FEMS Microbiology Ecology Advance Access published November 25, 2015

Pseudomonas aeruginosa facilitates Campylobacter jejuni growth in biofilms under oxic flow conditions

Alessandro Culotti1 ¶, Aaron I. Packman1 ¶ 1

Department of Civil and Environmental Engineering, McCormick School of Engineering, Downloaded from http://femsec.oxfordjournals.org/ by guest on November 28, 2015

Northwestern University – Evanston, IL. U.S.A.

Corresponding author: Aaron I. Packman Tel: 847-491-9902, Fax: 847-491-4011, E-mail: [email protected] Address: Department of Civil and Environmental Engineering, 2145 Sheridan Road, Evanston IL, 60208.

1

Abstract We investigated the growth of Campylobacter jejuni in biofilms with P. aeruginosa under oxic flow conditions. We observed the growth of C. jejuni in mono-culture, deposited on preestablished P. aeruginosa biofilms, and co-inoculated with P. aeruginosa. In mono-culture, C. jejuni was unable to form biofilms. However, deposited C. jejuni continuously grew on preestablished P. aeruginosa biofilms for a period of 3 days. The growth of scattered C. jejuni clusters was strictly limited to the P. aeruginosa biofilm surface, and no intergrowth was

in biofilms, with C. jejuni clusters developing on the surface of the P. aeruginosa biofilm. Dissolved oxygen measurements in the medium showed that P. aeruginosa biofilms depleted the effluent DO from 9.0 mg/L to 0.5 mg/L 24 hours after inoculation. The localized microaerophilic environment generated by P. aeruginosa promoted the persistence and growth of C. jejuni. Our findings show that P. aeruginosa not only prolongs the survival of C. jejuni under oxic conditions, but also enables the growth of C. jejuni on the surface of P. aeruginosa biofilms.

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observed. Co-culturing of C. jejuni and P. aeruginosa also enabled the growth of both organisms

Introduction

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Campylobacter jejuni is one of the most common causes of bacterial gastroenteritis worldwide

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(Allos, 2001). While most Campylobacter infections are related to poultry or other meat

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products, contaminated water has also been implicated in numerous disease outbreaks (Stehr-

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Green et al., 1991; Furtado et al., 1998; Kemp et al., 2005). C. jejuni has been isolated from a

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variety of water sources including sewage outflows, river water, streams, groundwater and ponds

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(Alary and Nadeau 1990; Stanley et al.1998; Waage et al. 1999; Jones 2001; Kemp et al. 2005).

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In Scandinavian countries such as Finland, Norway and Sweden, the regular consumption of

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untreated groundwater and stream water has led to numerous waterborne outbreaks of C. jejuni

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(Jones, 2001; Hänninen and Haajanen, 2003). In the United Kingdom, outbreaks of

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Campylobacter enteritis have been traced to contaminated drinking water from private water

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reservoirs in small rural systems (Anon, 2000; Jones, 2001). Campylobacter outbreaks from

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contaminated surface waters have also been reported in New Zealand, the United States, Canada,

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and Spain, amongst others (Vogt et al., 1982; Stehr-Green et al., 1991; Godoy et al., 2002; Clark

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et al., 2003). Previous findings suggest that C. jejuni survival in water distribution systems may

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be enhanced by the presence of environmental biofilms (Buswell et al., 1998; Zimmer et al.,

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2003; Ica et al., 2011). As a result, interactions between C. jejuni and local biofilm microflora in

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natural aquatic environments and drinking water distribution systems are a major concern for

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public health.

20 21

C. jejuni is a microaerophilic bacterium that requires sub-atmospheric levels of oxygen to grow

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(Kelly, 2001, 2008). Despite its sensitivity to atmospheric oxygen tension, C. jejuni has been

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found to survive in aquatic environments under a range of environmental conditions (Buswell et 3

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al., 1998; Zimmer et al., 2003; Hilbert et al., 2010). Temperature, bacterial predation, oxygen

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concentration, bacterial strain and the ability to enter a viable but non-culturable state (VBNC)

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have all been shown to influence the survival of C. jejuni in vitro (Rollins and Colwell, 1986;

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Korhonen and Martikainen, 1991; Buswell et al., 1998; Trachoo et al., 2002; Oliver, 2005;

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Hanning et al., 2008). Association with biofilms has also been found to affect the survival of C.

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jejuni in fully oxygenated systems (Buswell et al., 1998; Hilbert et al., 2010; Ica et al., 2011).

