The survival of Staphylococcus aureus in raw milk with different cell count

The antibacterial activity of the individual udder quarter milk sample cannot be evaluated by the number of somatic cells. Milk with the highest number of cells is not always the most bactericidal. However, when the data were obtained from a material large enough for statistical calculations, negative and highly significant correlation coefficient (-0.465, P less than or equal to 0.01) between number of somatic cells in milk and number of bacteria surviving in it, and negative and significant correlation coefficient (-0.203, P 0.05) between number of somatic cells in milk and number of bacteria surviving in whey, were found. Results of the analysis of variance demonstrated highly significant differences as regards number of cells and bacteria surviving in milk and in whey between cows, quarters and stimulation time. Nevertheless, among ten investigated cows, three cows with high cell count and high antibacterial activity in milk and whey, highly reacting to stimulant, were found.     

Cell-mediated immunoselection against cell-surface antigens of somatic cell hybrids.

Cytotoxic lymphoid cells derived from in vivo immunization of mice across H2 barriers were utilized in in vitro cytotoxicity assays. The target cells were somatic cell hybrids derived from parental cells differing at the H2 locus. The hybrid cells surviving cytotoxicity were grown to confluent populations and the H2 antigens selected against were no longer demonstrable by indirect immunofluorescence. Comparative karyology of hybrid cells expressing both parental H2 types before immunoselection with hybrid cells surviving immunoselection revealed a decrease in the number of murine chromosomes number 17, suggesting that those cells surviving cytotoxicity had spontaneously lost these chromosomes prior to the selection event. The possibility of immunoconstruction of somatic cell hybrids on the basis of their cell-surface antigens is discussed.     

Quantitative PCR for detection and discrimination of the bloodborne pathogen Staphylococcus epidermidis in platelet preparations using divIVA and icaA as target genes

Bacterial contamination of blood components is the major microbiological cause of transfusion-associated morbidity, with Staphylococcus epidermidis being the most frequently isolated organism from contaminated platelet preparations (PPs). We have recently shown that S. epidermidis forms biofilms during platelet storage, which might account for reported missed detection during routine screening. In this study, we developed a highly sensitive and specific multiplex quantitative PCR (QPCR) assay to detect S. epidermidis in PPs at levels of 10(2)-10(3) cfu/mL. A specific primer pair and hydrolysis probe were designed to amplify an internal region of the cell division divIVA gene that is unique to S. epidermidis. In addition, an internal sequence of the virulence gene icaA, which is involved in the synthesis of the S. epidermidis biofilm matrix, was selected to allow for differentiation of potentially biofilm-forming S. epidermidis isolates. A conserved region of the 8 alleles of the HLA-DQalpha1 locus present in residual white blood cells in PPs was selected as an internal control for the assay. The specificity of this assay was confirmed, as other staphylococcal species that were tested with the optimized parameters were not detected. This QPCR assay could be adaptable for the detection of other bloodborne bacterial pathogens.     

Proliferation of germ cells and somatic cells in first trimester human embryonic gonads as indicated by S and S+G2+M phase fractions

Objectives: The number of germ cells and somatic cells in human embryonic and foetal gonads has previously been estimated by stereological methods, which are time‐ and labour‐consuming with little information concerning cell proliferation. Here, we studied whether flow cytometry could be applied as an easier method, also enabling estimation of the fraction of cells in S or S+G₂+M (SG₂M) cell‐cycle phases as indicators of cell proliferation. Methods: Cell suspensions from 35 human embryonic gonads at days 37 to 68 post‐conception (pc) were immunomagnetically sorted into C‐KIT positive (germ) cells and negative (somatic) cells. They were stained for DNA content and analysed by flow cytometry. S and SG₂M fractions could be measured for 13 of the female and 20 of the male gonads. The number of cells was estimated using fluorescent reference beads. Results: During the period from 37 to 68 days pc, female germ and somatic cells had a stable S and SG₂M fractions indicating steady growth of both subpopulations, whereas they decreased in both male germ and somatic cells. The number of germ and somatic cells estimated by flow cytometry was significantly lower than in stereological estimates, suggesting loss of cells during preparation. Conclusions: Cell proliferation as indicated by S and SG₂M fractions could be estimated specifically for primordial germ and somatic cells. Estimation of total number of germ and somatic cells was not feasible.     

