Motility of Shewanella oneidensis MR-1 Allows for Nitrate Reduction in

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Ecotoxicology and Human Environmental Health

Motility of Shewanella oneidensis MR-1 Allows for Nitrate Reduction in the Toxic Region of a Ciprofloxacin Concentration Gradient in a Microfluidic Reactor Reinaldo Enrique Alcalde, Kyle Michelson, Lang Zhou, Emily Schmitz, Jinzi Deng, Robert A Sanford, Bruce W. Fouke, and Charles J. Werth Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b04838 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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Environmental Science & Technology

TITLE

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Motility of Shewanella oneidensis MR-1 Allows for Nitrate Reduction in the Toxic Region of a Ciprofloxacin

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Concentration Gradient in a Microfluidic Reactor

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Reinaldo E. Alcalde1, Kyle Michelson1, Lang Zhou1, Emily V. Schmitz2, Jinzi Deng3, Robert A. Sanford4, Bruce

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W. Fouke3,4,5, and Charles J. Werth1*

8 9 10

1Department

of Civil, Architectural, and Environmental Engineering, University of Texas at Austin, 301 E. Dean

Keeton Street, Austin, Texas 78712, United States

11 Department of Chemical Engineering, University of Texas at Austin, 200 E Dean Keeton St, Austin,

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2McKetta

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Texas 78712, United States

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3Carl

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Urbana, Illinois 61801 United States

R. Woese Institute of Genomic Biology, University of Illinois Urbana-Champaign, 1206 W Gregory Dr,

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4Department

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61801, United States

of Geology, University of Illinois at Urbana−Champaign, 1301 West Green Street, Urbana, Illinois

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5Department

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Urbana, Illinois 61801, United States

of Microbiology, University of Illinois at Urbana−Champaign, 601 South Goodwin Avenue,

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*Corresponding author: Charles J. Werth, [email protected]

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ABSTRACT

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Subsurface environments often contain mixtures of contaminants in which the microbial degradation of one

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pollutant may be inhibited by the toxicity of another. Agricultural settings exemplify these complex

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environments, where antimicrobial leachates may inhibit nitrate bio-reduction, and are the motivation to

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address this fundamental ecological response. In this study, a microfluidic reactor was fabricated to create

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diffusion-controlled concentration gradients of nitrate and ciprofloxacin under anoxic conditions in order to

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evaluate the ability of Shewanella oneidenisis MR-1 to reduce the former in the presence of the latter. Results

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show a surprising ecological response, where swimming motility allow S. oneidensis MR-1 to accumulate and

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maintain metabolic activity for nitrate reduction in regions with toxic ciprofloxacin concentrations (i.e. 50x

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Minimum Inhibitory Concentration, MIC), despite the lack of observed antibiotic resistance. Controls with

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limited nutrient flux and a non-motile mutant (∆flag) show that cells cannot colonize antibiotic rich

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microenvironments, and this results in minimal metabolic activity for nitrate reduction. These results

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demonstrate that under anoxic, nitrate-reducing conditions, motility can control microbial habitability and

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metabolic activity in spatially-heterogeneous toxic environments.

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ABSTRACT ART

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INTRODUCTION

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Antibiotics derived from animal, human and manufacturing waste are major pollutants found in many natural

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environments.1 As antibiotics are increasingly released into sediments, soils, surface water and groundwater,

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they exert growing deleterious effects on biogeochemical processes.2 An important example is the association

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between nitrates and antibiotics from agriculturally impacted soil and groundwater.3,4 Nitrate is the world’s

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most ubiquitous soil and groundwater contaminant stemming from the widespread use of synthetic and organic

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(i.e. biosolids) fertilizers for agricultural crops.5 Antibiotics are an agricultural contaminant resulting from land

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application of antibiotic rich animal manure and municipal biosolids.6 Ultimately both of these antibiotic sources

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represent a leaching hazard to soil and groundwater 7,8 with detected antibiotic concentrations ranging from

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ng/L to µg/L levels.9,10

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Alone, nitrate is readily reduced by microorganisms to either dinitrogen or ammonium.11,12 When mixed with

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antibiotics, nitrate reduction is shown to be inhibited in sediment,13–15 soil,16–19 and groundwater20,21 microcosm

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studies, and in some cases cause a shift in microbial community structure.22,23 A limitation of the previous

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studies is that they introduce well-mixed combinations of nitrate and antibiotics when they actually exhibit

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spatial heterogeneity in the field due to solute transport mechanisms . 24–26 For example, Smith et al. (1991)

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showed that nitrate concentrations decrease with diffusion into soil aggregates as redox potentials drop and

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more intra-aggregate reduction occurs.24 In addition, nitrate and antibiotic concentrations decrease away from

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groundwater plume centerlines, as transverse dispersion promotes mixing with surrounding groundwater.27 In

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these environments, microorganisms may be able to migrate either away or towards regions of stress or

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nutrients to optimize free energy yield28, and/or possibly adapt to successively higher concentrations of

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antibiotics along concentration gradients.29,30

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Numerous clinical studies have attempted to recreate antibiotic concentration gradients by exposing bacteria to

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successively higher concentrations over time, often resulting in the selection of resistant genotypes.31–34

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However, these temporal gradients are not good analogs for spatial gradients, because only in the latter do

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microorganisms have the option of adapting along a continuous and spatially varying gradient. Theoretical35

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and experimental29,30 models that considered spatially varying antibiotic concentration gradients suggest that ACS Paragon Plus Environment

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cell migration provides a competitive advantage in nutrient utilization. For example, in a study using soft agar