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Recent findings suggest that co-culture with aerobic bacteria may extend the survival of C. jejuni

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through depletion of oxygen in the biofilm (Hilbert et al., 2010; Ica et al., 2011). However, the

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factors that mediate local patterns of C. jejuni persistence are not well established. Further, prior

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studies have focused on the persistence of C. jejuni under bulk oxic conditions, but we

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hypothesized that association with environmental biofilms can also facilitate localized C. jejuni

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growth because of the high diversity of habitat conditions found within biofilms. A deeper

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understanding of C. jejuni interactions with biofilms is needed in order to identify environmental

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reservoirs of C. jejuni and conditions that can favor both the persistence and growth of this

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important pathogen in water systems.

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Here, we investigated the survival and growth of C. jejuni in mixed biofilms with Pseudomonas

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aeruginosa under oxic, oligotrophic conditions in flow cells. Immunofluorescent and nucleic

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acid stains were used to visualize and quantify the growth of C. jejuni and P. aeruginosa. We

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investigated the persistence and growth of C. jejuni deposited on pre-existing P. aeruginosa

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biofilms, as well as co-culture of C. jejuni and P. aeruginosa in biofilms. P. aeruginosa was

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chosen as the co-inoculated organism for this study because it is an important biofilm-forming

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organism in natural and engineered water systems, and has been found to co-occur with C. jejuni

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in such environments (Hilbert et al., 2010).

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Materials and Methods

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Flow Cells

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A polycarbonate flow cell (University of Iowa Medical Instruments Department) was used to

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study biofilm growth under a uniform velocity field. Each channel measures 35 mm x 4 mm x 1

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mm in size, with a single inlet and outlet port. A glass coverslip at the base of each channel

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allows in situ visualization of the biofilms.

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Bacterial Strains

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Flow cell experiments were conducted using a wild-type C. jejuni RM 1221 strain, and a

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laboratory P. aeruginosa PAO1 strain. C. jejuni RM 1221 was originally isolated from a chicken

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carcass (Miller et al., 2000; Poly et al., 2007) and was obtained from the USDA Agricultural

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Research Service, Albany, CA. Stock cultures of P. aeruginosa were streaked onto Luria-Bertani

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(LB) agar plates, and incubated for 24 hours at 37oC. Single colonies were transferred into

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separate tubes containing 3 mL of sterile LB broth and grown overnight at 37oC. Stock cultures

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of C. jejuni were streaked onto campy-cefex agar plates (Becton, Dickinson and Company), and

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incubated for 24 hours at 42oC in BD GasPak EZ Campy pouches (Becton, Dickinson and

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Company). Single colonies were then transferred into glass tubes containing 3 mL of Mueller-

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Hinton broth (BD Diagnostic Systems), and cultured overnight at 42oC (225 rpm) in BD GasPak

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EZ Campy pouches.

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Flow Cell Experimental Conditions

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Mono- and mixed-culture biofilms were grown in R2A medium at room temperature (24oC).

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R2A is commonly used for the growth and enumeration of microorganisms from drinking water

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systems (Reasoner and Geldreich, 1985). The flow was regulated to 0.2 mL/min for each flow

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cell chamber using Gilson Miniplus 3 peristaltic pumps. These pumps produce minimal flow

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pulsations and are well suited for the study of biofilms.

75 To investigate the growth of C jejuni biofilms in mono-culture, 1 mL of a stationary-phase

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culture of C. jejuni (OD600 = 0.13 ± 0.03) was injected into the flow cell chamber and allowed to

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deposit on the coverslip for a period of one hour under stagnant conditions. The flow of R2A

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medium was then resumed and maintained at a constant rate for the duration of the experiment.

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Results for each of the selected time points (1, 3 or 5 days) were obtained from independent flow

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cell runs. C. jejuni cultures were not diluted prior to inoculation to minimize oxygen exposure

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during inoculation. We found that dilution and other procedures that introduced oxygen during

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inoculation generally rendered C. jejuni non-culturable, as evidenced by an absence of any

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biofilm growth following these inoculations (results not shown).

