Effect of Milk on Fibronectin and Collagen Type I Binding to \u3ci\u3eStaphylococcus aureus\u3c/i\u3e and Coagulase-Negative Staphylococci Isolated from Bovine Mastitis

Tryptic soy broth (TSB)-grown cells of Staphylococcus aureus isolated from acute and chronic bovine mastitis bound mainly 125I-fibronectin (125I-Fn), whereas strains of nine species of coagulase-negative staphylococci showed a predominant interaction with 125I-collagen (125I-Cn) type I. A particle agglutination assay (PAA) was used to examine the interaction of coagulase-negative staphylococci with 1251-Fn and 125I-Cn immobilized on latex. All 368 coagulase-negative staphylococci demonstrated high 125I-Cn and moderate to low 125I-Fn interactions in the PAA. Cn-PAA reactivity was high among strains of Staphylococcus xylosus (84.2%), Staphylococcus simulans (77.8%), Staphylococcus epidermidis (76.7%), and Staphylococcus hyicus (74.3%), whereas all six Staphylococcus capitis strains clumped Cn-PAA reagent. Incubating TSB-grown cells in 10% skim milk for 1 h decreased the 125I-Fn- and 125I-Cn-binding affinity in most of the S. aureus and coagulase-negative staphylococci, while growth in 10% skim milk for 18 h resulted in more than 90% decrease or complete loss of interaction with these proteins. Decreased 1251-Fn binding in the presence of milk was correlated with protease production but not with 125I-Cn binding

Effect of Milk on Fibronectin and Collagen Type I Binding to \u3ci\u3eStaphylococcus aureus\u3c/i\u3e and Coagulase-Negative Staphylococci Isolated from Bovine Mastitis

Tryptic soy broth (TSB)-grown cells of Staphylococcus aureus isolated from acute and chronic bovine mastitis bound mainly 125I-fibronectin (125I-Fn), whereas strains of nine species of coagulase-negative staphylococci showed a predominant interaction with 125I-collagen (125I-Cn) type I. A particle agglutination assay (PAA) was used to examine the interaction of coagulase-negative staphylococci with 1251-Fn and 125I-Cn immobilized on latex. All 368 coagulase-negative staphylococci demonstrated high 125I-Cn and moderate to low 125I-Fn interactions in the PAA. Cn-PAA reactivity was high among strains of Staphylococcus xylosus (84.2%), Staphylococcus simulans (77.8%), Staphylococcus epidermidis (76.7%), and Staphylococcus hyicus (74.3%), whereas all six Staphylococcus capitis strains clumped Cn-PAA reagent. Incubating TSB-grown cells in 10% skim milk for 1 h decreased the 125I-Fn- and 125I-Cn-binding affinity in most of the S. aureus and coagulase-negative staphylococci, while growth in 10% skim milk for 18 h resulted in more than 90% decrease or complete loss of interaction with these proteins. Decreased 1251-Fn binding in the presence of milk was correlated with protease production but not with 125I-Cn binding

PHYSIOLOGY AND MANAGEMENT Bovine Lactoferrin Receptors in Staphylococcus aureus Isolated from Bovine Mastitis

ABSTRACT A total of 103 Staphylococcus a w e w strains isolated from bovine mastitis were tested for bovine lactoferrin binding in a 1251-labeled protein binding assay. More than 85% of the strains demonstrated high to moderate and a few showed little or no binding. Bovine lactoferrin binding to S. aureus cells was high when grown on blood, nutrient, or proteose-peptone agar, but the binding capacity was low with cells grown on salt rich media, in skim milk, or in broth. The kinetics of 1251-labeled bovine lactoferrin binding required approximately 90 min for complete saturation with optimal interaction in the pH range 4.0 to 7.0. The lactoferrin-staphylococci interaction was specific with a high affinity (association constant, K, 14 x 106 L/mol). Scatchard plot analysis estimated the number of binding sites per cell at 7200 on strain SA-340. Unlabeled bovine lactoferrin effectively displaced the binding of the labeled ligand to strain SA-340 in a dosedependent manner. Abbreviation key: bLf = bovine lactoferrin, hLf = human lactoferrin, HRPO = horseradish peroxidase, Lf = lactoferrin, PBS = phosphatebuffered saline, PMN = polymorphonuclear leukocytes

