Escherichia coli response to subinhibitory concentration of Colistin: Insights from study of membrane dynamics and morphology.

Prevalence of wide spread bacterial infections bring forth a critical need in understanding the molecular mechanisms of the antibiotics as well as the bacterial response to those antibiotics. Improper usage of antibiotics, which can be in sub-lethal concentrations is one among the multiple reasons for acquiring antibiotic resistance which makes it vital to understand the bacterial response towards sub-lethal concentrations of antibiotics. In this work, we have used colistin, a well-known membrane active antibiotic used to treat severe bacterial infections and explored the impact of its subminimum inhibitory concentration (MIC) on the lipid membrane dynamics and morphological changes of E. coli. Upon investigation of live cell membrane properties such as lipid dynamics using fluorescence correlation spectroscopy, we observed that colistin disrupts the lipid membrane at sub-MIC by altering the lipid diffusivity. Interestingly, filamentationlike cell elongation was observed upon colistin treatment which led to further exploration of surface morphology with the help of atomic force spectroscopy. The changes in the surface roughness upon colistin treatment provides additional insight on the colistin-membrane interaction corroborating with the altered lipid diffusion. Although altered lipid dynamics could be attributed to an outcome of lipid rearrangement due to direct disruption by antibiotic molecules on the membrane or an indirect consequence of disruptions in lipid biosynthetic pathways, we were able to ascertain that altered bacterial membrane dynamics is due to direct disruptions. Our results provide a broad overview on the consequence of the cyclic polypeptide, colistin on membrane specific lipid dynamics and morphology of a live Gram-negative bacterial cell.

The Rhizobium-Legume Symbiosis: Co-opting Successful Stress Management

The interaction of bacteria with plants can result in either a positive, negative, or neutral association. The rhizobium-legume interaction is a well-studied model system of a process that is considered a positive interaction. This process has evolved to require a complex signal exchange between the host and the symbiont. During this process, rhizobia are subject to several stresses, including low pH, oxidative stress, osmotic stress, as well as growth inhibiting plant peptides. A great deal of work has been carried out to characterize the bacterial response to these stresses. Many of the responses to stress are also observed to have key roles in symbiotic signaling. We propose that stress tolerance responses have been co-opted by the plant and bacterial partners to play a role in the complex signal exchange that occurs between rhizobia and legumes to establish functional symbiosis. This review will cover how rhizobia tolerate stresses, and how aspects of these tolerance mechanisms play a role in signal exchange between rhizobia and legumes.

Bacteria-derived peptidoglycan triggers an NF-kB dependent response in Drosophila gustatory neurons

Probing the external world is essential for eukaryotes to distinguish beneficial from pathogenic microorganisms. If it is clear that this task falls to the immune cells, recent work shows that neurons can also detect microbes, although the molecules and mechanisms involved are less characterized. In Drosophila, detection of bacteria-derived peptidoglycan by pattern recognition receptor (PRR) of the PGRP family expressed in immune cells, triggers NF-kB/IMD dependent signaling. We show here that one PGRP protein, called PGRP-LB, is expressed in some proboscis's bitter taste neurons. In vivo calcium imaging reveals that the PGRP/IMD pathway is cell-autonomously required in these neurons to transduce the PGN signal. We finally show that NF-kB/IMD pathway activation in bitter neurons influences fly behavior. This demonstrates that flies use the same bacterial elicitor and signaling module to sense bacterial presence via the peripheral nervous system and trigger an anti-bacterial response in immune-competent cells.

Predicting Antimicrobial Activity at the Target Site: Pharmacokinetic/Pharmacodynamic Indices versus Time–Kill Approaches

Antibiotic dosing strategies are generally based on systemic drug concentrations. However, drug concentrations at the infection site drive antimicrobial effect, and efficacy predictions and dosing strategies should be based on these concentrations. We set out to review different translational pharmacokinetic-pharmacodynamic (PK/PD) approaches from a target site perspective. The most common approach involves calculating the probability of attaining animal-derived PK/PD index targets, which link PK parameters to antimicrobial susceptibility measures. This approach is time efficient but ignores some aspects of the shape of the PK profile and inter-species differences in drug clearance and distribution, and provides no information on the PD time-course. Time–kill curves, in contrast, depict bacterial response over time. In vitro dynamic time–kill setups allow for the evaluation of bacterial response to clinical PK profiles, but are not representative of the infection site environment. The translational value of in vivo time–kill experiments, conversely, is limited from a PK perspective. Computational PK/PD models, especially when developed using both in vitro and in vivo data and coupled to target site PK models, can bridge translational gaps in both PK and PD. Ultimately, clinical PK and experimental and computational tools should be combined to tailor antibiotic treatment strategies to the site of infection.