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swarming motility allowed cells to migrate from low to high antibiotic concentrations as mutations emerged that

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promoted resistance in order to access limited nutrients.29 But swarming motility and high cell densities have

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also been shown to induce collective antibiotic tolerance, independent of mutational resistance.36 In another

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study, spatial concentration gradients of antibiotics were established in a microfluidic device, where mutation-

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driven antibiotic resistance of an E. coli strain developed within 10 hours to optimize nutrient utilization.30 E. coli

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appeared to grow in antibiotic concentrations as high as 200x MIC (minimum inhibitory concentration) in the

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microfluidic device, while cells extracted from the device had significant growth at 2x MIC and slight growth at

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up to 20x MIC (MIC of extracted cells was not reported). These results call into question whether cells must

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develop mutation-driven antibiotic resistance to access nutrients in antibiotic rich zones along spatial

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concentration gradients. Or conversely, whether other mechanisms, such as cell motility, might allow access to

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nutrients without the development of antibiotic resistance.

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The objective of the present study is to evaluate the effects of cell motility on nitrate bio-reduction during

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chronic exposure to and along a continuous spatial gradient of the antibiotic ciprofloxacin by the bacterium

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Shewanella oneidensis MR-1 in a microfluidic gradient chamber (MGC). Ciprofloxacin was selected as a

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representative fluoroquinolone antibiotic. Fluoroquinolones are environmentally recalcitrant, as they persist in

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livestock manure and municipal biosolids that are used for land-application.37–39 Ciprofloxacin is a broad-

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spectrum antibiotic that inhibits the DNA gyrase, an enzyme that catalyzes DNA double helix de-coiling and

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promotes cell division.40 It is both a biostatic and biocidal antibiotic with biocidal effects at elevated

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concentrations due to DNA fragmentation.41 S. oneidensis MR-1 is a gram-negative sediment isolate42 that is

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motile43 and undergoes respiratory nitrate ammonification.44 The MGC contains a rectangular array of 850

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interconnected hexagonal wells and is an idealized representation of environmental spatial heterogeneity in

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porous networks (Figure 1). The array is bounded on either side by flow channels, which contain set

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concentrations of solutes. Diffusion controls mass transport through the well array, and this along with flow

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channel concentrations establishes nitrate and ciprofloxacin concentration profiles. The well array was

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inoculated with S. oneidensis MR-1, and the growth, location, and activity of these bacteria were monitored

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over time to evaluate their response to different nitrate and ciprofloxacin concentration gradients. The ACS Paragon Plus Environment


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experiments were designed to test for the first time the hypothesis that cell motility allows non-resistant

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microorganisms to access and reduce nitrate in the toxic region along a spatial gradient of ciprofloxacin without

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the development of antibiotic resistance.

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MATERIALS AND METHODS

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Bacterial Strains and Media

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Three bacterial strains of Shewanella oneidensis MR-1 were used in this study. They include a wild-type S.

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oneidensis MR-1 strain (ATTC 700550), a flagellum deficient non-motile S. oneidensis MR-1 mutant (∆flag)

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with and an antibiotic resistant S. oneidensis MR-1 mutant (SP-1), generated from serial passage experiments

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(details in section ‘serial passage experiments’ below). For all experiments, cells were initially streaked on

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standard LB agar plates (Fisher BioReagents: LB Agar, Miller) from a -80˚ C clonal stock and incubated

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overnight at 30˚C. One colony was selected and transferred to anoxic media (described below), incubated

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overnight at 30˚C, and then aliquoted for batch or microfluidic experiments.

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All strains were cultured in PIPES-buffered anoxic media adapted from Myers and Nealson (1988)42 unless

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otherwise specified. The media consisted of the following per liter of distilled water: 0.027 g NH4Cl, 0.068 g

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KH2PO4, 0.2 g MgCl2·6H2O, 0.15 g CaCl2·2H2O, 3 g NaCl, 1g yeast extract, 10 ml of 10× Wolfe’s vitamin

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solution (ATCC MD-VS), 10 mL of 10× Wolfe’s mineral solution (ATCC MD-TMS) and 9 g PIPES (piperazine-

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N,N′-bis (2-ethanesulfonic acid) buffer (adjusted to 7.1 with 10 M NaOH). The media was amended as needed

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with 4 mM sodium DL-lactate as the carbon source and electron donor, and with 2 mM sodium nitrate as the

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electron acceptor. A separate buffer-only solution was prepared for use in selected MGC experiments; it

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contained all media constituents except yeast; it did not include lactate or nitrate. The media and buffer

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solutions were purged with N2 gas to remove O2,45 stored in anaerobic culture tubes (28mL, Chemglass) or

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serum vials (125mL, Wheaton) sealed with butyl rubber stoppers and aluminum crimp caps, and autoclaved

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prior to use. Some experiments were conducted with a bicarbonate buffered media, rather than PIPES,

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consisting of 30mM NaHCO3 and an atmosphere of N2:CO2 (80:20). No difference in growth was observed for

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cells grown in the two media.

