Antibiotic susceptibility of coagulase-negative staphylococci isolated from very low birth weight babies: comprehensive comparisons of bacteria at different stages of biofilm formation

Background: Coagulase-negative staphylococci are major causes of bloodstream infections in very low birth weight babies cared for in Neonatal Intensive Care Units. The virulence of these bacteria is mainly due to their ability to form biofilms on indwelling medical devices. Biofilm-related infections often fail to respond to antibiotic chemotherapy guided by conventional antibiotic susceptibility tests.Methods: Coagulase-negative staphylococcal blood culture isolates were grown in different phases relevant to biofilm formation: planktonic cells at mid-log phase, planktonic cells at stationary phase, adherent monolayers and mature biofilms and their susceptibilities to conventional antibiotics were assessed. The effects of oxacillin, gentamicin, and vancomycin on preformed biofilms, at the highest achievable serum concentrations were examined. Epifluorescence microscopy and confocal laser scanning microscopy in combination with bacterial viability staining and polysaccharide staining were used to confirm the stimulatory effects of antibiotics on biofilms.Results: Most coagulase-negative staphylococcal clinical isolates were resistant to penicillin G (100%), gentamicin (83.3%) and oxacillin (91.7%) and susceptible to vancomycin (100%), ciprofloxacin (100%), and rifampicin (79.2%). Bacteria grown as adherent monolayers showed similar susceptibilities to their planktonic counterparts at mid-log phase. Isolates in a biofilm growth mode were more resistant to antibiotics than both planktonic cultures at mid-log phase and adherent monolayers; however they were equally resistant or less resistant than planktonic cells at stationary phase. Moreover, for some cell-wall active antibiotics, concentrations higher than conventional MICs were required to prevent the establishment of planktonic cultures from biofilms. Finally, the biofilm-growth of two S. capitis isolates could be enhanced by oxacillin at the highest achievable serum concentration.Conclusion: We conclude that the resistance of coagulase-negative staphylococci to multiple antibiotics initially remain similar when the bacteria shift from a planktonic growth mode into an early attached mode, then increase significantly as the adherent mode further develops. Furthermore, preformed biofilms of some CoNS are enhanced by oxacillin in a dose-dependent manner

Biological Effects of a De Novo Designed Myxoma Virus Peptide Analogue: Evaluation of Cytotoxicity on Tumor Cells

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A bioactive peptide analogue for myxoma virus protein with a targeted cytotoxicity for human skin cancer <it>in vitro</it>

Abstract Background Cancer is an international health problem, and the search for effective treatments is still in progress. Peptide therapy is focused on the development of short peptides with strong tumoricidal activity and low toxicity. In this study, we investigated the efficacy of a myxoma virus peptide analogue (RRM-MV) as a candidate for skin cancer therapy. RRM-MV was designed using the Resonant Recognition Model (RRM) and its effect was examined on human skin cancer and normal human skin cells in vitro. Methods Cell cultures were treated with various concentrations of the peptides at different incubation intervals. Cellular morphological changes (apoptosis and necrosis) were evaluated using confocal laser scanning microscopy. The cytotoxic effects of RRM-MV on human skin cancer and normal human skin cells were quantitatively determined by cytotoxicity and cell viability assays. The effect on human erythrocytes was also determined using quantitative hemolysis assay. DNA fragmentation assay was performed to detect early apoptotic events in treated cancer cells. Furthermore, to investigate the possible cell signalling pathway targeted by the peptides treatment, the levels of p-Akt expression in skin cancer and normal cells were detected by immunoblotting. Results Our results indicate that RRM-MV has a dose-dependent toxic effect on cancer cells only up to 18 h. The immunoblotting results indicated that the RRM-MV slightly increased p-Akt expression in melanoma and carcinoma cells, but did not seem to affect p-Akt expression in normal skin cells. Conclusions RRM-MV targets and lethally harms cancer cells and leaves normal cells unharmed. It is able to reduce the cancer cell viability, disrupting the LDH activity in cancer cells and can significantly affect cancer progression. Further investigation into other cell signalling pathways is needed in the process leading to the in vivo testing of this peptide to prove its safety as a possible effective treatment for skin cancer.</p

CLSM micrographs for apoptosis/necrosis assay with annexin V-Alexa Fluor 488 (green fluorescence) and propidium iodide (red fluorescence) in mouse melanoma cell line (B16F0).

<p>After 3 h incubation with DMEM only in <b>A</b> (blank), 3 h incubation with 800 ng/ml RRM-C in <b>B</b>, and with 800 ng/ml RRM-MV in C. Cytotoxic changes including detachment of confluent layer, apoptotic cells (green) and necrotic cells (red) and are obvious in <b>C</b> when compared with A and B. Longer treatment periods 6 h; 9 h; and 18 h in (<b>D–F</b> respectively) with increased levels of necrosis and cellular detachment when B16F0 cell cultures were treated with (800 ng/ml) of RRM-MV. (200× magnification).</p

Multiple cross-spectral function of myxoma virus proteins (10 sequences alignment).

<p>Multiple cross-spectral function of myxoma virus proteins (10 sequences alignment).</p

CLSM micrographs for human squamous carcinoma COLO 16 cell line.

<p>Cell cultures were treated with 50 ng/ml, 100 ng/ml and 200 ng/ml of RRM-MV for 3 h in <b>A</b>, <b>B</b>, and <b>C</b> respectively. Cell culture in <b>D</b> was treated with 200 ng/ml of RRM-C, while cell cultures in <b>E</b> were similarly incubated without any treatment. More necrotic cells and detachment can be seen in B and C as compared with A indicating dose-dependent cytotoxic effect of RRM-MV. No cellular detachment can be seen in the cell culture treated with 200 ng/ml of the negative control RRM-C in D or in the non-treated cell culture in E.</p

CLSM micrographs for the apoptosis/necrosis assay in three normal cell lines after 3 h incubation with 800 ng/ml of RRM-MV; or 800 ng/ml of RRM-C.

<p>Mouse skin fibroblasts in <b>A</b>; mouse macrophages J774 in <b>B</b>; and CHO in <b>C</b>. No significant cytotoxic effects (apoptosis, necrosis and cellular detachment) were detected in all cell cultures treated with RRM-MV or RRM-C as compared with the non-treated cell cultures similarly incubated in DMEM, indicating the minimal cytotoxic effect of RRM-MV on the 3 normal cell lines.</p

Cytotoxic effect of RRM peptide analogues on normal and cancer cells measured by LDH assay.

<p>Cells (3×10⁵) were incubated for 3 h with control peptide (RRM-C), or with RRM-MV at 400 ng/ml (for COLO16) and 1600 ng/ml (for CHO, J774A.1 and B16-F0). Cells without treatment were similarly incubated for 3 h (blank). Each bar represents mean ± standard errors of 3 separate experiments in triplicate. Data values that are significantly altered (ANOVA and Dunnett's post-hoc analysis) are indicated by <b>*</b> (when compared to control treated cells) and <b>+</b> (when compared to untreated cells) at a significant level of <i>p</i><0.05.</p