Insight Into the Anti-staphylococcal Activity of JBC 1847 at Sub-Inhibitory Concentration

Multidrug-resistant pathogens constitute a serious global issue and, therefore, novel antimicrobials with new modes of action are urgently needed. Here, we investigated the effect of a phenothiazine derivative (JBC 1847) with high antimicrobial activity on Staphylococcus aureus, using a wide range of in vitro assays, flow cytometry, and RNA transcriptomics. The flow cytometry results showed that JBC 1847 rapidly caused depolarization of the cell membrane, while the macromolecule synthesis inhibition assay showed that the synthesis rates of DNA, RNA, cell wall, and proteins, respectively, were strongly decreased. Transcriptome analysis of S. aureus exposed to sub-inhibitory concentrations of JBC 1847 identified a total of 78 downregulated genes, whereas not a single gene was found to be significantly upregulated. Most importantly, there was downregulation of genes involved in adenosintrifosfat (ATP)-dependent pathways, including histidine biosynthesis, which is likely to correlate with the observed lower level of intracellular ATP in JBC 1847–treated cells. Furthermore, we showed that JBC 1847 is bactericidal against both exponentially growing cells and cells in a stationary growth phase. In conclusion, our results showed that the antimicrobial properties of JBC 1847 were primarily caused by depolarization of the cell membrane resulting in dissipation of the proton motive force (PMF), whereby many essential bacterial processes are affected. JBC 1847 resulted in lowered intracellular levels of ATP followed by decreased macromolecule synthesis rate and downregulation of genes essential for the amino acid metabolism in S. aureus. Bacterial compensatory mechanisms for this proposed multi-target activity of JBC 1847 seem to be limited based on the observed very low frequency of resistance toward the compound

GFP fusions of Sec-routed extracellular proteins in Staphylococcus aureus reveal surface-associated coagulase in biofilms

Staphylococcus aureus is a major human pathogen that utilises many surface-associated and secreted proteins to form biofilms and cause disease. However, our understanding of these processes is limited by challenges of using fluores-cent protein reporters in their native environment, because they must be ex-ported and fold correctly to become fluorescent. Here, we demonstrate the feasibility of using the monomeric superfolder GFP (msfGFP) exported from S. aureus. By fusing msfGFP to signal peptides for the Secretory (Sec) and Twin Arginine Translocation (Tat) pathways, the two major secretion pathways in S. aureus, we quantified msfGFP fluorescence in bacterial cultures and cell-free supernatant from the cultures. When fused to a Tat signal peptide, we detect-ed msfGFP fluorescence inside but not outside bacterial cells, indicating a fail-ure to export msfGFP. However, when fused to a Sec signal peptide, msfGFP fluorescence was present outside cells, indicating successful export of the msfGFP in the unfolded state, followed by extracellular folding and maturation to the photoactive state. We applied this strategy to study coagulase (Coa), a secreted protein and a major contributor to the formation of a fibrin network in S. aureus biofilms that protects bacteria from the host immune system and increases attachment to host surfaces. We confirmed that a genomically inte-grated C-terminal fusion of Coa to msfGFP does not impair the activity of Coa or its localisation within the biofilm matrix. Our findings demonstrate that msfGFP is a good candidate fluorescent reporter to consider when studying proteins secreted by the Sec pathway in S. aureus

Systemic thioridazine in combination with dicloxacillin against early aortic graft infections caused by Staphylococcus aureus in a porcine model: In vivo results do not reproduce the in vitro synergistic activity.

INTRODUCTION:Conservative treatment solutions against aortic prosthetic vascular graft infection (APVGI) for inoperable patients are limited. The combination of antibiotics with antibacterial helper compounds, such as the neuroleptic drug thioridazine (TDZ), should be explored. AIM:To investigate the efficacy of conservative systemic treatment with dicloxacillin (DCX) in combination with TDZ (DCX+TDZ), compared to DCX alone, against early APVGI caused by methicillin-sensitive Staphylococcus aureus (MSSA) in a porcine model. METHODS:The synergism of DCX+TDZ against MSSA was initially assessed in vitro by viability assay. Thereafter, thirty-two pigs had polyester grafts implanted in the infrarenal aorta, followed by inoculation with 106 CFU of MSSA, and were randomly administered oral systemic treatment with either 1) DCX or 2) DCX+TDZ. Treatment was initiated one week postoperatively and continued for a further 21 days. Weight, temperature, and blood samples were collected at predefined intervals. By termination, bacterial quantities from the graft surface, graft material, and perigraft tissue were obtained. RESULTS:Despite in vitro synergism, the porcine experiment revealed no statistical differences for bacteriological endpoints between the two treatment groups, and none of the treatments eradicated the APVGI. Accordingly, the mixed model analyses of weight, temperature, and blood samples revealed no statistical differences. CONCLUSION:Conservative systemic treatment with DCX+TDZ did not reproduce in vitro results against APVGI caused by MSSA in this porcine model. However, unexpected severe adverse effects related to the planned dose of TDZ required a considerable reduction to the administered dose of TDZ, which may have compromised the results

Antimicrobial agents and dosages in the main trial.

<p>Antimicrobial agents and dosages utilized in the main trial. Equivalent dosages in mice and humans are listed.</p><p>Antimicrobial agents and dosages in the main trial.</p

A-D: Main trial: Box-whisker plots of bacterial quantities displayed as ln(CFU/mL) in all treatments groups sorted by bacteriological endpoints–(A) P-flush, (B) Spleen, (C) Kidney, and (D) Total.

<p>The filled dots indicate outliers. DCX: Dicloxacillin; TDZ: Thioridazine; VAN: Vancomycin; SALINE: Isotonic saline.</p

A + B: Main trial: (A) Development in mean weight displayed as a percentage of baseline weight (100%) within each treatment group during the 48-hour trial period. DCX: Dicloxacillin; TDZ: Thioridazine; VAN: Vancomycin; SALINE: Isotonic saline.

<p><b>(B)</b> Scatter plot with a predicted linear regression line and a 95% CI (grey zone) of the predicted mean showing the correlation between the total quantity of bacteria and total change in weight during the entire trial period displayed as a percentage of baseline weight (100%).</p

IP trial: Box-whisker plot of pooled bacteriological endpoints related to treatment groups in the IP-trial.

<p>The filled dots indicate outliers. DCX_ip: Dicloxacillin administered IP; TDZ_ip: Thioridazine administered IP; VAN_ip: Vancomycin administered IP.</p