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MIC Determination

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The minimum inhibitory concentration (MIC) of ciprofloxacin for all S. oneidensis MR-1 strains used in this

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study was determined under nitrate reducing conditions using 96-well microtiter plates following previous

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methods. 30,46 The 96-well plates were prepared and incubated at 25˚C in an anaerobic chamber containing a

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N2:H2 (98.5:1.5) atmosphere. Each well contained 200 µL PIPES-buffered media amended with lactate (4mM)

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and nitrate (2mM), and 10 µL of cell inoculum, yielding a final concentration of 1*106 cells/mL. The wells were

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then amended with ten different concentrations of ciprofloxacin, each in triplicate: 0 ng/mL, 10 ng/mL, 20

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ng/mL, 30 ng/mL, 40 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, 1000 ng/mL, 5000 ng/mL and

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10000 ng/mL, and allowed to incubate for 24 hours. The MIC was determined at the concentration of

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ciprofloxacin that inhibited visible growth at 24 hrs. Optical density at 24 hrs was measured using a microtiter

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plate reader (Biotech NeoS2.). The MIC of S. oneidensis MR-1 wild-type cells with no prior exposure to

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ciprofloxacin was designated MICWT.

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Batch Nitrate Reduction Experiments and Kill Curve

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Nitrate reduction tests were conducted in anaerobic culture tubes containing 10 mL of PIPES-buffered media

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amended with lactate (4mM) and nitrate (2mM), and incubated at 25 ˚C. Four conditions were tested to assess

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the impact of ciprofloxacin concentrations on nitrate removal: wild-type cells at 106 cells/mL, with either 0

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µg/mL, 0.05 µg/mL (1x MICWT), or 2.5 µg/mL (50x MICWT) ciprofloxacin, and wild-type cells at 107 cells/mL with

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2.5 µg/mL (50x MICWT) ciprofloxacin. Note, an inoculum of 107 cells/mL was used to explore effects of cell

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density on nitrate reduction in the presence of ciprofloxacin. Nitrate reduction and cell growth were monitored

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for 48 hrs. Triplicate samples were collected and filtered (0.2 µm filter) at 0, 2, 4, 8, 24 and 48hrs for analysis of

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nitrate, nitrite, and ammonia.

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A time-kill study that distinguishes biostatic versus biocidal effects of antibiotics was conducted following

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previous protocols.41 Serum bottles containing 50 mL of PIPES-buffered media amended with lactate (4mM)

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and nitrate (2mM), and incubated at 25 ˚C. Ciprofloxacin concentrations tested included 0 µg/mL, 0.025 µg/mL

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(1/2x MIC), 0.05 µg/mL (1x MICWT), 0.25 µg/mL (5x MICWT), or 2.5 µg/mL (50x MICWT). Again, for 50x MIC

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inoculums of 106 cells/mL and 107 cells/mL were used. Samples were collected at 0, 4, 8, 24, 48hrs and plated ACS Paragon Plus Environment


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on LB agar plates to determine viable cell counts. Three analytical replicates were measured for each single

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experimental sample. The detection limit for the kill-curve was 1*103 cells/mL due to required dilutions.

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Batch Serial Passage for Mutant Generation

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Batch serial passage experiments were conducted in anaerobic culture tubes containing 10 mL of bicarb-

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buffered media amended with lactate (4mM) and nitrate (2mM) and incubated at 30˚C. A culture was initially

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grown and incubated overnight. After incubation, 10% of the solution with cells was transferred to new tubes

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containing fresh media and 10 ng/mL of ciprofloxacin (equivalent to 0.2x MICWT), and incubated for 24hrs.

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Next, 10% of the solution with cells was transferred to a new tube with fresh media and incubated for 24hrs

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and the transfer process was repeated. With each transfer, the ciprofloxacin was increased by 20 ng/mL per

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day (0.4x MICWT/day). This was repeated over 18 days, such that the final antibiotic concentrations S.

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oneidensis MR-1 was exposed to was 350 ng/mL (7x MICWT). The serial passage experiment was performed

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in triplicate, and a control samples with no antibiotics were also transferred over 18 days. Aliquots were taken

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daily from each anaerobic culture tube, and either analyzed for cell density, or amended with glycerol and

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frozen for stock storage. A Nanodrop 2000C (Thermo Fisher Scientific) was used to obtain OD600

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measurements for each sample. After the experiment, samples were revived and tested for new MICs to

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determine antibiotic resistance.

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MGC Design and Fabrication

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The microfluidic gradient chamber (MGC) consists of a regular array of 850-interconnected hexagonal wells,

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etched 10µm deep into a silicon wafer. Each of the wells has a side length of 200µm, supporting a volume of

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1nL, and is connected to six surrounding wells by channels that are 200µm long, 10µm long, and 10µm deep

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(Figure 1a-c). Channels bound either side of the well-array, and each is 100 µm wide by 10µm deep. These

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boundary channels are connected to adjacent wells by barrier walls containing 200nm deep pores that allow

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solute diffusion between boundary channels and the well array but prevent advective transport into the well

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array, and passage of S. oneidensis MR-1 from wells to channels. Overall, the hexagonal well-array is 2 by

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1.25 cm with a total volume of 850nL.


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The MGC was fabricated on a silicon substrate using standard photolithography and inductively coupled

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plasma reactive ion etching (ICP-RIE) as described in the Supporting Information. Inlet and outlet ports (~

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1mm diameter) on either ends of the boundary channels, and for the well array, were ultrasonically drilled. The

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etched and drilled silicon wafer was sealed by anodically bonding a glass wafer (Borofloat 33) to the silicon,

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etch-side up, at 900V and 400˚C. PEEK NanoPort assemblies (IDEX Corporatin) were attached (LOCTITE HP-

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60 epoxy) to the drilled ports and connected to 0.005” ID PEEK tubing. PEEK and ETFE fittings connected the

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tubing to gastight syringes (Hamilton 1000 series, 5mL). A microfluidic syringe pump (Cole-Parmer) controlled

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the injection of solutions into the MGC. The entire assembly is shown in Figure S1.

