagulating staphylococci (Staphylococcus aureus) and coagulase-negative Staphylococcus epidermidis organisms. The two Staphylococcus species were isolated from cows with clinical and subclinical mastitis. The division between pathogenic and nonpathogenic Staphylococcus strains by the plasma-coagulating symptom proved impossible, and this made it necessary to use other tests for pathogenicity. It became evident that the thing Staph. aureus and Staph. epidermidis had in common when isolated from cows with mastitis was the production of a gold-like pigment and delta hemolysin. Similarly to Staph. aureus isolated animals, the bovine Staph. epidermidis organisms did not possess fibrinolysin and rarely produced hemolysin. The isolated organisms belonging to the coagulase-positive staphylococci corresponded by their basic properties to Staphylococcus aureus var. bovis as described in the literature. The cultures of Staphylococcus epidermidis isolated under similar conditions showed in a considerable per cent of the cases somewhat different behaviour.     

Botulinum toxin A for hyperkinetic facial lines: results of a double-blind, placebo-controlled study

Although enterotoxins have been implicated in disease states such as food poisoning and toxic shock syndrome, their role in infectious arthritis is not known. To study the arthritogenic properties of toxic shock syndrome toxin 1 (TSST-1), two pairs of S. aureus strains isogenic for TSST-1 production were injected intravenously into healthy Swiss mice. Mice injected with TSST-1-secreting staphylococcal strains developed more frequent and more severe arthritis than did mice inoculated with the isogenic TSST-1-deficient counterparts. Immunohistochemical analysis of arthritic joints revealed an equal number of infiltrating phagocytes in both groups; however, mice inoculated with TSST-1-producing staphylococci had significantly more (P < .01) interleukin-2 receptor-expressing cells in the inflamed synovium than did mice that received the isogenic counterpart. Thus, TSST-1 is a virulence determinant in S. aureus arthritis in mice. The precise mechanism by which this toxin contributes to the development and progression of arthritis needs further investigation.     

Aspirin and platelets: the antiplatelet action of aspirin and its role in thrombosis treatment and prophylaxis

The aim of this study was to investigate the influence of iron present in the growth medium of Staphylococcus aureus on the bacterial adhesion to collagen. The experiments were extended to determinate the siderophore production and to examine the S. aureus isolates surface hydrophobicity. The addition of iron to metal deficient defined medium causes the change in hydrophobicity of the examined S. aureus strains surfaces from hydrophilic to hydrophobic. The presence of iron in staphylococcal growth medium alters also the adhesion to the surface covered with collagen. Four out of six S. aureus strains adhere to collagen weaker when cells come from iron-rich medium. Majority of tested strains produce markedly less of siderophores in media containing the excess of iron (1 and 10 microM Fe) and there is no staphylococcal siderophore activity in the growth medium with a very high concentration of this compound (120 microM Fe). The obtained results indicate that the iron-stressed conditions influence the staphylococcal adhesion to collagen.     

Diversity of adenosine degradation in staphylococci

Products of the early steps of adenosine catabolism by resting cells of staphylococci were chromatographically separated. Under the assay conditions employed, adenosine deaminase activity was associated with S. intermedius and S. epidermidis but it was absent from S. aureus and S. auricularis. Adenosine nucleosidase activity was associated with S. epidermidis, S. aureus and S. auricularis but it was absent from S. intermedius.     

Antibiotic sensitivity of various Staphylococcus species isolated from newborn infants with purulent septic infections

Sensitivity of 120 staphylococcal strains belonging to different species isolated from newborn infants with pyoseptic infections was studied with respect to 13 antibiotics. Methicillin, cephaloridin, rifampicin, ristomycin, gentamicin and novobiocin were mainly highly active against the isolates, while some of the strains were resistant. The coagulase negative species were as a whole characterized by higher resistance levels with respect to some antibiotics, as compared to Staph. aureus. The predominant part of the coagulase negative strains with exceedingly high resistance levels did not belong to Staph. epidermidis. The sensitivity of staphylococci depended on the source of their isolation. At large the strains isolated from purulent excretions of localized foci were characterized by lower levels of resistance to a number of antibiotics (benzylpenicillin, tetracycline, erythromycin, methicillin and others) as compared to the strains isolated from the blood.     

Toxin involvement in staphylococcal scalded skin syndrome

The production of staphylococcal exfoliative toxin A (ETA) and toxin B (ETB), toxic shock syndrome toxin (TSST-1), and enterotoxins A-E was analyzed in 60 Staphylococcus aureus strains isolated from children with scalded skin syndrome (15 with generalized exfoliative syndrome, 28 with bullous impetigo, and 17 with staphylococcal scarlet fever). All strains isolated from patients with generalized exfoliative syndrome or bullous impetigo produced ETA and/or ETB and caused a Nikolsky's sign when injected subcutaneously into newborn mice. In contrast, exfoliative toxin was detected in an S. aureus strain from only one of 17 case of staphylococcal scarlet fever; the 16 other S. aureus strains produced TSST-1 and/or an enterotoxin. In conclusion, enterotoxins or TSST-1 are more frequently associated with staphylococcal scarlet fever than are exfoliative toxins. Hence staphylococcal scarlet fever may well represent an abortive form of toxic shock syndrome rather than a milder form of staphylococcal scalded skin syndrome.