Acidic Microenvironment Determines Antibiotic Susceptibility and Biofilm Formation of Pseudomonas aeruginosa

Pseudomonas aeruginosa is the most prevalent bacterial species that contribute to cystic fibrosis (CF) respiratory failure. The impaired function of CF transmembrane conductance regulator leads to abnormal epithelial Cl–/HCO3– transport and acidification of airway surface liquid. However, it remains unclear why the CF lung is most commonly infected by Pseudomonas aeruginosa versus other pathogens. We carried out studies to investigate if lower pH helps Pseudomonas aeruginosa adapt and thrive in the CF-like acidic lung environment. Our results revealed that Pseudomonas aeruginosa generally forms more biofilm, induces antibiotic resistance faster in acidic conditions, and can be reversed by returning the acidic environment to physiologically neutral conditions. Pseudomonas aeruginosa appears to be highly adaptive to the CF-like acidic pH environment. By studying the effects of an acidic environment on bacterial response, we may provide a new therapeutic option in preventing chronic Pseudomonas aeruginosa infection and colonization.

Bacterial response to spatial gradients of algal-derived nutrients in a porous microplate

Photosynthetic microalgae are responsible for 50% of the global atmospheric CO2 fixation into organic matter and hold potential as a renewable bioenergy source. Their metabolic interactions with the surrounding microbial community (the algal microbiome) play critical roles in carbon cycling, but due to methodological limitations, it has been challenging to examine how community development is influenced by spatial proximity to their algal host. Here we introduce a copolymer-based porous microplate to co-culture algae and bacteria, where metabolites are constantly exchanged between the microorganisms while maintaining physical separation. In the microplate, we found that the diatom Phaeodactylum tricornutum accumulated to cell abundances ~20 fold higher than under normal batch conditions due to constant replenishment of nutrients through the porous structure. We also demonstrate that algal-associated bacteria, both single isolates and complex communities, responded to inorganic nutrients away from their host as well as organic nutrients originating from the algae in a spatially predictable manner. These experimental findings coupled with a mathematical model suggest that host proximity and algal culture growth phase impact bacterial community development in a taxon-specific manner through organic and inorganic nutrient availability. Our novel system presents a useful tool to investigate universal metabolic interactions between microbes in aquatic ecosystems.

Antarctic Bacteria, Sea Ice Ecosystem Dynamics, and Global Climate Change

<p>Productivity in the Southern Ocean reflects both the spatial and temporal dynamics of the sea ice ecosystem, as well as the complex cycling of energy through the microbial community. Marine bacteria are thought to be integral to trophodynamics and the functioning of a microbial loop within the ice matrix, but there is no clear understanding of the distribution and diversity of bacteria or the importance of bacterial production. Understanding the bacterial response to environmental change in the sea ice ecosystem may provide an insight into the potential changes to the physical oceanography and ecology of the Southern Ocean. In this study, a multivariate statistical approach was used to compare the distribution and abundance of bacteria occurring in pack ice at the tongue of the Mertz Glacier (George V Coast, Antarctica) with bacteria from fast ice at Cape Hallett (Victoria Land coastline, Antarctica). Estimates of bacterial abundance were derived using both epifluorescence microscopy and flow cytometry and correlated with algal and chlorophyll a data. Significant differences in the vertical distribution of cells within the ice were observed between the Mertz Glacier and Cape Hallett, but no overall difference in cell abundance was found between the two locations with 7.6 ± 1.2 x 109 cells per m2 and 8.7 ± 1.6 x 109 cells per m2 respectively. Bacteria and algae were positively correlated in pack ice of the Mertz Glacier indicating a functional microbial loop, but no discernable relationship was exhibited in multiyear ice at Cape Hallett. These findings support the general consensus that the generation of bacterial biomass from algal-derived dissolved organic matter is highly variable across seasons and habitats. The tetrazolium salt 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was used to investigate the bacterial response to experimentally induced changes in light and salinity in fast ice at Cape Hallett. Two distinct assemblages were examined; the brine channel assemblage near the surface of the ice and the interstitial or bottom assemblage. This study presents preliminary evidence that the metabolic activity of brine bacteria is influenced by light stimulus, most likely as a response to increased levels of algal-derived dissolved organic matter. No cells were deemed to be metabolically active when incubated in the dark, while on average thirty-eight percent of the cells incubated at 150 =mol photons m-2 s-1 were metabolically active. Additional results indicate that salt concentration is more significant than light irradiance in influencing the metabolic response of cells present in the interstitial region of the sea ice profile. When acclimated over a period of eight hours, cells exhibited a tolerance to changing saline concentrations, but after a further eight hours there is some evidence to suggest activity is reduced at either end of the saline regime. Bacterial metabolic activity in each assemblage is thus thought to reflect the fundamentally different light and saline environments within the sea ice. Metabolic probes such as CTC will prove useful in providing a mechanistic understanding of productivity and trophodynamics in the Antarctic coastal ecosystem, and may contribute to prognostic models for qualifying the resilience of the microbial community to climate change.</p>