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MGC Tracer Experiment

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Diffusion of a conservative tracer across the MGC was monitored over a 96hr period. First, the entire well array

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and both boundary channels were purged with PIPES-buffered media. Next, the well array inlet and outlet

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ports were sealed, and the media amended with 10µM fluorescein dye was continuously purged through one

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boundary channel (BC-1), and media alone was continuously purged through the other boundary channel (BC-

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2). Fluorescent dye diffused into and across the well array, and intensity in the well array was monitored over

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time. Depth-averaged dye intensity values across the well array were fit using a non-steady state 1-D

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analytical diffusion equation,47 and a best-fit effective diffusion coefficient was obtained. A tortuosity value was

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obtained from this parameter, and this was used to estimate concentration profiles for ciprofloxacin in the well

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array in subsequent experiments.

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MGC Nitrate Reduction Experiment

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Prior to each experiment, the MGC was sterilized by flushing the boundary channels and well array with 70%

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ETOH and sterile distilled water. Media or buffer was then feed to the boundary channels. Cells (i.e., WT,

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∆flag, SP-1) grown in PIPES-buffered media overnight were diluted to 1*106 cells/mL in new PIPES-buffered

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media containing lactate (0.4mM) and nitrate (0.2mM) and continuously flowed through the hexagonal well-

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array at 100µL/hr for 15 minutes for inoculation; at this cell density, there is roughly one microbial cell per

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hexagonal well before growth begins.



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Experiments were conducted for 5 or 30 days at 25 ±1˚C. During this period, there was no flow through the

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well array (diffusion only). Media with lactate, nitrate, and antibiotics was continuously supplied through BC-1

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at 30 µL/hr, and media with lactate and nitrate, or a buffer only, was continuously supplied to BC-2 at 30µL/hr.

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Effluent from the boundary channels was collected every 24hrs for analysis of nitrate, nitrite, and ammonia. At

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the conclusion of each experiment, cells were extracted by flushing the well array with 1mL of media containing

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lactate (4mM) and nitrate (2mM). Extracted cells were then incubated for 8hrs, and the new minimum inhibitory

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concentration (MIC) of ciprofloxacin was determined and compared to wild-type cells never exposed the

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antibiotic (MICWT). In a second MIC method, the 1 ml of extracted cells was divided into 200 µl aliquots that

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were directly amended (without dilution) with different ciprofloxacin concentrations (i.e., 0, 1, 2, 3, and 5x

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MICWT) and run in the 96 well plate. The total solution volume in each well was 210ul (10ul of antibiotic and

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200ul cells extracted from MGC). Several other methods to extract and test MICs were attempted with the

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same results (see Supporting Information). Note that the 8hr incubation susceptibility assay was conducted for

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all experiments and the direct susceptibility assay was only conducted for WT-H and FLA-H experiments. Each

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experiment performed is listed in Table 1.

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Image Acquisition and Processing

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All images were acquired with a Nikon Eclipse Ti-E system inverted microscope using reflected brightfield and

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epi-fluorescent microscopy. The microscope was equipped with a monochrome digital charge couple device

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camera, and automated stage with pre-calibrated auto z-focus (depth focus). The fluorescent tracer was

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imaged using an FITC filter cube (C-FL B-2E/C) and 4x objective lens. Microbial cells were imaged using

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reflected brightfield with a 15x objective lens (NA = 0.3, WD = 10.1 mm). At this magnification, individual cells

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could be identified via thresholding, and individual cells counts and microbial dimensions were determined.

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Similar to OD600 measurements, there is no discrimination between live and dead cell counts with this

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method. When cell densities were too high and individual cells were not able to be distinguished, cell density

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was averaged by area coverage. Each image captured only a portion of the well array, so many individual

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images were taken and montaged with 5% overlap to capture the entire array. All image processing was done

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with NIS Elements.


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RESULTS

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Ciprofloxacin Inhibits S. oneidensis MR-1 Growth and Nitrate Reduction, and Promotes Antibiotic

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Resistance in Batch Reactors

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Wild-type S. oneidensis MR-1 was exposed to distinct concentrations of ciprofloxacin in 96-well plate

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experiments to determine the minimum inhibitory concentration (MICWT) (Figure S2). As expected, cell growth

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decreases with increasing ciprofloxacin concentration, and as a result cell doubling-times increase. The

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MICWT, was 50 ng/mL of ciprofloxacin. The same MIC, via an endpoint measurement only, was obtained for the

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flagella deficient mutant (∆flag). Besides the lack of flagella, a constitutively expressed mutation in the

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chromosome, ∆flag behaves similarly to its wild-type counterpart (Figure S3). Although an MIC of ciprofloxacin

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for S. oneidensis MR-1 in anoxic settings has not been previously reported, the value of ~50 ng/mL is in the

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range of MICs observed for E. coli under aerobic conditions.30 Nitrate reduction to nitrite and ammonia by wild-

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type S. oneidensis MR-1 in the presence of different antibiotic concentrations was monitored in batch reactors

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to probe reduction inhibition. Nitrite and ammonium production rates decrease with lower initial cell density,

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and with greater ciprofloxacin concentrations (Figure S4). At high cell density (107 cells/ml) and 50x MICWT,

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nitrate is almost entirely degraded over 24 hours, but nitrite persists after this time period. At 50x MICWT, a

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time-kill curve (Figure S5) confirms that nitrate (and by association nitrite) reduction inhibition is attributed to

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the biocidal effects of high ciprofloxacin concentrations.

