Neuropathogenicity of non-viable Borrelia burgdorferi ex vivo

Even after appropriate treatment, a proportion of Lyme disease patients suffer from a constellation of symptoms, collectively called Post-Treatment Lyme Disease Syndrome (PTLDS). Brain PET scan of patients with PTLDS have demonstrated likely glial activation indicating persistent neuroinflammatory processes. It is possible that unresolved bacterial remnants can continue to cause neuroinflammation. In previous studies, we have shown that non-viable Borrelia burgdorferi can induce neuroinflammation and apoptosis in an oligodendrocyte cell line. In this follow-up study, we analyze the effect of sonicated remnants of B. burgdorferi on primary rhesus frontal cortex (FC) and dorsal root ganglion (DRG) explants. Five FC and three DRG tissue fragments from rhesus macaques were exposed to sonicated B. burgdorferi and analyzed for 26 inflammatory mediators. Live bacteria and medium alone served as positive and negative control, respectively. Tissues were also analyzed for cell types mediating inflammation and overall apoptotic changes. Non-viable B. burgdorferi induced significant levels of several inflammatory mediators in both FC and DRG, similar to live bacteria. However, the levels induced by non-viable B. burgdorferi was often (several fold) higher than those induced by live ones, especially for IL-6, CXCL8 and CCL2. This effect was also more profound in the FC than in the DRG. Although the levels often differed, both live and dead fragments induced the same mediators, with significant overlap between FC and DRG. In the FC, immunohistochemical staining for several inflammatory mediators showed the presence of multiple mediators in astrocytes, followed by microglia and oligodendrocytes, in response to bacterial remnants. Staining was also seen in endothelial cells. In the DRG, chemokine/cytokine staining was predominantly seen in S100 positive (glial) cells. B. burgdorferi remnants also induced significant levels of apoptosis in both the FC and DRG. Apoptosis was confined to S100 + cells in the DRG while distinct neuronal apoptosis was also detected in most FC tissues in response to sonicated bacteria. Non-viable B. burgdorferi can continue to be neuropathogenic to both CNS and PNS tissues with effects likely more profound in the former. Persistence of remnant-induced neuroinflammatory processes can lead to long term health consequences.

Evaluating H2O2 in Living Bacteria by Ratiometric Fluorescent Probe

Hydrogen peroxide (H2O2) is a significant signal molecule in physiological and pathological processes. Levels of H2O2 in bacteria are proved to be a key factor in immune response. To sum up, detection of H2O2 levels in living bacteria is remarkable for further study of its physiological and pathological effects. Herein, we propose a novel ratiometric fluorescent probe (Nahp) to detect H2O2 in living cells and bacteria. In addition, based on boronate, Nahp has satisfactory selectivity and sensitivity toward H2O2 (LOD = 0.158 μM). Furthermore, with excellent detection performance to H2O2, Nahp is successfully used for fluorescent bioimaging of H2O2 and measuring H2O2 accumulation in bacteria. Most importantly, the probe was also used to image H2O2 in three Gram-negative bacteria, clearly revealing for the first time significant differences in H2O2 expression levels in live bacteria.

A target engagement assay to assess uptake, potency and retention of antibiotics in living bacteria

A major challenge in antibiotics drug discovery is to turn potent biochemical inhibitors of essential bacterial components into effective antimicrobials. This difficulty is underpinned by a lack of methods to investigate the physicochemical properties needed for candidate antibiotics to permeate the bacterial cell envelope and avoid clearance by the action of bacterial efflux pumps. To address these issues, here we used a target engagement assay to measure the equilibrium and kinetics binding parameters of antibiotics to their molecular targets in live bacteria. We validated this approach for a known antibiotic target, dihydrofolate reductase, using the Gram-negative bacteria Escherichia coli and the emerging human pathogen Mycobacterium abscessus. We expect the use of similar target engagement assays to expedite the discovery and progression of novel, cell-permeable antibiotics with on-target activity.

Metabolic Processing of Selenium-based Bioisostere of meso-diaminopimelic Acid in Live Bacteria

The bacterial cell wall supports cell shape and prevents lysis due to internal turgor pressure. A primary component of all known bacterial cell walls is the peptidoglycan (PG) layer, which is comprised of repeating units of sugars connected to short and unusual peptides. The various steps within PG biosynthesis are often the target of antibiotics as they are essential for cellular growth and survival. Synthetic mimics of PG have proven to be indispensable tools to study bacterial cell growth and remodeling. Yet, a common component of PG, meso-diaminopimelic acid (m-DAP) at the third position of the stem peptide, remains challenging to build synthetically and is not commercially available. Here, we describe the synthesis and metabolic processing of a selenium-based bioisostere of a m-DAP analogue, selenolanthionine. We show that selenolanthionine is installed within the PG of live bacteria by the native cell wall crosslinking machinery in several mycobacteria species. We envision that this probe will supplement the current methods available for investigating PG crosslinking in m-DAP containing organisms.