Antarctic Bacteria, Sea Ice Ecosystem Dynamics, and Global Climate Change

<p>Productivity in the Southern Ocean reflects both the spatial and temporal dynamics of the sea ice ecosystem, as well as the complex cycling of energy through the microbial community. Marine bacteria are thought to be integral to trophodynamics and the functioning of a microbial loop within the ice matrix, but there is no clear understanding of the distribution and diversity of bacteria or the importance of bacterial production. Understanding the bacterial response to environmental change in the sea ice ecosystem may provide an insight into the potential changes to the physical oceanography and ecology of the Southern Ocean. In this study, a multivariate statistical approach was used to compare the distribution and abundance of bacteria occurring in pack ice at the tongue of the Mertz Glacier (George V Coast, Antarctica) with bacteria from fast ice at Cape Hallett (Victoria Land coastline, Antarctica). Estimates of bacterial abundance were derived using both epifluorescence microscopy and flow cytometry and correlated with algal and chlorophyll a data. Significant differences in the vertical distribution of cells within the ice were observed between the Mertz Glacier and Cape Hallett, but no overall difference in cell abundance was found between the two locations with 7.6 ± 1.2 x 109 cells per m2 and 8.7 ± 1.6 x 109 cells per m2 respectively. Bacteria and algae were positively correlated in pack ice of the Mertz Glacier indicating a functional microbial loop, but no discernable relationship was exhibited in multiyear ice at Cape Hallett. These findings support the general consensus that the generation of bacterial biomass from algal-derived dissolved organic matter is highly variable across seasons and habitats. The tetrazolium salt 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was used to investigate the bacterial response to experimentally induced changes in light and salinity in fast ice at Cape Hallett. Two distinct assemblages were examined; the brine channel assemblage near the surface of the ice and the interstitial or bottom assemblage. This study presents preliminary evidence that the metabolic activity of brine bacteria is influenced by light stimulus, most likely as a response to increased levels of algal-derived dissolved organic matter. No cells were deemed to be metabolically active when incubated in the dark, while on average thirty-eight percent of the cells incubated at 150 =mol photons m-2 s-1 were metabolically active. Additional results indicate that salt concentration is more significant than light irradiance in influencing the metabolic response of cells present in the interstitial region of the sea ice profile. When acclimated over a period of eight hours, cells exhibited a tolerance to changing saline concentrations, but after a further eight hours there is some evidence to suggest activity is reduced at either end of the saline regime. Bacterial metabolic activity in each assemblage is thus thought to reflect the fundamentally different light and saline environments within the sea ice. Metabolic probes such as CTC will prove useful in providing a mechanistic understanding of productivity and trophodynamics in the Antarctic coastal ecosystem, and may contribute to prognostic models for qualifying the resilience of the microbial community to climate change.</p>

Oxalate Alters Cellular Bioenergetics, Redox Homeostasis, Antibacterial Response, and Immune Response in Macrophages

Individuals with calcium oxalate (CaOx) kidney stones can have secondarily infected calculi which may play a role in the development of recurrent urinary tract infection (UTI). Uropathogenic Escherichia coli (UPEC) is the most common causative pathogen of UTIs. Macrophages play a critical role in host immune defense against bacterial infections. Our previous study demonstrated that oxalate, an important component of the most common type of kidney stone, impairs monocyte cellular bioenergetics and redox homeostasis. The objective of this study was to investigate whether oxalate compromises macrophage metabolism, redox status, anti-bacterial response, and immune response. Monocytes (THP-1, a human monocytic cell line) were exposed to sodium oxalate (soluble oxalate; 50 µM) for 48 hours prior to being differentiated into macrophages. Macrophages were subsequently exposed to calcium oxalate crystals (50 µM) for 48 hours followed by UPEC (MOI 1:2 or 1:5) for 2 hours. Peritoneal macrophages and bone marrow-derived macrophages (BMDM) from C57BL/6 mice were also exposed to oxalate. THP-1 macrophages treated with oxalate had decreased cellular bioenergetics, mitochondrial complex I and IV activity, and ATP levels compared to control cells. In addition, these cells had a significant increase in mitochondrial and total reactive oxygen species levels, mitochondrial gene expression, and pro-inflammatory cytokine (i.e. Interleukin-1β, IL-1β and Interleukin-6, IL-6) mRNA levels and secretion. In contrast, oxalate significantly decreased the mRNA levels and secretion of the anti-inflammatory cytokine, Interleukin-10 (IL-10). Further, oxalate increased the bacterial burden of primary macrophages. Our findings demonstrate that oxalate compromises macrophage metabolism, redox homeostasis, and cytokine signaling leading to a reduction in anti-bacterial response and increased infection. These data highlight a novel role of oxalate on macrophage function.