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Anoxic serial passage experiments were conducted to generate an antibiotic resistant strain of S. oneidensis

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MR-1 for MGC experiments (Figure S6). Subsequent MIC assays show that cells had greater resistance with

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increasing time (i.e., with exposure to higher ciprofloxacin concentrations). In all cases, new MIC (i.e., MICSP)

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levels always exceed serial passage exposure concentrations, with an MICSP of 2.5 µg/mL (50x MICWT)

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obtained for cultures exposed to 7x MICWT (i.e., Day 18 sample). A single colony from a day 18 sample was

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selected as the resistant mutant (i.e., SP-1 mutant) for use in MGC experiments.

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Tracer Test Confirms Diffusion Controls Transport in the MGC Well Array

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Fluorescein dye mass transport across the MGC was imaged over time (Figure 1d). Fluorescent dye intensity

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varies mainly with depth between the boundary channels, indicating the concentration in BC-1 is constant

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along its length. Depth averaged cross sectional profiles of relative intensity from the experiment were

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compared to profiles of relative concentration from the 1D diffusion model (Figure 1e). Experimental profile

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shapes match the model, with a best-fit apparent diffusion coefficient (Dapp) of 1.6x10-6 cm2/s. The molecular

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diffusion coefficient for fluorescein in water (Dmol,aq) is 4.25 *10-6 cm2/s.48 Assuming Dapp=Dmol,aq/, where

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=tortuosity, then  =2.56. This is similar to values measured for natural porous media,49 and indicates

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diffusion controls mass transport in the well array. The value of Dmol,aq for ciprofloxacin is within 6% of that for

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fluorescein, thus diffusion time scales for both chemicals are similar. Diffusion profiles change markedly over

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24 hours, and wells adjacent to BC-1 reach 95% of steady state values at this time. Hence, ciprofloxacin

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concentrations in wells adjacent to BC-1 reach 95% of their steady state values after 24 hours in MGC

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experiments with S. oneidensis MR-1.

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Cell Motility and Migration Allow for Accumulation and Persistence of Cells in High Ciprofloxacin

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Concentration Regions of the MGC

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The sequence of MGC experiments performed are listed in Table 1; they are the base case experiment for

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wild-type S. oneidensis MR-1 cells with a high nutrient flux (WT-H), a similar experiment with the flagella

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deficient strain (∆flag) and a high nutrient flux (FLA-H), an experiment with the wild-type cells and low nutrient

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flux (WT-L), and an experiment with the ciprofloxacin resistant mutant and low nutrient flux (SP-L). In the base

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case experiment, we first inoculated the MGC with a wild-type strain of S. oneidensis MR-1 to query its

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response to spatial gradients of ciprofloxacin. The experiment, termed WT-H, ran for 120hrs and consisted of

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media containing nitrate (2mM), lactate (4mM), and ciprofloxacin (2.5 µg/ml, 50x MICWT) in boundary channel 1

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(BC-1) and media with nitrate (2mM) and lactate (4mM) in BC-2. Figure 2a shows time-lapse images of cells at

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6, 24, and 48hrs for a single well adjacent to both BC-1 and BC-2 and Figure 2b shows the cell density

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measurements over time in wells across the MGC. The same results are also presented as cell density across

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the antibiotic gradient in the MGC in Figure 3a, and these will be compared to other experiments in later

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sections. A video clip of cells at BC-1 also provides a visualization of cell motility. (Movie S1). To our surprise,

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a dense population of S. oneidensis MR-1 cells persisted at wells adjacent to BC-1 from start to finish of ACS Paragon Plus Environment

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experimental runs. At 24 hours, the cell density at wells adjacent to BC-1 (row 1) was 1190  138 cells/well

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(1.19 *109 cells/ml) and reached a sustained steady-state concentration, thereafter. Cells in row 2 continue to

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increase in density until 96 hrs, where they reach a similar steady-state concentration. At 24 hours, the cell

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density in wells adjacent to BC-2 was 4275  575 cells/well (4.27 *109 cells/ml) and slightly increased to 7946 

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556 cells/well (7.95 *109 cells/ml) by the end of the 120hr experiment. At 24 hours, cells adjacent to BC-1, but

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not BC-2, were elongated and filamentous (Figure S7a). After the first 24hrs, cells began to revert back to their

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original size. Cells extracted from the MGC at the conclusion of the experiment were assessed for antibiotic

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resistance; they acquired resistance of only 0-1x MICwt among three replicates. We repeated WT-H but ran the

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experiment for 30 days to determine if prolonged exposure would promote antibiotic resistance, but a small

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resistance of only 1x MICwt was acquired.

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Overall, cell densities in the MGC (~109 cells/mL) were greater than batch controls without ciprofloxacin at

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24hrs (~108 cells/mL). This is likely due to the more favorable growth conditions in the MGC, where the

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boundary channels provide a continuous source of fresh nutrients and a sink for metabolic by-products.