Extraction and determination of adenosine 5'-triphosphate in bovine milk by the firefly luciferase assay

The release of ATP from somatic cells in milk with the detergent Triton X-100 was optimized for assay with firefly luciferase. A small volume of milk (40 microliters) is added to 0.8 ml of 0.2% Triton X-100 in 100 mM Tris, 4 mm EDTA, pH 7.8. After approximately 1 min, 0.2 ml of luciferase reagent is added and the emission of light is measured in a luminometer. Results are calibrated with an ATP standard. This single method gave high yields of ATP from somatic cells in milk without interference from bacterial ATP. Extracts could be stored or transported prior to assay without deterioration of results. A close correlation was found between somatic cell count and ATP in milk samples collected at a farm as well as in milk samples from a cow with experimental mastitis. Results are promising for future use for diagnosis of mastitis but further work and field testing has to be done before it can be used on a wider scale.     

TNF-α reduces the level of Staphylococcus epidermidis internalization by bovine endothelial cells

Staphylococcus epidermidis is an environmental opportunistic pathogen associated with bovine intramammary infections. In bacterial infections, the endothelial tissue plays an important role during inflammation and it is the target of proinflammatory cytokines such as tumor necrosis factor α (TNF-α). Therefore, this work was designed to explore the effect of TNF-α on the interaction of S. epidermidis with bovine endothelial cells (BEC). We show that cell signaling activated by TNF-α caused a marked reduction in the number of intracellular S. epidermidis , suggesting that molecules participating in this pathway were involved in the internalization of this bacterium. We also found that S. epidermidis internalization was not associated with basal levels of nuclear factor kappa B (NF-κB) activity because the intracellular number of bacteria recovered after treating BEC with the NF-κB inhibitors, SN50 or BAY 11–7083, was similar to that of the untreated control. Interestingly, inhibition of the basal activity of JNK with SP600125 and p38 with SB203580 caused a decrease in the number of intracellular S. epidermidis . These results suggest that activation of the signaling pathway initiated by TNF-α could play an important role in the phagocytosis of this bacterium. However, the basal activity of NF-κB was shown not to be important for the internalization process of S. epidermidis .     

Flow Cytometric Measurement of Bovine Milk Neutrophil Phagocytosis

A flow cytometric method for the evaluation of the phagocytic capacity of bovine milk polymorphonuclear neutrophils (PMN) is described. Milk PMN were isolated from stripping milk collected from udder quarters fitted with abraded intramammary devices (AIMD). A significant increase in the milk somatic cell count was observed in the stripping milk after the insertion of AIMD (308×103 and 1447×l03 cells/ml milk before respectively after the insertion of the AIMD, p < 0.001). PMN were also isolated from blood by a discontinous gradient of Percoli. Blood and milk PMN were incubated for 15 min with FITC-labeled bacteria in a ratio of 1 PMN:20 bacteria and a final serum concentration of 10 %. The number of extracellular bacteria and the percentage of phagocytic cells were measured by a flow cytometer. Percentage of phagocytized bacteria by milk PMN was significantly lower than that by blood PMN (p < 0.05). A smaller number of active phagocytes was present among cells isolated from milk than among cells isolated from blood. The phagocytic capacity of milk PMN reflects that of blood PMN in the same animal. A large variation in the phagocytic capacity of blood and milk PMN among animals was observed.     

Influence of Milk Components on the Injury, Repair of Injury, and Death of Salmonella anatum Cells Subjected to Freezing and Thawing

Fast freezing and slow thawing Salmonella anatum cells in various milk components inactivated from 20 to 98% of the cells and damaged 40 to 90% of the cells surviving the treatments. Injured cells failed to form colonies on a selective medium (xylose-lysine-peptone agar with 0.2% sodium deoxycholate) but did form colonies on a nonselective plating medium (xylose-lysine-peptone agar). The major milk components-lactose, milk salts, casein, and whey proteins-influenced the extent of injury, repair of injury, and death. The percentages of cells injured and inactivated were decreased by the presence of any milk components except whey proteins. Also, repair of injury was promoted by the presence of each milk component except whey proteins, which in contrast inhibited repair. Phosphate was the most influential milk salts component that protected the cells and promoted repair of injury. These individual milk components may have decreased the extent of freezing-induced death and cellular damage by stabilizing the S. anatum cell envelope.     

