Comparative in vitro efficacy of antibiotics against the intracellular reservoir of Staphylococcus aureus.

Staphylococcus aureus (SA) is a leading cause of bloodstream infection. The liver represents the sentinel immune organ for clearance of bloodstream pathogens and eradication of intracellular SA from liver-resident macrophages (Kupffer cells, KCs) eliminates the likely pathogenic reservoir that contributes to persistent bacteraemia. OBJECTIVES: We assessed antimicrobial activity at phagolysosome-mimicking pH, intracellular penetration, and SA eradication within KCs in vitro for clinically prescribed antistaphylococcal agents alone or in combination: vancomycin, daptomycin, ceftaroline, ceftobiprole, oritavancin, oxacillin, cefazolin; rifampin and fosfomycin. METHODS: pH-adjusted broth microdilution assays, intracellular bioaccumulation assays, and intracellular killing assays against clinical bloodstream isolates were performed using a murine KC line with study agents. RESULTS: Rifampin and ß-lactams exhibited enhanced activity [2- to 16-fold minimum inhibitory concentrations (MIC) decrease] at phagolysosomal pH while vancomycin, oritavancin, daptomycin and fosfomycin demonstrated reduced activity (2- to 32-fold MIC increase in order of least to greatest potency reduction). All agents evaluated had poor to modest intracellular to extracellular concentration ratios (0.024-7.8), with exceptions of rifampin and oritavancin (intracellular to extracellular ratios of 17.4 and 78.2, respectively). Finally, we showed that the first-line treatment for SA bacteraemia (SAB), vancomycin, performed worse than all other tested antibiotics in eradicating intracellular SA at human Cmax concentration (0.20 log cfu decrease), while oritavancin performed better than all other agents alone (2.05 versus 1.06-1.36 log cfu decrease). CONCLUSIONS: Our findings raise concerns about the efficacy of commonly prescribed antibiotics against intracellular SA reservoirs and emphasize the need to consider targeting pathogen eradication from the liver to achieve early control of SAB.

Machine Learning with Alpha Toxin Phenotype to Predict Clinical Outcome in Patients with Staphylococcus aureus Bloodstream Infection.

Staphylococcus aureus bloodstream (SAB) infection remains a leading cause of sepsis-related mortality. Yet, current treatment does not account for variable virulence traits that mediate host dysregulated immune response, such as SA α-toxin (Hla)-mediated thrombocytopenia. Here, we applied machine learning (ML) to bacterial growth images combined with platelet count data to predict patient outcomes. We profiled Hla phenotypes of SA isolates collected from patients with bacteremia by taking smartphone images of beta-hemolytic growth on sheep blood agar (SBA). Electronic medical records were reviewed to extract relevant laboratory and clinical data. A convolutional neural network was applied to process the plate image data for input along with day 1 patient platelet count to generate ML-based models that predict thrombocytopenia on day 4 and mortality. A total of 229 patients infected with SA strains exhibiting varying zone sizes of beta-hemolysis on SBA were included. A total of 539 images of bacterial growth on SBA were generated as inputs for model development. One-third of patients developed thrombocytopenia at onset, with an overall mortality rate of 18.8%. The models developed from the ML algorithm showed strong performance (AUC 0.92) for predicting thrombocytopenia on day 4 of infection and modest performance (AUC 0.711) for mortality. Our findings support further development and validation of a proof-of-concept ML application in digital microbiology, with a measure of bacterial virulence factor production that carries prognostic significance and can help guide treatment selection.

Nano- and Macroscale Imaging of Cholesterol Linoleate and Human Beta Defensin 2-Induced Changes in Pseudomonas aeruginosa Biofilms.

The biofilm production of Pseudomonas aeruginosa (PA) is central to establishing chronic infection in the airways in cystic fibrosis. Epithelial cells secrete an array of innate immune factors, including antimicrobial proteins and lipids, such as human beta defensin 2 (HBD2) and cholesteryl lineolate (CL), respectively, to combat colonization by pathogens. We have recently shown that HBD2 inhibits biofilm production by PA, possibly linked to interference with the transport of biofilm precursors. Considering that both HBD2 and CL are increased in airway fluids during infection, we hypothesized that CL synergizes with HBD2 in biofilm inhibition. CL was formulated in phospholipid-based liposomes (CL-PL). As measured by atomic force microscopy of single bacteria, CL-PL alone and in combination with HBD2 significantly increased bacterial surface roughness. Additionally, extracellular structures emanated from untreated bacterial cells, but not from cells treated with CL-PL and HBD2 alone and in combination. Crystal violet staining of the biofilm revealed that CL-PL combined with HBD2 effected a significant decrease of biofilm mass and increased the number of larger biofilm particles consistent with altered cohesion of formed biofilms. These data suggest that CL and HBD2 affect PA biofilm formation at the single cell and community-wide level and that the community-wide effects of CL are enhanced by HBD2. This research may inform future novel treatments for recalcitrant infections in the airways of CF patients.

The Antimicrobial Peptide Human Beta-Defensin 2 Inhibits Biofilm Production of Pseudomonas aeruginosa Without Compromising Metabolic Activity.

Biofilm production is a key virulence factor that facilitates bacterial colonization on host surfaces and is regulated by complex pathways, including quorum sensing, that also control pigment production, among others. To limit colonization, epithelial cells, as part of the first line of defense, utilize a variety of antimicrobial peptides (AMPs) including defensins. Pore formation is the best investigated mechanism for the bactericidal activity of AMPs. Considering the induction of human beta-defensin 2 (HBD2) secretion to the epithelial surface in response to bacteria and the importance of biofilm in microbial infection, we hypothesized that HBD2 has biofilm inhibitory activity. We assessed the viability and biofilm formation of a pyorubin-producing Pseudomonas aeruginosa strain in the presence and absence of HBD2 in comparison to the highly bactericidal HBD3. At nanomolar concentrations, HBD2 - independent of its chiral state - significantly reduced biofilm formation but not metabolic activity, unlike HBD3, which reduced biofilm and metabolic activity to the same degree. A similar discrepancy between biofilm inhibition and maintenance of metabolic activity was also observed in HBD2 treated Acinetobacter baumannii, another Gram-negative bacterium. There was no evidence for HBD2 interference with the regulation of biofilm production. The expression of biofilm-related genes and the extracellular accumulation of pyorubin pigment, another quorum sensing controlled product, did not differ significantly between HBD2 treated and control bacteria, and in silico modeling did not support direct binding of HBD2 to quorum sensing molecules. However, alterations in the outer membrane protein profile accompanied by surface topology changes, documented by atomic force microscopy, was observed after HBD2 treatment. This suggests that HBD2 induces structural changes that interfere with the transport of biofilm precursors into the extracellular space. Taken together, these data support a novel mechanism of biofilm inhibition by nanomolar concentrations of HBD2 that is independent of biofilm regulatory pathways.