Joint genomic and proteomic analysis identifies meta-trait characteristics of virulent and non-virulent Staphylococcus aureus strains

Staphylococcus aureus is an opportunistic pathogen of humans and warm-blooded animals and presents a growing threat in terms of multi-drug resistance. Despite numerous studies, the basis of staphylococcal virulence and switching between commensal and pathogenic phenotypes is not fully understood. Using genomics, we show here that S. aureus strains exhibiting virulent (VIR) and non-virulent (NVIR) phenotypes in a chicken embryo infection model genetically fall into two separate groups, with the VIR group being much more cohesive than the NVIR group. Significantly, the genes encoding known staphylococcal virulence factors, such as clumping factors, are either found in different allelic variants in the genomes of NVIR strains (compared to VIR strains) or are inactive pseudogenes. Moreover, the pyruvate carboxylase and gamma-aminobutyrate permease genes, which were previously linked with virulence, are pseudogenized in NVIR strain ch22. Further, we use comprehensive proteomics tools to characterize strains that show opposing phenotypes in a chicken embryo virulence model. VIR strain CH21 had an elevated level of diapolycopene oxygenase involved in staphyloxanthin production (protection against free radicals) and expressed a higher level of immunoglobulin-binding protein Sbi on its surface compared to NVIR strain ch22. Furthermore, joint genomic and proteomic approaches linked the elevated production of superoxide dismutase and DNA-binding protein by NVIR strain ch22 with gene duplications

Development of a reference data set for assigning Streptococcus and Enterococcus species based on next generation sequencing of the 16S-23S rRNA region

Background: Many members of Streptococcus and Enterococcus genera are clinically relevant opportunistic pathogens warranting accurate and rapid identification for targeted therapy. Currently, the developed method based on next generation sequencing (NGS) of the 16S-23S rRNA region proved to be a rapid, reliable and precise approach for species identification directly from polymicrobial and challenging clinical samples. The introduction of this new method to routine diagnostics is hindered by a lack of the reference sequences for the 16S-23S rRNA region for many bacterial species. The aim of this study was to develop a careful assignment for streptococcal and enterococcal species based on NGS of the 16S-23S rRNA region. Methods: Thirty two strains recovered from clinical samples and 19 reference strains representing 42 streptococcal species and nine enterococcal species were subjected to bacterial identification by four Sanger-based sequencing methods targeting the genes encoding (i) 16S rRNA, (ii) sodA, (iii) tuf and (iv) rpoB; and NGS of the 16S-23S rRNA region. Results: This study allowed obtainment and deposition of reference sequences of the 16S-23S rRNA region for 15 streptococcal and 3 enterococcal species followed by enrichment for 27 and 6 species, respectively, for which reference sequences were available in the databases. For Streptococcus, NGS of the 16S-23S rRNA region was as discriminative as Sanger sequencing of the tuf and rpoB genes allowing for an unambiguous identification of 93% of analyzed species. For Enterococcus, sodA, tuf and rpoB genes sequencing allowed for identification of all species, while the NGS-based method did not allow for identification of only one enterococcal species. For both genera, the sequence analysis of the 16S rRNA gene was endowed with a low identification potential and was inferior to that of other tested identification methods. Moreover, in case of phylogenetically related species the sequence analysis of only the intergenic spacer region was not sufficient enough to precisely identify Streptococcus strains at the species level. Conclusions: Based on the developed reference dataset, clinically relevant streptococcal and enterococcal species can now be reliably identified by 16S-23S rRNA sequences in samples. This study will be useful for introduction of a novel diagnostic tool, NGS of the 16S-23S rRNA region, which undoubtedly is an improvement for reliable culture-independent species identification directly from polymicrobially constituted clinical samples