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Fluorescein tracer data (Figure 1e) indicates that at 6hrs and 24hrs, ciprofloxacin concentrations in wells

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adjacent to BC-1 were at 76% and 95% of the steady state values (50x MIC). In batch, cells exposed to 50x

352

MIC (conditions in BC-1) had very little activity for nitrite reduction after 24hrs; furthermore, they died off by 24

353

hours due to the biocidal effects of ciprofloxacin at 50x MIC41 (Figure S5). Therefore, it is unclear why cells

354

persist adjacent to this boundary given the noted lack of acquired antibiotic resistance. Ciprofloxacin appears

355

to have caused elongation of cells near BC-1. This is not surprising, given that filamentation upon exposure to

356

ciprofloxacin is well documented50,51 and is caused by induction of division inhibitors (e.g. SulA in e. coli).52,53

357

However, it is not clear why cells revert back to their original size in wells adjacent to BC-1, which has

358

previously been reported as an indication of resistant genotypes in E. coli. 54 Note that other studies have

359

correlated obtained genetic antibiotic resistance with increases in MIC measurements,, thus a post MIC test

360

infers but does not negate a lack of genetically encoded resistance.55

361

362

The accumulation of cells near the nutrient rich boundaries in WT-H indicates that random migration of cells,

363

as suggested elsewhere,56 is not responsible for the occurrence of bacteria in regions with high ciprofloxacin ACS Paragon Plus Environment


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364

concentrations. This is not surprising given that S. oneidensis MR-1 has 3 chemotaxis genes and 27

365

chemoreceptors, implying that migration toward favorable niches is an essential survival strategy.57 Nitrate

366

likely served as a chemoattractant for S. oneidensis MR-1, in line with previous research showing chemotaxis

367

towards nitrate in the absence of alternative electron acceptors.58 The lack of antibiotic resistance measured in

368

cells extracted from WT-H suggest that a step-wise resistance up the antibiotic gradient is not responsible for

369

the observed increase in cell density in wells near BC-1. We hypothesized that cell motility plays a key role in

370

the accumulation of S. oneidensis MR-1 in wells adjacent to BC-1 in WT-H, so we inoculated a new MGC with

371

a flagella deficient strain (∆flag), which renders all the cells immobile. Results for this experiment, termed FLA-

372

H, with boundary conditions identical to WT-H, are shown in Figure 3b. Cells did not emerge in wells adjacent

373

to BC-1 throughout the experiment; rather, cell density across the reactor was inversely correlated to the

374

concentration of ciprofloxacin. Also, cell elongation was more evident and sustained over 120 hours in wells

375

closer to BC-1 (Figure S7b), indicating a stress response to elevated ciprofloxacin concentrations. FLA-H

376

experiments suggest that the apparent initial cell elongation and later reversion back to originally sized cells in

377

WT-H is due to an influx of healthy cells migrating towards the ciprofloxacin boundary for nutrient utilization.

378

Cells extracted from FLA-H at the conclusion of the experiment were assessed for antibiotic resistance; they

379

showed consistent resistance to 5x MICwt (2 replicates), higher than in WT-H but still lower than the

380

ciprofloxacin concentrations in wells adjacent to BC-1. It is likely higher than in WT-H because FLA-H cells

381

could not migrate from well to well, and thus a selective pressure resulted in either resistance or death, similar

382

to the conditions presented in the serial passage batch experiment.

383

384

The results from WT-H and FLA-H indicate that cell motility rather than antibiotic resistance is playing a key

385

role in accumulation of cells in wells adjacent to BC-1 in the WT-H experiment. We hypothesized that in WT-H,

386

cells were only growing in wells adjacent to BC-2 and migrating toward BC-1, to metabolize the high

387

concentrations of nitrate and lactate near this boundary without antibiotic resistance. This is attainable as S.

388

oneidensis MR-1 has swimming speed of 67.7  4.3 µm/s. 43 Therefore, we inoculated a new MGC with the

389

wild-type strain, but delivered nitrate (2mM) and lactate (4mM) with ciprofloxacin (50x MIC) at BC-1, and only

390

buffer with no nutrients to BC-2. Results for this experiment, termed WT-L, are shown in Figure 3c. In the first

391

24hrs, cell density reached 950 cells/well (9.5 *108 cells/ml) but rapidly declined thereafter near BC-1 wells, ACS Paragon Plus Environment

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392

and at 72hrs cells began to accumulate near BC-2 wells only. The initial spike in cell density at BC-1, similar to

393

that of WT-H, is attributed to initial growth from the inoculum. The subsequent decline in cell density adjacent

394

to BC-1 is likely due to chronic exposure and biocidal effects of ciprofloxacin at high concentrations,41 and the

395

lack of cells migrating up gradient from BC-2. The increase in cell density adjacent to BC-2 at 72hrs is

396

attributed to the eventual diffusion of nutrients toward this boundary, and the corresponding low antibiotic

397

concentration. Cells extracted from WT-L showed no increase in antibiotic resistance. We repeated WT-L, but

398

ran the experiment for 30 days, to determine if cells could either build up sufficient biomass at BC-2 to allow

399

migration toward BC-1 to metabolize the higher nutrient concentrations without resistance or if cells could gain

400

resistance to ciprofloxacin, step-wise, up gradient and eventually grow in wells adjacent to BC-1. Cell did not

401

migrate up gradient nor did they accumulate in wells adjacent to BC-1. Cells extracted from the 30-day

402

experiment were resistant to 5x MICWT.