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To investigate the persistence and colonization of C. jejuni in pre-existing P. aeruginosa

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biofilms, 1 mL of a stationary-phase culture of P. aeruginosa (OD600 = 0.1) was first injected

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into each chamber and allowed to deposit on flow cell coverslips under stagnant conditions for

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one hour. The flow of R2A medium was then initiated and maintained at a constant rate for 3

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days. On day 3, the flow cells containing pre-established P. aeruginosa biofilms were inoculated

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with a stationary-phase C. jejuni culture (OD600 = 0.13 ± 0.03), and the flow was halted for 30

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min to facilitate C. jejuni deposition. Again, C. jejuni cultures were not diluted prior to

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inoculation so as to avoid excess oxygen exposure. Following C. jejuni inoculation, the inflow of

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medium was resumed and maintained at a constant rate for an additional 1, 2 or 3 days.

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Independent flow cells were run for each of the listed time points.

96 In co-inoculation experiments, 0.5 mL of stationary-phase cultures of C. jejuni (OD600 = 0.13 ±

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0.03) and P. aeruginosa (OD600 = 0.1) were mixed and inoculated into the flow cell chambers.

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The cells were allowed to deposit on the coverslip for a 1-h period under stagnant conditions,

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and the flow was then resumed for 3 days.

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All experiments were conducted in triplicate using three independent flow cells run in parallel. In

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each independent experiment, three images of the biofilm were obtained near the center of each

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of the three flow cell chambers using methods described below, yielding nine unique

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observations under each test condition.

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Imaging Procedures

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Polyclonal IgG rabbit anti-C. jejuni antibody conjugated to Fluorescein Isothiocyanate Isomer

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(FITC) (AbD SeroTec) was used to image C. jejuni within biofilm. SYTO 62, a cell-permeant

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nucleic acid stain (Life Technologies), was used to counter-stain the biofilms and image P.

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aeruginosa. A solution containing both SYTO 62 (50 M) and fluorescently labeled C. jejuni

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antibodies (50 g/mL) was introduced into the flow chamber and allowed to bind under stagnant

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conditions for 30 minutes in the dark. The flow was then resumed for 25 min in order to wash

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out unbound stains before imaging. This type of immunofluorescence staining has been

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previously demonstrated to fully resolve populations of C. jejuni, Legionella pneumophila and

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Cryptosporidium oocysts distributed throughout mixed-species biofilms (Buswell et al., 1998;

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Kuiper et al., 2004; Searcy et al., 2006; Ammann et al., 2013; Koh et al., 2013)

118 Biofilm micrographs were obtained using a Leica SP2 confocal laser scanning microscope. The

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three-dimensional images were generated using the image processing software VOLOCITY

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(Improvision, Inc.). Quantitative analysis of the biofilm structures was conducted using the

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COMSTAT image processing software (Heydorn et al., 2000).

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Dissolved Oxygen Concentration Measurements

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Dissolved oxygen (DO) concentrations were measured in the influent and effluent of the flow

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cell over 3-days of mono-cultured P. aeruginosa and C. jejuni biofilm growth. To facilitate these

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measurements, compression union tees (1/8”) were installed immediately upstream and

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downstream of the flow cell chambers. Thermolite septa (ResTek) were secured unto the union

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tees in order to allow for repeated insertions of the microelectrode. P. aeruginosa was inoculated

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following the procedures described previously. Following inoculation, an oxygen needle

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microelectrode (Unisense) was introduced into each union tee after 1, 4, 8, 24, 48 and 72 hours.

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DO concentration measurements were collected using a 2-channel picoammeter (PA2000,

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Unisense) and recorded with the program Sensor Trace PRO 3.0 (Unisense). The DO

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microelectrode was calibrated using a stock solution of sodium metabisulfite to generate a

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standard calibration curve at 22oC.

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Results

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Growth of C. jejuni in mono-culture. C. jejuni was unable to form biofilms in flow cells under

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oxic, oligotrophic conditions (Fig. 1A). Changes in the average biomass (Fig. 1B) of C. jejuni

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over a 5-day period following inoculation were not statistically significant (t-test P > 0.45),

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suggesting that cell growth did not occur (Fig. 1B). Scattered C. jejuni cells remained present

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and attached to the flow cell coverslips throughout each experiment (Fig. 1A). Approximately

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10% of the surface-attached cells exhibited active surface motility in the form of rotation around

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a fixed point of contact at the pole of the cell (results not shown). Such motility has been

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attributed to flagellar motion while cells are attached to the surface (Toutain et al., 2007; Tran et

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al., 2011). These observations demonstrate that C. jejuni was incapable of forming biofilms

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under the imposed oxic, oligotrophic conditions, but was still able to persist on the coverslip

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surface for 5 days.