Violagabriellae variant of Staphylococcus epidermidis on normal human skin

Twenty-seven strains of a reddish violet pigment-producing variety of Staphylococcus epidermidis have been recovered from normal human skin. They closely resemble the previously unique Castellani strain, named Micrococcus violagabriellae, which was placed by K. J. Steel in S. epidermidis. These strains are classified as S. epidermidis by using the Baird-Parker schema; however, besides the pigment produced, they also differ from S. epidermidis in proteolytic activity and effect on litmus milk. The variety seems to be part of the normal flora of the human axilla.     

Is the GehD lipase from Staphylococcus epidermidis a collagen binding adhesin?

The opportunistic human pathogen Staphylococcus epidermidis is the major cause of nosocomial biomaterial infections. S. epidermidis has the ability to attach to indwelling materials coated with extracellular matrix proteins such as fibrinogen, fibronectin, vitronectin, and collagen. To identify the proteins necessary for S. epidermidis attachment to collagen, we screened an expression library using digoxigenin-labeled collagen as well as two monoclonal antibodies generated against the Staphylococcus aureus collagen-adhesin, Cna, as probes. These monoclonal antibodies recognize collagen binding epitopes on the surface of S. aureus and S. epidermidis cells. Using this approach, we identified GehD, the extracellular lipase originally found in S. epidermidis 9, as a collagen-binding protein. Despite the monoclonal antibody cross-reactivity, the GehD amino acid sequence and predicted structure are radically different from those of Cna. The mature GehD circular dichroism spectra differs from that of Cna but strongly resembles that of a mammalian cell-surface collagen binding receptor, known as the alpha(1) integrin I domain, suggesting that they have similar secondary structures. The GehD protein is translated as a preproenzyme, secreted, and post-translationally processed into mature lipase. GehD does not have the conserved LPXTG C-terminal motif present in cell wall-anchored proteins, but it can be detected in lysostaphin cell wall extracts. A recombinant version of mature GehD binds to collagens type I, II, and IV adsorbed onto microtiter plates in a dose-dependent saturable manner. Recombinant, mature GehD protein and anti-GehD antibodies can inhibit the attachment of S. epidermidis to immobilized collagen. These results provide evidence that GehD may be a bi-functional molecule, acting not only as a lipase but also as a cell surface-associated collagen adhesin.     

Identification of a novel antigen from Staphylococcus epidermidis

A genomic DNA library of Staphylococcus epidermidis NCTC 11047 was constructed, using the Lambda Zap Express cloning vector, and screened with serum collected from a patient with S. epidermidis endocarditis. Sequence analysis of a 30 kDa cloned protein, termed staphylococcal secretory antigen, SsaA, identified a novel protein not previously reported in S. epidermidis. SsaA showed strong homology with two other staphylococcal proteins: SceB from Staphylococcus carnosus and a staphyloxanthin biosynthesis protein from Staphylococcus aureus. Further investigation revealed SsaA to be a highly antigenic protein that was expressed in vivo and could be recovered from whole cells and from the culture supernatant. A combination of Western blot analysis and PCR screening identified SsaA or a homologue in 103/103 staphylococcal strains. SsaA-like genes were not detected in other Gram-positive bacteria of medical importance or a number of Gram-negative organisms. Elevated anti-SsaA IgG antibody levels were detected in sera of five patients with S. epidermidis endocarditis but not in patients with other S. epidermidis infections, endocarditis of other aetiologies or patients with no evidence of infection. The expression of SsaA during episodes of S. epidermidis endocarditis suggests a virulence role specific to the pathogenesis of this infectious disease.     

Staphylococcal and other Bacterial Species Associated with Intramammary Infections in Danish Dairy Herds

