Effect of antimicrobial peptides on planktonic growth, biofilm formation and biofilm-derived bacterial viability of Streptococcus pneumoniae

Streptococcus pneumoniae is a leading cause of pneumonia mortality globally. Pneumococcal disease is often associated with prolonged colonisation of hosts and this process is facilitated by biofilm formation that is largely resistant to conventional antibiotics. We investigated the effects of antimicrobial peptides (AMPs) lysozyme, lactoferrin, LL37 and a combination of all three on planktonic growth, biofilm formation and biofilm-derived bacterial viability by S. pneumoniae, serotype 23F. Planktonic growth and biofilm-derived bacterial viability were determined using standard colony-forming techniques, while biofilm formation was measured using a crystal violet based spectrophotometric method. Relative to controls, lysozyme significantly reduced biofilm formation (0.08 OD vs. 0.10 OD at 570 nm, p = 0.01), while LL37 and the AMP combination increased biofilm formation (0.14 OD vs. 0.10 OD at 570 nm, p = 0.01). The combination of AMPs significantly decreased planktonic growth (1.10 × 108 colony-forming units per millilitres [CFU/ mL] vs. 2.13 × 108 CFU/mL, p = 0.02). Biofilm-derived bacterial viability was greatly reduced by exposure to a combination of AMPs (1.05 × 105 CFU/mL vs. 1.12 × 106 CFU/mL, p = 3.60 × 10−8). Streptococcus pneumoniae displays marked resistance to the individual AMPs. A combination of lysozyme, lactoferrin and LL37 effectively inhibited planktonic growth and biofilm-derived bacterial viability; however, persister cell growth was still evident after exposure.The Medical Research Council (MRC) Unit for Inflammation and Immunity as well as the National Research Foundation (NRF).https://sajid.co.za/index.php/sajiddm2022Internal Medicin

Exposure of a 23F serotype strain of <i>Streptococcus pneumoniae</i> to cigarette smoke condensate is associated with selective upregulation of genes encoding the two-component regulatory system 11 (TCS11)

Alterations in whole genome expression profiles following exposure of the pneumococcus (strain 172, serotype 23F) to cigarette smoke condensate (160 μg/mL) for 15 and 60 min have been determined using the TIGR4 DNA microarray chip. Exposure to CSC resulted in the significant (P &#60; 0.014–0.0006) upregulation of the genes encoding the two-component regulatory system 11 (TCS11), consisting of the sensor kinase, hk11, and its cognate response regulator, rr11, in the setting of increased biofilm formation. These effects of cigarette smoke on the pneumococcus may contribute to colonization of the airways by this microbial pathogen

Overview of community-acquired pneumonia and the role of imflammatory mechanisms in the immunopathogenesis of severe pneumococcal disease

Community-acquired pneumonia (CAP) remains a leading cause of morbidity and mortality among the infectious diseases.Despite the implementation of national pneumococcal polyvalent vaccine-based immunisation strategies targeted at high-risk groups, Streptococcus pneumoniae (the pneumococcus) remains the most common cause of CAP. Notwithstanding the HIV pandemic, major challenges confronting the control of CAP include the range of bacterial and viral pathogens causing this condition, the ever-increasing problem of antibiotic resistance worldwide, and increased vulnerability associated with steadily aging populations in developed countries.These and other risk factors, as well as diagnostic strategies, are covered in the first section of this review. Thereafter, the review is focused on the pneumococcus, specifically the major virulence factors of this microbial pathogen and their role in triggering overexuberant inflammatory responses which contribute to the immunopathogenesis of invasive disease. The final section of the review is devoted to a consideration of pharmacological, anti-inflammatory strategies with adjunctive potential in the antimicrobial chemotherapy of CAP. This is focused on macrolides, corticosteroids, and statins with respect to their modes of anti-inflammatory action, current status, and limitations.http://www.hindawi.comam201

Protein kinase C promotes restoration of calcium homeostasis to platelet activating factor-stimulated human neutrophils by inhibition of phospholipase C