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[Insert Figure 1]

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Persistence and growth of C. jejuni in pre-established P. aeruginosa biofilms. The growth of

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C. jejuni deposited on 3-day old pre-established P. aeruginosa biofilms is shown in Figure 2.

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One day after C. jejuni inoculation, small C. jejuni biofilm clusters were observed on the surface

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of P. aeruginosa biofilms (Fig. 2A, B). C. jejuni grew only on the exterior surface of the P.

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aeruginosa biofilms, and was unable to penetrate into the base of the biofilm. Three days after

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inoculation, C. jejuni continued to grow in small clusters on the biofilm surface (Fig. 2C, D).

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Average C. jejuni biomass increased (Fig. 2E) continuously during the period of observation

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(day 1 to day 2 (P < 0.05), and day 2 to day 3 (P < 0.05)). No differences in P. aeruginosa

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biofilm morphology were observed after the introduction and subsequent growth of C. jejuni

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(results not shown). These findings demonstrate that pre-existing P. aeruginosa biofilms not only

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enhance the survival of C. jejuni under bulk oxic conditions, but also enable C. jejuni to grow

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within the biofilm.

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[Insert Figure 2]

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Co-inoculation of P. aeruginosa and C. jejuni. The growth of co-inoculated C. jejuni and P.

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aeruginosa biofilms is presented in Figure 3. After 3 days, C. jejuni growth occurred exclusively

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on the surface of P. aeruginosa biofilms. Scattered, discrete C. jejuni colonies were present over

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the entire biofilm surface, but no intergrowth of the two strains was observed within the biofilm.

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The morphology of the P. aeruginosa biofilms once again remained unchanged in the presence

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of co-inoculated C. jejuni, and exhibited the same characteristics as typical mono-cultured P.

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aeruginosa (Zhang et al., 2013; Culotti and Packman, 2014). The overall biomass accumulation

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of C. jejuni in biofilms in monoculture, deposited on pre-existing P. aeruginosa biofilms, and in

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co-culture with P. aeruginosa is presented in Figure 4. These results show that both co-

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inoculation with P. aeruginosa and deposition on pre-established P. aeruginosa biofilms

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facilitate C. jejuni growth in biofilms.

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[Insert Figure 3]

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[Insert Figure 4]

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Dissolved oxygen concentrations. To clarify the mechanism by which co-culture and deposition

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on existing biofilms facilitated C. jejuni growth, we measured DO concentrations in the effluents

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of flow cells with P. aeruginosa biofilms. DO concentrations became progressively depleted in

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the effluent over time. The influent concentration was 9.0 mg/L (22oC) upstream of the flow

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chamber, and the effluent concentration matched this before and just after inoculation (Fig. 5).

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Effluent DO decreased as the biofilm developed. Eight hours after inoculation, the effluent DO

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concentration decreased to 7.9 mg/L. After 24 hours, the DO concentration dropped to 0.5 mg/L

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and then remained constant for the remainder of the experiment. The results demonstrate that P.

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aeruginosa biofilms consumed approximately 95% of the DO in the flow cell chamber 24 hours

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after inoculation.

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[Insert Figure 5]

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Discussion

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We investigated the persistence and growth of C. jejuni in biofilms under flow with bulk oxic

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influent conditions. We observed C. jejuni in monoculture, deposited on pre-established P.

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aeruginosa biofilms, and co-inoculated with P. aeruginosa. We found that association with P.

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aeruginosa consistently enabled growth of C. jejuni in mixed biofilms, without regard to the

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timing of introduction of the two organisms. In prior studies, interactions with other biofilm-

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forming microorganisms have resulted in an increased persistence of C. jejuni under oxic

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conditions (Buswell et al., 1998; Sanders et al., 2007; Hilbert et al., 2010; Ica et al., 2011).

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Buswell et al. (1998) reported that the survival of C. jejuni increased in pre-established biofilms

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consisting of standardized autochthonous water microflora isolated from potable water. Sanders

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et al. (2007) reported that growth within mixed populations of bacteria collected from a

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defeathered poultry carcass enhanced C. jejuni survival under aerobic conditions. Hilbert et al.

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(2010) showed that C. jejuni inoculated with various Pseudomonas strains survived for more

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than 48 hours under aerobic conditions. However, detailed information on the spatial

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distribution, biofilm morphology and biomass growth of C. jejuni within mixed-species biofilms

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remains largely absent from the literature.