Four thousand six hundred forty– five quarter milk samples from 1179 cows from 20 commercial dairy herds were examined in order to determine the prevalence of bacterial species. A total of 859 isolates from 839 (18.1%) culture positive samples could be assigned to 34 different species and subspecies. Diagnostics of staphylococcal species was based on conventional procedures able to differentiate between all 36 species and subspecies presently acknowledged. Staphylococcus aureus was found in 10.2% of the samples and was the most common species isolated. Streptococcus dysgalactiae (1.6%) and Streptococcus uberis (1.4%) were the second and third most common species isolated. Seventeen different coagulase negative staphylococcal species (CNS) were found in 4.1% of the samples. The most frequently isolated CNS were S. epidermidis (1.3%), S. chromogenes (1.0%) and S. simulans (0.7%). Isolates of S. aureus were phage typed, and isolates of S. epidermidis were investigated by phage typing, antibiogram typing, and biotyping. A total of 378 (79.9%) isolates of S. aureus could be typed by phages, assigning them to 18 different phage types. However, 6 phage types accounted for 92.1% of the typable isolates. One to 2 phage types predominated within each herd. Eleven (18%) isolates of S. epidermidis could be typed by phages, assigning the isolates to 3 different types. Biotyping of S. epidermidis produced a total of 8 different types, the most common accounting for 29.5% of the isolates. A total of 6 different antibiogram types were observed among all isolates of S. epidermidis. Resistance towards penicillin (36.1%), tetracycline (9.8%) and streptomycin (9.8%), were recorded in the isolates of S. epidermidis. However, 35 (57.4%) of the isolates were susceptible to all 12 antibiotics tested.     

Hypothesis: cell volume limits cell divisions

Mammalian somatic cells and also cells of the yeast Saccharomyces cerevisiae are capable of undergoing a limited number of divisions. Reaching the division limit is referred to, apparently not very fortunately, as replicative aging. A common feature of S. cerevisiae cells and fibroblasts approaching the limit of cell divisions in vitro is attaining giant volumes. In yeast cells this phenomenon is an inevitable consequence of budding so it is not causally related to aging. Therefore, reaching a critically large cell volume may underlie the limit of cell divisions. A similar phenomenon may limit the number of cell divisions of cultured mammalian cells. The term replicative (generative) aging may be therefore illegitimate.     

Intramammary immunization with live-attenuated Staphylococcus aureus protects mice from experimental mastitis

Female mice were immunized by the intramammary route with live-attenuated Staphylococcus aureus according to different schedules and challenged with virulent S. aureus. Immunization in late pregnancy or early lactation induced a significant decrease (P<0.05) in the number of S. aureus CFU recovered from glands after the challenge and a significant increase (P<0.05) in the levels of milk and serum specific IgG and IgA antibodies. Mice immunized before pregnancy were not protected from S. aureus challenge. Immunization did not increase the number of somatic cells in milk when compared with control mice. Protection from S. aureus intramammary infection may be achieved if mice are locally immunized during late pregnancy or early lactation.     

Flow Cytometry Approach to Quantify the Viability of Milk Somatic Cell Counts after Various Physico-Chemical Treatments

Flow cytometry has been used as a routine method to count somatic cells in milk, and to ascertain udder health and milk quality. However, few studies investigate the viability of somatic cells and even fewer at a subpopulation level to follow up how the cells can resist to various stresses that can be encountered during technological processes. To address this issue, a flow cytometry approach was used to simultaneously identify cell types of bovine milk using cell-specific antibodies and to measure the cell viability among the identified subpopulations by using a live/dead cell viability kit. Confirmation of the cell viability was performed by using conventional microscopy. Different physico-chemical treatments were carried out on standardized cell samples, such as heat treatment, various centrifugation rates and storage in milk or in PBS pH 7.4 for three days. Cytometry gating strategy was developed by using blood cell samples stored at 4°C in PBS and milk cell samples heat-treated at 80°C for 30 min as a control for the maximum (95.9%) and minimum (0.7%) values of cell viability respectively. Cell viability in the initial samples was 39.5% for all cells and varied for each cell population from 26.7% for PMNs, to 32.6% for macrophages, and 58.3% for lymphocytes. Regarding the physico-chemical treatments applied, somatic cells did not sustain heat treatment at 60°C and 80°C in contrast to changes in centrifugation rates, for which only the higher level, i.e. 5000×g led to a cell viability decrease, down to 9.4%, but no significant changes within the cell subpopulation distribution were observed. Finally, the somatic cells were better preserved in milk after 72h storage, in particular PMNs, that maintained a viability of 34.0 ± 2.9% compared to 4.9±1.9% in PBS, while there was almost no changes for macrophages (41.7 ± 5.7% in milk vs 31.2 ± 2.4% in PBS) and lymphocytes (25.3 ± 3.0% in milk vs 11.4 ± 3.1% in PBS). This study provides a new array to better understand milk cell biology and to establish the relationship between the cell viability and the release of their endogenous enzymes in dairy matrix.     