403 404

A control experiment, termed SP-L, with the same boundary conditions as WT-L, but with the ciprofloxacin

405

resistant mutant (SP-1) in place of the wild-type cells, was run, and results are shown in Figure 3d. At 24 hrs,

406

the cell density in wells adjacent to BC-1 reached 380 cells/well (3.8 *108 cells/mL). After 24hrs and throughout

407

the 120hr experimental run, cell density increased at BC-1 with a final cell density of 1030 cells/well (1.03*109

408

cells/mL). Hence, cells persist and appear to continuously grow in wells adjacent to BC-1 if a resistant strain is

409

present, unlike WT-L which dies off at wells adjacent to BC-1. Interestingly, a large portion of the SP-1 mutant

410

remained elongated (Figure S7c), further suggesting that the reduction in elongation for cells adjacent to BC-1

411

in WT-H is a result of migration. Cells extracted from SP-L at the conclusion of the experiment were assessed

412

for antibiotic resistance and they maintained their resistance of 50x MICWT

413 414

Cell Motility and Migration Allow for Nitrate Reduction in the Presence of High Ciprofloxacin

415

Concentration in the MGC

416

MGC results suggest that S. oneidensis MR-1 in wells adjacent to BC-1 in the WT-H experiment were

417

metabolizing nitrate. In contrast, the lack of cells adjacent to this boundary in the FLA-H experiment suggested

418

the opposite. To confirm, daily effluent samples from each boundary channel were collected from replicate

419

MGC experiments over a seven-day period and analyzed for nitrate, nitrite, and ammonium. Flow rates during ACS Paragon Plus Environment


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420

the first three days in these replicates matched the original experiments, but on day four the flow rate was

421

reduced by 3x in order to increase fluid residence time in the boundary channels and allow greater buildup of

422

nitrate reduction products (i.e., nitrite, ammonium). Results of replicate measurements on days 4-7 are shown

423

in Figure 4. Similar levels of nitrate conversion to nitrite and ammonium were observed in BC-2 effluent of both

424

experiments. However, in BC-1, 10.9 times more nitrate was reduced to nitrite (p=0.016) and 1.04 times more

425

nitrite was reduced to ammonium (p=0.038) when comparing WT-H to FLA-H. The greater conversion to nitrite

426

in BC-1 effluent from WT-H indicates that cells in wells adjacent to this boundary were actively metabolizing

427

nitrate. These results validate the role of S. oneidensis MR-1 motility on enhancing nitrate reduction in the

428

antibiotic ciprofloxacin. We delivered the RedoxSensor Green dye into a replicate of WT-H after 120 hours to

429

provide additional evidence that cells adjacent to BC-1 were metabolically active. This dye is an indicator for

430

metabolic redox activity and does not inhibit cell growth.59,60 The majority of cells at wells adjacent to BC-1 in

431

the WT-H experiment show fluorescence at the conclusion of the experiment (Figure S8).

432 433

The previously presented batch tests for nitrate reduction demonstrate that reduction occurs only over limited

434

time periods in the presence of elevated ciprofloxacin concentrations. The unique aspect here is that in the

435

MGC for WT-H experiments, nitrate reduction is persistent throughout the experimental run (120hrs), whereas

436

in batch at 50x MIC, nitrate reduction ceases in a short time period (8-24hrs). The persistent biotransformation

437

of a contaminant in the presence of lethal antibiotic concentrations with what seems to be primarily a

438

phenotypic ecosystem response has not been previously observed. This is to say that S. oneidensis MR-1’s

439

phenotypic traits (i.e., motility, migration, cell density, chemotaxis) are responsible for the persistent metabolic

440

activity in the toxic regions of the ciprofloxacin concentration gradient.

441 442

Implications of Antibiotic Gradients in Natural Porous Networks

443

The MGC experimental results support the hypothesis that chemotactically-directed motility from high cell

444

density regions is the dominant factor influencing sustained habitability and metabolic activity in highly toxic

445

microenvironments, independent of antibiotic resistance development. They do not support alternative

446

mechanisms proposed by others: (a) Random cell migration;56 (b) rapid development of localized antibiotic

447

resistance in toxic regions;30 or (c) step-wise, directed migration up the antibiotic gradient due to ACS Paragon Plus Environment

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448

chemoattraction (i.e., nitrate), as antibiotic resistance is attained.35 Previous studies have observed similar

449

behavior as ours with cells lacking antibiotic resistance. Butler et al., (2010) shows that swarming motility and

450

high cell density allowed Salmonella cells to migrate to antibiotic-containing solid environments (swarming

451

agar). 36 Additionally, Hol et al., (2016) report that high-density and swimming motility allow E. coli populations

452

to tolerate a lethal dose of kanamycin.55 By using a motility deficient mutant, our work expands on these

453

studies by explicitly demonstrating the dependence on motility regardless of high or low cell-density.

454

Furthermore, our results demonstrate sustained habitability in toxic regions beyond the time period probed in

455

previous studies (5-30 days vs 16-20hrs); this was done not only through image processing like the previous

456

studies, but also by quantifying metabolic by-products. Last, this is the first study to show that the phenomenon

457

presented occurs under anoxic, nitrate-reducing conditions.

458 459

The results suggest that in soil, sediment, and groundwater environments, S. oneidensis MR-1 can reduce

460

nitrate in the presence of ciprofloxacin at levels above the MIC due to motility and migration. Cells are able to

461

grow away from toxic ciprofloxacin concentrations and migrate towards nitrate in the presence of toxic

462

ciprofloxacin concentrations. Batch results indicate that cells are temporarily active (e.g., up to 24 hours at 107

463

cell/ml and 50x MICWT) at high ciprofloxacin concentrations; therefore, if no mutations are occurring, it is

464

plausible that a constant migration of new cells from non-toxic regions can result in continued nitrate reduction

465

in the presence of elevated fluoroquinolone antibiotics in natural systems. This bodes well for nitrate depletion

466

in soil aggregates, where anaerobic microenvironments persist where both nitrate and antibiotic concentration

467

may be elevated. It also bodes well for groundwater plumes containing both nitrate and antibiotics, because

468

microbes can be metabolically active near the plume margins where the electron donor is high, and gradients

469

of nitrate and ciprofloxacin are present. Our results provide insight into the microbial populations that may

470

dominate antibiotic contaminated environments based on their phenotypic characteristics. Lastly, they provide

471

an advancement in understanding biogeochemical cycling under the effects of stressors compounds (e.g.,

472

antibiotics) at the mixing zones of natural environments and broaden our understanding of the mechanisms

473

that allow for such processes to prevail.