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Here, we used immunofluorescence staining and confocal microscopy to directly observe

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distributions of C. jejuni in mixed-species biofilms. This imaging approach offers several

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advantages over other commonly used detection and quantification methods. The average

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survival of C. jejuni within mixed biofilms is frequently determined by culturing methods in

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coliform-forming units per milliliter (CFU/mL). Tracking fluctuations of C. jejuni cell

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populations using a standard plate counting approach can be challenging, as C. jejuni is known to

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enter a viable but nonculturable state as a response to environmental stresses (Oliver, 2005; Ica et

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al., 2011). VBNC cells exhibit low metabolic activity and cannot be detected using conventional

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culture techniques (Oliver, 2005) With immunofluorescence and nucleic acid staining, we were

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able to detect C. jejuni both alone and in mixed biofilms, avoiding the limitations of VBNC cell

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detection. This approach also allowed us to directly determine the morphology of C. jejuni

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microclusters, the spatial distribution of these clusters, and the overall growth of C. jejuni

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biomass in mixed-culture biofilms with P. aeruginosa.

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We found that P. aeruginosa biofilms promoted C. jejuni biofilm growth over a period of 3 days

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under oxic flow conditions. In mono-culture, C. jejuni was incapable of forming biofilms after 5

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days, though individual cells remained attached to the glass cover slip and exhibited motility on

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the surface throughout this period of observation. Motile cells rotated about a fixed attachment

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point located at the pole of the cell, demonstrating that the cells were alive but unable to form

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biofilms. However, C. jejuni deposited on pre-established 3-day old P. aeruginosa biofilms

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yielded continuous growth of C. jejuni in the biofilm. Scattered C. jejuni clusters formed on the

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surface of the pre-existing biofilm within one day of inoculation and continued to grow for the

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remainder of the experiment (3 days). C. jejuni colonies were strictly limited to the biofilm

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surface, and no intergrowth of the two organisms was observed within the biofilm. Average C.

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jejuni biomass increased significantly over the 3-day period of observation following inoculation

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(P < 0.005). C. jejuni co-inoculated with P. aeruginosa onto a bare surface also formed mixed-

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species biofilms. C. jejuni again grew exclusively on the surface of the lawn-like P. aeruginosa

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biofilms, despite the absence of a base biofilm at the time of C. jejuni inoculation. C. jejuni

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deposition onto pre-existing P. aeruginosa biofilms led to an increase in C. jejuni biomass of

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~80x, while co-cultured growth resulted in a biomass increase of ~20x compared to mono-

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culture experiments. By contrast, the introduced C. jejuni did not alter the morphology of the P.

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aeruginosa biofilms during both C. jejuni deposition and co-inoculated experiments. The P.

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aeruginosa biofilms exhibited an architecture typical of mono-species P. aeruginosa growth and

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multi-species biofilms where P. aeruginosa is the dominant organism (Zhang et al., 2013;

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Culotti and Packman, 2014). These results demonstrate that P. aeruginosa can not only extend

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the survival time of C. jejuni, but also promote the growth of C. jejuni in biofilms under bulk

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oxic flow conditions.

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The prolonged survival of C. jejuni in mixed culture with P. aeruginosa has been previously

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reported to occur as a result of favorable conditions generated by the local consumption of

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oxygen by P. aeruginosa (Ica et al., 2011). We monitored dissolved oxygen concentrations in the

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effluent of flow cells with P. aeruginosa biofilms and found that P. aeruginosa rapidly depleted

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oxygen in the medium, yielding a decrease in the effluent DO from 9.0 mg/L to 0.5 mg/L 24

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hours after inoculation. By comparison, the effluent remained oxygenated when C. jejuni was

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introduced alone, indicating that aerobic metabolism by P. aeruginosa is responsible for the

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observed oxygen depletion. Our findings indicate that P. aeruginosa is able to generate a

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localized microaerophilic environment that promotes the persistence and growth of C. jejuni.

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These results have important implications for the transmission of C. jejuni in aquatic systems.

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Biofilms are ubiquitous in water distribution networks and animal husbandry and processing

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facilities, and have been implicated as an important reservoir of C. jejuni (Zimmer et al., 2003;

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Sanders et al., 2007; Hilbert et al., 2010). Prior studies have demonstrated that biofilms can

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increase the persistence of C. jejuni under aerobic conditions (Buswell et al., 1998; Zimmer et

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al., 2003; Reeser et al., 2007; Bui et al., 2012). Here, we showed that heterotrophic bacterial

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biofilms are capable of locally generating oxygen-depleted environments that facilitate C. jejuni

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growth under bulk oxic influent conditions. Our results demonstrate that C. jejuni can actively

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replicate within environmental biofilms, making these biofilms not only passive reservoirs of C.