Amphiphilic Carboxymethyl Dextran Nanomicelles for Antibacterial Coating on Titanium

Peri-implantitis occurs at a significant rate, which is the leading cause of implant failure. The main reason for this unwanted complication is bacterial invasion and biofilm formation. To reduce the incidence of peri-implantitis, we constructed a carboxymethyl dextran (CMD) based nanomicelles antibacterial coating on microarc-oxidized titanium (MAO-Ti) surface. After cross-linking, the drug-loaded nanomicelles were spherical with a particle size of 130nm and uniformly dispersed. Zeta potential was negative, and the absolute value was greater than 10 mV, effectively avoiding micelles aggregation. It was observed by dynamic light scattering (DLS) that the stability of nanomicelles was significantly improved after cross-linking. The hemolysis rate of micelles was less than 5%, and the overall survival rate of human umbilical vein endothelial cells was more than 90%. After being coated on MAO-Ti surface, the cumulative drug release rate of drug-loaded nanomicelles reached 86.6% after 360 hours. Fluorescence staining of immobilized bacteria showed more dead bacteria on the coating surface, and the number of live bacteria was significantly reduced. It was concluded that dextran-based nanomicelles, which showed long-term drug release properties and excellent biocompatibility, are potential drug carriers for fabricating antibacterial coating on titanium surfaces.

Bacterial Characteristics of Dust Particle Saltation in Gobi Dust Sites, Mongolia

The Gobi Desert is a major source of Asian dust events, and the resulting health hazards have increased significantly in recent years. We reported that a variety of live bacteria were distributed in the Gobi Desert in relation to land use. Bacterial distribution was confirmed in the environment and on the land used by animals; however, bacterial saltation due to dust events has not been investigated in detail. In this study, to understand the distribution of surface bacteria in the atmosphere by dust saltation, live bacteria in four dust-generating areas in the Gobi area were monitored using an artificial dust generating device. The live bacteria were detected by experimental saltation at a wind speed of 6.5–8 m/s in all areas. A certain number of live bacteria are constantly saltated by dust events, and these bacteria depend on land use. Moreover, the bacterial saltation strain depended on land use and diversity, indicating that live bacteria are lifted into the environment by dust events. These findings indicate that dust events saltate environmental bacteria on the ground, suggest the risk of animal-derived bacterial saltation affected by land use, and present cross-border public health challenges to be considered in the future.

Novel Nematode-Killing Protein-1 (Nkp-1) from a Marine Epiphytic Bacterium Pseudoalteromonas tunicata

Drug resistance among parasitic nematodes has resulted in an urgent need for the development of new therapies. However, the high re-discovery rate of anti-nematode compounds from terrestrial environments necessitates a new repository for future drug research. Marine epiphytes are hypothesised to produce nematicidal compounds as a defence against bacterivorous predators, thus representing a promising yet underexplored source for anti-nematode drug discovery. The marine epiphytic bacterium Pseudoalteromonas tunicata is known to produce several bioactive compounds. Screening heterologously expressed genomic libraries of P. tunicata against the nematode Caenorhabditis elegans, identified as an E. coli clone (HG8), shows fast-killing activity. Here we show that clone HG8 produces a novel nematode-killing protein-1 (Nkp-1) harbouring a predicted carbohydrate-binding domain with weak homology to known bacterial pore-forming toxins. We found bacteria expressing Nkp-1 were able to colonise the C. elegans intestine, with exposure to both live bacteria and protein extracts resulting in physical damage and necrosis, leading to nematode death within 24 h of exposure. Furthermore, this study revealed C. elegans dar (deformed anal region) and internal hatching may act as a nematode defence strategy against Nkp-1 toxicity. The characterisation of this novel protein and putative mode of action not only contributes to the development of novel anti-nematode applications in the future but reaffirms the potential of marine epiphytic bacteria as a new source of novel biomolecules.

Challenges in the Microbiological Diagnosis of Implant-Associated Infections: A Summary of the Current Knowledge

Implant-associated infections are characterized by microbial biofilm formation on implant surface, which renders the microbiological diagnosis challenging and requires, in the majority of cases, a complete device removal along with a prolonged antimicrobial therapy. Traditional cultures have shown unsatisfactory sensitivity and a significant advance in the field has been represented by both the application of the sonication technique for the detachment of live bacteria from biofilm and the implementation of metabolic and molecular assays. However, despite the recent progresses in the microbiological diagnosis have considerably reduced the rate of culture-negative infections, still their reported incidence is not negligible. Overall, several culture- and non-culture based methods have been developed for diagnosis optimization, which mostly relies on pre-operative and intra-operative (i.e., removed implants and surrounding tissues) samples. This review outlines the principal culture- and non-culture based methods for the diagnosis of the causative agents of implant-associated infections and gives an overview on their application in the clinical practice. Furthermore, advantages and disadvantages of each method are described.