474 475

ACKNOWLEDGEMENTS ACS Paragon Plus Environment


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This work was supported by the National Aeronautics and Space Administration (NASA) through the NASA

477

Astrobiology Institute under Cooperative Agreement No. NNA13AA91A issued through the Science Mission

478

Directorate. This work is supported by the National Science Foundation Graduate Research Fellowship

479

Program (NSF GRFP) under Grant No. DGE-1610403 to Reinaldo E Alcalde. Any opinion, findings, and

480

conclusions or recommendations expressed in this work are those of the authors and do not necessarily reflect

481

the views of the NASA Astrobiology Institute and National Science Foundation. We thank Dr. Kai Thormann,

482

Justus Liebig University Giessen) for kindly providing the S. oneidensis MR-1 ∆flag mutant.

483 484

SUPPORTING INFORMATION

485

Supporting information and methods; Pages S2-S4: Chemicals, MGC Fabrication, MGC Cleaning and

486

Sterilization, Analytical Methods, MGC Cell Extraction and Susceptibility Assay Methods. Microfluid assembly

487

(Figure S1). Kinetic run for MIC determination (Figure S2). Kinetic run comparing WT and ∆flag (Figure S3),

488

Comparison of NO3-, NO2- , and NH4+ concentrations over time in batch cultures under ammonifying conditions

489

(Figure S4). Viable cell counts for cultures of WT S. oneidensis MR-1 vs time (Figure S5). SP-1 mutant

490

generation through a serial passage experiment (figure S6). Relative frequency (%) of cell lengths vs time (hr)

491

for MGC experiments (figure S7). Epi-fluorescent microscopy image at 120hr of a single well for experiment

492

WT-H (figure S8). Video clip of cell motility at BC-1 for WT-H. (Movie S1)

493

494

495

496

497

498

499

ACS Paragon Plus Environment

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500


Table 1: Summary of experiments conducted and respective experimental conditions Boundary Channel 2 BC-2

Code Name

S. Oneidensis MR-1 strain

Boundary Channel 1 BC-1

WT-H

Wild-type Strain

NO3 = 2 mM Lac = 4 mM CIP = 50x MICWT

NO3 = 2 mM Lac = 4 mM


NO3 = 2 mM Lac = 4 mM

FLA-H

∆flag strain

WT-L


Buffer only

Wild-type Strain


Buffer only

SP-L

Lab generated mutant SP-1

Initial Condition

MGC Exp. Duration

Post MGC Resistance

NO3 = 0.2 mM Lac = 0.4 mM Inoc= 106 cells/mL

5-day (n=3) -----------------30-day (n=1)

0-1x MIC -----------0-1x MIC


5-day (n=2)

5x MIC


5-day (n=1) -----------------30-day (n=1)

0x MIC ----------0-1x MIC


5-day (n=1)

50x MIC

501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 ACS Paragon Plus Environment


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518 519 520 521 522 523 524 525 526 527 528 529 530 531 532

Figure 1: (a) Illustration of the MGC. Yellow arrows represent continuous flow of solution through boundary

533

channels. Row 1 arrow represents the row of wells adjacent to BC-1. Row 41 arrow represents the row of wells

534

adjacent to BC-2. Red square represents magnification for fig 1b.(b) SEM image depicting wells, well chanells

535

and boundary channels. (c) SEM images depicting 200nm deep nanoporous barrier. (d) Montaged fluorescence

536

microscopy image depicting gradient establishment throughout the MGC. (e) Experimental and modeled tracer

537

concentration profiles across the MGC for various time points.

538 539 540 541 542 543 ACS Paragon Plus Environment

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544 545 10 5

546 Cell Density (cells/well)

547 548 549

WT-H row 1 row 2 row 10 row 20 row 30 row 41

b

10 4

10 3

10 2

550

20

551

40

60 Time (hr)

80

100

120

552

Figure 2: (a) Time-lapsed brightfield microscopy images of a single hexagonal well adjacent to either boundary channel for

553

WT-H experiment. (b) Plot of the cell density vs time for WT-H experiment (error bars are as standard deviation of separate

554

regions perpendicular to gradient formation in MGC, n=4).

555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 ACS Paragon Plus Environment


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570 571

575 576 577

a

2000

10

578 3000

582 583 584 585

Cell Density (cells/well)

580 581

24 hr 48 hr 72 hr 96 hr 120 hr

1000

0

579

3000

20 30 Distance (rows) WT-L

c

1000

0

10

20 30 Distance (rows)

40

b

2000

1000

3000

2000

FLA-H

0

40


574


573

WT-H


3000

572

10


40

SP-L d

2000

1000

0

10


40

586 587 588

Figure 3: Cell density across antibiotic gradient in MGC at 24 hr intervals for (a) WT-H (b) FLA-H (c)

589

WT-L and (d) SP-L. Row 1 corresponds to the wells adjacent to boundary channel 1, BC-1. Row 41

590

corresponds to the wells adjacent to boundary channel 2, BC-2. Arrows indicate increase or decrease of

591

cell density overtime near the boundary channels.

592 593 594 595 ACS Paragon Plus Environment

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596 597 598

30 a

599

602 603 604

+ NH Concentration, mM 4

601

0.95

p

Motility of Shewanella oneidensis MR-1 Allows for Nitrate Reduction in

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