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jejuni delivered from animal sources, but also direct environmental sources of C. jejuni. Because

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we observed that C. jejuni only replicated in association with P. aeruginosa biofilms, controlling

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primary surface colonizers such as P. aeruginosa may limit the presence of C. jejuni in water

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reservoirs and distribution networks.

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272 In conclusion, we demonstrated that P. aeruginosa biofilms not only prolong the survival of C.

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jejuni, but also promote the growth of C. jejuni under bulk oxic flow conditions. C. jejuni

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immunofluorescence was used as an effective, non-invasive imaging technique for the

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observation and quantification of C. jejuni in mixed biofilms with P. aeruginosa under flow. C.

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jejuni formed scattered clusters exclusively on the surface of P. aeruginosa biofilms, and no

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intergrowth of the two organisms was observed. Further, we showed that P. aeruginosa depletes

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oxygen locally, generating favorable conditions that enable the growth of C. jejuni colonies on

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the biofilm surface.

281 282

Acknowledgements

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The authors would like to thank Emma Yee and Dr. William Miller (United States Department of

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Agriculture) for providing the wild-type C. jejuni RM 1221 strain. Imaging work was performed

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at the Northwestern University Biological Imaging Facility generously supported by the

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Northwestern University Office for Research.

287 288

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Figure Legends

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Figure 1. Persistence (but not growth) of C. jejuni was observed in mono-culture under

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flow. A) C. jejuni cells attached to the flow cell coverslip after a period of 1, 3 and 5 days. B)

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Average C. jejuni biomass. Results are presented as mean values (± one standard deviation) of

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nine unique observations of biofilm biomass.

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Figure 2. Growth of C. jejuni deposited on pre-established P. aeruginosa biofilms. P.

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aeruginosa appears red and C. jejuni appears green. A) 3-D micrograph showing C. jejuni

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persistence on P. aeruginosa biofilms 1 day after C. jejuni inoculation (grid unit is 23.8 m). B)

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Horizontal section near the base of the biofilm and vertical sections of the biofilm shown in

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panel A (scale bar = 20 μm). C) 3-D micrograph showing C. jejuni persistence on P. aeruginosa

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biofilms 3 days after C. jejuni inoculation (grid unit is 23.8 m). D) Horizontal section near the

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base of the biofilm and vertical sections of the biofilm shown in panel C (scale bar = 15 μm). E)

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Growth of C. jejuni following deposition on 3-day old pre-established P. aeruginosa biofilms in

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R2A medium. Results are presented as mean values (± one standard deviation) of nine unique

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observations of biofilm biomass.

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Figure 3. Co-development of C. jejuni and P. aeruginosa in biofilms in R2A medium. P.

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aeruginosa appears red and C. jejuni appears green. A) 3-day old co-inoculated biofilm show

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discrete clusters of C. jejuni growing on the surface of a lawn of P. aeruginosa (grid unit is 23.8

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m). B) Horizontal section near the base of the biofilm and vertical sections of the biofilm

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shown in panel A (scale bar = 15 μm).

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Figure 4. Biomass of C. jejuni after 3 days of growth in biofilms in mono-culture, deposited

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on pre-existing P. aeruginosa biofilms, and in co-culture with P. aeruginosa. A) Biomass of

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3-day old, mono-cultured C. jejuni biofilms. B) C. jejuni biomass 3 days after deposition on pre-

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existing P. aeruginosa biofilms. C) Biomass of 3-day old C. jejuni biofilms co-cultured with P.

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aeruginosa. Results are presented as mean values (± one standard deviation) of nine unique

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observations of biofilm biomass.

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Figure 5. Dissolved oxygen concentrations in the flow cell effluent following P. aeruginosa

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inoculation. The DO concentration decreased from 9.0 mg/L to 0.50 mg/L after 24 hours (22oC),

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and remained at that level for the remainder of the experiment.

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Pseudomonas aeruginosa facilitates Campylobacter jejuni growth in biofilms under oxic flow conditions.

We investigated the growth of Campylobacter jejuni in biofilms with Pseudomonas aeruginosa under oxic flow conditions. We observed the growth of C. je...
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