[Selected parameters of the cellular and humoral immunity in atopic dermatitis. Relationship to the severity of the disease]

Despite the great progress, our understanding of the pathogenesis of atopic dermatitis (AD) is still incomplete. In particular, the clinical importance of various changes of the immune system parameters is unclear. Accordingly we have undertaken the study to compare selected parameters of cellular and humoral immunity between AD subjects (n = 26) and healthy controls (n = 10). These parameters included immunoglobulin levels (IgE in particular), neutrophil respiratory, oxygen burst, peripheral blood lymphocyte phenotype and response to mitogens. We also analysed the relationship between these parameters and clinical severity of skin lesions. RESULTS Mean total immunoglobulin E levels were very significantly increased in the AD group (1563 +/- 459 vs 35.5 +/- 12.1 IU/ml; p = 0.001). Simultaneously total serum IgE levels varied extensively between individual subjects with AD and were significantly correlated to clinical severity of the disease (Rs = 0.44; p = 0.02). Atopic dermatitis was also associated with the increase in the number of CD4+ and simultaneous decrease in the CD8+ lymphocytes causing statistically significant difference in CD4:CD8 ratio compared to the control group. We also observed changes of proliferation indices to phytohaemagglutinin (decrease) and increase of responses to anti CD3 mAb (OKT-3) and staphylococcal enterotoxin B. None of these immune parameters however, appeared to be statistically correlated to clinical status. CONCLUSIONS We find that atopic dermatitis is associated with significant changes of several important indices of cellular and humoral immunity including increased IgE levels and altered peripheral lymphocyte proliferation capacity and phenotype. Change of total IgE levels appears to be the most important from clinical point of view

[Influence of treating atopic dermatitis with oral antihistamine and topical steroids on selected parameters of cell and humoral immunity]

Pathogenesis of atopic dermatitis (AD) is multifactorial, therefore despite many different treatment protocols none of the available methods is effective enough. In the present study we analysed the effects of oral antihistamines and topical steroids on selected parameters of humoral (immunoglobulin levels) and cellular (lymphocyte proliferation index, granulocyte oxidative burst, lymphocyte phenotype CD4:CD8) immunity. These parameters were analysed in 34 AD subjects during the exacerbation of the disease and after 4 and 12 weeks of the treatment. RESULTS Along with the clinical status improvement (change form 3.17 +/- 0.1 to 1.3 +/- 0.2; 0-4 scale; p < 0.001), total serum IgE levels decreased significantly from 1563 +/- 459 to 1266 +/- +/- 364 IU/ml (p < 0.05). Other immunoglobulin levels did not change. Total blood chemiluminescence in response to latex stimulation was relatively higher during exacerbation than during remission, but this difference was not significant (p = 0.1). Mean spontaneous or PMA stimulated chemiluminescence did not differ either. CD4:CD8 index decreased significantly during the treatment (from 2.7 +/- 0.3 to 1.8 +/- 0.2; p = 0.03). Lymphocyte proliferation in response to PHA was significantly lower during exacerbation than during remission (10.4 +/- 2.8 vs. 27.1 +/- +/- 5.2; p < 0.03) and the improvement was observed after first 4 weeks of the treatment reaching the levels found in healthy controls. Proliferation index in response to anti-CD3 mAb (OKT-3) was in turn significantly higher during exacerbation (19.1 +/- 3.4 vs. 11.2 +/- 2.0; p = 0.04). CONCLUSIONS Oral antihistamine in combination with topical steroids leads to several significant changes in the immune parameters (IgE levels, CD4:CD8 and proliferation indexes) which may explain its high effectiveness in majority of patients with AD