In vitro method to assess the antimicrobial activity and potential efficacy of novel types of wound dressings

AIMS: To develop a simple, reproducible in vitro static diffusion method using cellulose disks and defined species to test antimicrobial efficacy of wound dressings. METHODS AND RESULTS: Cellulose disks were inoculated by immersion in cell suspensions of target species Staphylococcus epidermidis, Candida albicans and Fusobacterium nucleatum. Test and control wound dressings were cut into equal sized squares (25 x 25 mm) and applied to the surface of 10-mm thick tryptone yeast extract agar on test beds. Following a 2-h equilibration period, inoculated cellulose disks were inserted (one per dressing) at the interface between dressing and agar surface and a small weight applied over each square. At various sampling times, disks were removed and surviving cells enumerated by viable counts. Disk to disk variation for microbial loading was assessed using S. epidermidis for both initial (n = 16) and standard treatment (n = 16) conditions. The coefficient of variation was low (<5%) indicating good reproducibility for cell loading and treatment position on the test bed. Replicate assays (n = 6) using S. epidermidis and oxyzyme gels produced similar kill rates with low scatter (R2 > 0.9) indicating good reproducibility between assays. Significant differences (P < 0.05) in kill rates were observed for different target species, types of dressing and test bed conditions (+/-blood and nutrients). CONCLUSIONS: The method is reproducible and useful in tracking the death kinetics of test species, enabling the comparison of different types of dressing. SIGNIFICANCE AND IMPACT OF THE STUDY: The reported method has significant advantages over established test procedures; it can be applied equally across a wide range of target species (including anaerobes and yeasts), a wide range of conditions, and different types of surface dressings, including those relying upon oxygen diffusion.     

The influence of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis>-derived surfactants on staphylococcal adhesion and biofilm formation

The ability of surfactants obtained from three Lactobacillus acidophilus strains to inhibit Staphylococcus aureus and S. epidermidis biofilms was evaluated. Their influence was determined on bacterial initial adhesion, biofilm formation and dispersal using MTT-reduction assay, confocal laser scanning microscopy and image PHLIP analysis. The number of adhering S. aureus and S. epidermidis cells after a 3-h co-incubation with biosurfactants was reduced by 5-56 % in a strain-and dose-dependent manner. S. epidermidis-and, to a lower extent, in S. aureus-biofilm formation was also inhibited in the presence of the tested surfactants. The addition of surfactants to preformed mature biofilms accelerated their dispersal, and changed the parameters of biofilm morphology. The L. acidophilus-derived surfactants inhibit bacterial deposition rate and biofilm development (and also its maturation) without affecting cell growth probably due to the influence on the cell-surface hydrophobicity of staphylococci.     

Characteristics of the composition of higher fatty acids in methicillin-resistant and methicillin-sensitive Staphylococci

The composition of fatty acids of methicillin-resistant (MR) and methicillin-sensitive (MS) strains of Staph. aureus and Staph. epidermidis was determined with the method of reactive gas liquid chromatography. The MS staphylococci of the above species differed by the content of acids with branched chains of iso- and anteisostructures and straight chains. Anteisoacids in the cells of Staph. epidermidis amounted almost to 80 per cent of the total number of the acids, while in the cells of Staph. aureus, their total number amounted only to a half of the fatty acid pool. Comparison of the composition of the fatty acids of the MS and MR strains of Staph. aureus revealed differences in the proportions of the anteiso- and isoacids. The total number of the long-chain C20.0 acid in the cells of Staph. epidermidis resistant to methicillin was lower as compared to that in the sensitive cells.     

In vitro measurement of the adherence of Staphylococcus epidermidis to plastic by using cellular urease as a marker.

A rapid and sensitive in vitro assay was developed to quantitatively assess the adherence of Staphylococcus epidermidis to a hydrophobic plastic surface. The assay is based upon the detection of cell-associated urease activity as a marker of bacteria remaining adherent to the polystyrene microwells of flat-bottomed, 96-well tissue culture plates. Using ATCC 35984, a slime-producing strain of S. epidermidis, the assay could detect as few as 3 x 10(3) bacteria and was linear to 3.5 x 10(7) bacteria. The adherence of both slime-positive and slime-negative coagulase-negative staphylococci could be evaluated by using this method. This assay could be used to examine factors which influence the adherence of individual S. epidermidis strains to hydrophobic surfaces and to develop agents or coating materials which suppress the adherence of coagulase-negative staphylococci to biomedical implants.