More than 10% of the global human population is now afflicted with kidney stones, which are commonly associated with other significant health problems including diabetes, hypertension and obesity. Nearly 70% of these stones are primarily composed of calcium oxalate, a mineral previously assumed to be effectively insoluble within the kidney. This has limited currently available treatment options to painful passage and/or invasive surgical procedures. We analyze kidney stone thin sections with a combination of optical techniques, which include bright field, polarization, confocal and super-resolution nanometer-scale auto-fluorescence microscopy. Here we demonstrate using interdisciplinary geology and biology (geobiology) approaches that calcium oxalate stones undergo multiple events of dissolution as they crystallize and grow within the kidney. These observations open a fundamentally new paradigm for clinical approaches that include in vivo stone dissolution and identify high-frequency layering of organic matter and minerals as a template for biomineralization in natural and engineered settings.
A microfluidic
gradient chamber (MGC) and a homogeneous batch culturing
system were used to evaluate whether spatial concentration gradients
of the antibiotic ciprofloxacin allow development of greater antibiotic
resistance in Escherichia coli strain
307 (E. coli 307) compared to exclusively
temporal concentration gradients, as indicated in an earlier study.
A linear spatial gradient of ciprofloxacin and Luria–Bertani
broth (LB) medium was established and maintained by diffusion over
5 days across a well array in the MGC, with relative concentrations
along the gradient of 1.7–7.7× the original minimum inhibitory
concentration (MICoriginal). The E. coli biomass increased in wells with lower ciprofloxacin concentrations,
and only a low level of resistance to ciprofloxacin was detected in
the recovered cells (∼2× MICoriginal). Homogeneous
batch culture experiments were performed with the same temporal exposure
history to ciprofloxacin concentration, the same and higher initial
cell densities, and the same and higher nutrient (i.e., LB) concentrations
as in the MGC. In all batch experiments, E. coli 307 developed higher ciprofloxacin resistance after exposure, ranging
from 4 to 24× MICoriginal in all replicates. Hence,
these results suggest that the presence of spatial gradients appears
to reduce the driving force for E. coli 307 adaptation to ciprofloxacin, which suggests that results from
batch experiments may over predict the development of antibiotic resistance
in natural environments.
We fabricated a microfluidic reactor
with a nanoporous barrier
to characterize electron transport between Shewanella
oneidensis MR-1 and the metal oxide birnessite across
a physical separation. Real-time quantification of electron flux across
this barrier by strains with different electron transfer capabilities
revealed that this bacterium exports flavins to its surroundings when
faced with no direct physical access to an electron acceptor, allowing
it to reduce metals at distances exceeding 60 μm. An energy
balance indicates that flavins must be recycled for S. oneidensis MR-1 to yield energy from lactate oxidation
coupled to flavin reduction. In our system, we find that flavins are
recycled between 24 and 60 times depending on flow conditions. This
energy saving strategy, which until now had not been systematically
tested or captured in environmentally relevant systems, suggests that
electron shuttling microorganisms have the capacity to access and
reduce metals in physically distant or potentially toxic microenvironments
(i.e., pores with soluble and transiently sorbed toxins) where direct
contact is limited or unfavorable. Our results challenge the prediction
that diffusion-based electron shuttling is only effective across short
distances and may lead to improved bioremediation strategies or advance
biogeochemical models of electron transfer in anaerobic sediments.
Subsurface environments often contain mixtures of contaminants in which the microbial degradation of one pollutant may be inhibited by the toxicity of another. Agricultural settings exemplify these complex environments, where antimicrobial leachates may inhibit nitrate bioreduction, and are the motivation to address this fundamental ecological response. In this study, a microfluidic reactor was fabricated to create diffusioncontrolled concentration gradients of nitrate and ciprofloxacin under anoxic conditions in order to evaluate the ability of Shewanella oneidenisis MR-1 to reduce the former in the presence of the latter. Results show a surprising ecological response, where swimming motility allow S. oneidensis MR-1 to accumulate and maintain metabolic activity for nitrate reduction in regions with toxic ciprofloxacin concentrations (i.e., 50× minimum inhibitory concentration, MIC), despite the lack of observed antibiotic resistance. Controls with limited nutrient flux and a nonmotile mutant (Δf lag) show that cells cannot colonize antibiotic rich microenvironments, and this results in minimal metabolic activity for nitrate reduction. These results demonstrate that under anoxic, nitrate-reducing conditions, motility can control microbial habitability and metabolic activity in spatially heterogeneous toxic environments.
Spatial concentration gradients of antibiotics are prevalent in the natural environment. Yet, the microbial response in these heterogeneous systems remains poorly understood. We used a microfluidic reactor to create an artificial microscopic ecosystem that generates diffusive gradients of solutes across interconnected microenvironments. With this reactor, we showed that chemotaxis toward a soluble electron acceptor (nitrate) allowed Shewanella oneidensis MR-1 to inhabit and sustain metabolic activity in highly toxic regions of the antibiotic ciprofloxacin (>80× minimum inhibitory concentration, MIC). Acquired antibiotic resistance was not observed for cells extracted from the reactor, so we explored the role of transient adaptive resistance by probing multidrug resistance (MDR) efflux pumps, ancient elements that are important for bacterial physiology and virulence. Accordingly, we constructed an efflux pump deficient mutant (∆mexF) and used resistance-nodulation-division (RND) efflux pump inhibitors (EPIs). While batch results showed the importance of RND efflux pumps for microbial survival, microfluidic studies indicated that these pumps were not necessary for survival in antibiotic gradients. Our work contributes to an emerging body of knowledge deciphering the effects of antibiotic spatial heterogeneity on microorganisms and highlights differences of microbial response in these systems versus well-mixed batch conditions.
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