<p>Abstract</p> <p>Background</p> <p>The role of protein kinase C (PKC) in regulating the activity of phospholipase C (PLC) in neutrophils activated with the chemoattractant, platelet-activating factor (PAF, 20 and 200 nM), was probed in the current study using the selective PKC inhibitors, GF10903X (0.5 - 1 μM) and staurosporine (400 nM).</p> <p>Methods</p> <p>Alterations in cytosolic Ca²⁺, Ca²⁺influx, inositol triphosphate (IP₃), and leukotriene B₄production were measured using spectrofluorimetric, radiometric and competitive binding radioreceptor and immunoassay procedures, respectively.</p> <p>Results</p> <p>Activation of the cells with PAF was accompanied by an abrupt increase in cytosolic Ca²⁺followed by a gradual decline towards basal levels. Pretreatment of neutrophils with the PKC inhibitors significantly increased IP₃production with associated enhanced Ca²⁺release from storage vesicles, prolongation of the peak cytosolic Ca²⁺transients, delayed clearance and exaggerated reuptake of the cation, and markedly increased synthesis of LTB₄. The alterations in Ca²⁺fluxes observed with the PKC inhibitors were significantly attenuated by U73122, a PLC inhibitor, as well as by cyclic AMP-mediated upregulation of the Ca²⁺-resequestering endomembrane ATPase.</p> <p>Taken together, these observations are compatible with a mechanism whereby PKC negatively modulates the activity of PLC, with consequent suppression of IP₃production and down-regulation of Ca²⁺mediated pro-inflammatory responses of PAF-activated neutrophils.</p> <p>Conclusion</p> <p>Although generally considered to initiate and/or amplify intracellular signalling cascades which activate and sustain the pro-inflammatory activities of neutrophils and other cell types, the findings of the current study have identified a potentially important physiological, anti-inflammatory function for PKC, at least in neutrophils.</p

Investigation of biofilm formation on a charged intravenous catheter relative to that on a similar but uncharged catheter

Catheter-related blood stream infections increase morbidity, mortality, and costs. This study investigated whether Certofix® protect antimicrobial catheters carry a surface charge and whether this inhibits biofilm formation. The capacitance of the catheter surfaces was measured and, to determine if the catheters released ions, distilled water was passed through and current measured as a function of voltage. With probes touching the inner and outer surfaces, capacitance was not voltage-dependent, indicating surfaces were uncharged or carried a similar charge. When one probe penetrated the catheter wall, capacitance was weakly voltage-dependent, indicating the presence of a surface charge. Standard and charged catheters were also exposed to phosphate buffered saline as controls or 2×106 colony forming units/mL (in phosphate buffered saline) of six different microorganisms for 60 or 120 minutes. When the growth of detached bacteria was measured, biofilm formation was significantly reduced, (P<0.05), for charged catheters for all organisms.B Braun Melsungen AG (Melsungen, Germany) supplied the catheters and sufficient funds to perform the biofilm studies in the form of an unrestricted grant. RC is supported by the National Research Foundation of South Africa. RM is supported by the National Research Foundation (SA) under the Nanotechnology Flagship Project grant and the University Research Council (Witwatersrand)http://www.dovepress.com/medical-devices-evidence-and-research-journalhb201

Investigation of the anti-mycobacterial mechanism of action of 7-methyljuglone

Although the naphthoquinone, 7-methyljuglone (7-MJ), is active against Mycobacterium tuberculosis (MTB) in vitro, neither the cellular site nor mechanism of anti-mycobacterial action of this agent has been identified. The primary objective of the current study was to investigate the mycobacterial outer membrane as a potential target of 7-MJ by measuring the effects of this agent (0.023 - 1.5 mg/L) on microbial ATP levels and uptake of K+. Methods: Bioluminescence and radiometric (uptake of 86Rb+) procedures were used to assay microbial ATP levels and K+ trans-port respectively. Results: Exposure of MTB (strain H37Rv) to 7-MJ for 60 min resulted in dose-related decreases in both microbial ATP levels and uptake of 86Rb+ which achieved statistical significance (P < 0.05) at concentrations of 0.4 and 0.1 mg/L respectively. Conclusions: These observations are compatible with the mycobacterial membrane as being the putative site of action of 7-MJ, targeting microbial energy metabolism and K+ transport.The National Research Foundation and the Medical Research Council, Pretoria, South Africa.http://www.SciRP.org/journal/ojrd)am2016ImmunologyPlant Scienc

