PHYSIOLOGICAL RESPONSE OF NITRATE REDUCING MICROORGANISMS TO STEEP ANTIBIOTIC CONCENTRATION GRADIENTS

Sedimentary rock deposits are strongly heterogeneous at the microscopic scale, creating a diverse and complexly distributed array of stress-promoting chemical and physical microenvironments that may prove to be advantageous for accelerated microbial adaptation and evolution. This phenomena was initially probed by Zhang et. al. (2011) in an experiment where bacterial cells (E. coli) grown in a stress gradient of Ciprofloxacin (Cipro) developed antibiotic resistance within 10 hours. To further address this phenomena and additionally assess its rates on nitrogen cycling (i.e., nitrate reduction), we are conducting controlled experiments in which strategically chosen microorganisms are subjected to stressor gradients in a microfluidic gradient cell (MGC). Shewanella oneidensis MR-1 was chosen as an environmentally relevant target organism as it allows us to explore this phenomenon under anaerobic ammonifying conditions. The MGC was fabricated on a silicon wafers using standard photolithography methods and was anodically bonded to a glass cover slip. This configuration is gas impermeable and thus allows anaerobic conditions to be maintained. It consists of a hexagonal well array with interconnecting channels and nanoporous barrier separating boundary channel from wells. The nanoporous barrier allows for diffusion of solutes into the well array. Experimental images were captured with a Nikon Eclipse Ti-E system, Epifluorescence microscope. S. oneidensis was subjected to Cipro at 4X and 10X the minimum inhibitory concentration (MIC). In these experiments, strong physiological responses to Cipro are evident. Stressed cells undergo elongation and show promising results of adaptation and evolution as elongation decreases and cell activity increases. The next step in this study is to extract samples of the stressed cells and to compare their metabolism and antibiotic resistance to the true wild-type S. oneidensis MR-1. In addition, genomic changes will be addressed by conducting directed DNA analysis on specific DNA segments where single nucleotide polymorphism (SNPs) mutations due to ciprofloxacin resistance could occur (e.g., gyrA, mexF).