Calcium-dependent potentiation of the pro-inflammatory functions of human neutrophils by tigecycline in vitro

OBJECTIVES: Tigecycline is the prototype of the recently introduced, intravenously administered glycylcycline class of antibiotics, developed in response to the increasing problem of antibiotic resistance in Gram-positive bacteria, especially Staphylococcus aureus, as well as Gram-negative bacteria and anaerobes. However, relatively little is known about the immunomodulatory potential of tigecycline, specifically its interactions with human neutrophils. In the current study we investigated the effects of tigecycline at therapeutically relevant concentrations and greater (0.625–10 mg/L) on alterations in cytosolic Ca2+ concentrations, generation of antimicrobial reactive oxygen species (ROS) and release of granule proteases [elastase, matrix metalloproteinase- 8 (MMP-8) and matrix metalloproteinase-9 (MMP-9)] by human blood neutrophils activated with the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 1 mM). METHODS: Cytosolic Ca2+ concentrations were measured using fura-2/AM-based spectrofluorimetry and radiometric procedures, generation of ROS by oxygen consumption and myeloperoxidase-mediated auto-iodination, and protease release by ELISA procedures. RESULTS: Exposure of the cells to fMLP resulted in activation of the generation of ROS, as well as release of the granule proteases, all of which were significantly increased by pre-incubation of the cells with tigecycline in a dose-dependent manner. Tigecycline-mediated enhancement of these neutrophil functions was associated with elevations in the concentrations of cytosolic Ca2+, which appeared to result from the Ca2+ ionophore activity of tigecycline. CONCLUSIONS: Tigecycline, by functioning as a Ca2+ ionophore, and independent of antimicrobial activity, potentiates the pro-inflammatory functions of human neutrophils in vitro.The South African Medical Research Council as well as by a research grant (grant number 35733) from Wyeth Pharmaceuticalshttp://jac.oxfordjournals.org

Effects of clarithromycin at sub-minimum inhibitory concentrations on early ermB gene expression, metabolic activity and growth of an erm(B)-expressing macrolide-resistant strain of Streptococcus pneumoniae

AIM: To investigate the effects of exposure of a macrolide-resistant [erm(B)-expressing] strain of Streptococcus pneumo- niae (strain 2507) to clarithromycin (0.5 and 5 mg/L) added at the outset and 6 hours after initiation of culture on early gene expression, energy metabolism, and growth. METHODS: Bacterial growth was determined by turbidometric and colony counting procedures, energy metabolism by measurement of ATP, while analysis of gene expression was per- formed using reverse transcription-PCR and sequencing. RESULTS: Addition of clarithromycin, at either concentration, at the outset of culture, caused transient suppression of growth of 10 - 12 hours duration, while delayed addition of antibi-otic (during the logarithmic phase) resulted in an abrupt halt in growth followed by recovery. These inhibitory effects of clarithromycin on bacterial growth were associated with up-regulation of expression of erm(B), decreased ATP and protein synthesis, and were unaffected by inclusion of either catalase (500 and 1000 kunits/L), or compe- tence-stimulating peptide (CSP-1, 0.5 mg/L). The inhibitory effects could, however, be overcome by pre-exposure of the bacteria to the antibiotic. Moreover, clarithromycin appeared to potentiate the antimicrobial actions of ceftriaxone, at sub-MIC concentrations, for strain 2507. CONCLUSIONS: Unlike several other common bacterial pathogens, the full expression of erm(B)-mediated macrolide resistance by the pneumococcus has a slow onset, which is associated with transient susceptibility to macrolides and inhibition of growth.CF is supported by the National Research Foundation of South Africa.http://www.SciRP.org/journal/ojr