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Suppression of Enteric Bacteria by Bacteriophages: Importance of Phage Polyvalence in the Presence of Soil Bacteria Pingfeng Yu, Jacques Mathieu, Yu Yang, and Pedro J. J. Alvarez Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b00529 • Publication Date (Web): 17 Apr 2017 Downloaded from http://pubs.acs.org on April 18, 2017

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Environmental Science & Technology

Suppression of Enteric Bacteria by Bacteriophages: Importance of Phage Polyvalence in the Presence of Soil Bacteria

Pingfeng Yu, Jacques Mathieu, Yu Yang and Pedro J.J. Alvarez* Department of Civil and Environmental Engineering, Rice University, Houston, TX 77005

RUNNING TITLE: Growth Suppression by polyvalent phages and competitive exclusion *

Corresponding Author: Phone: 713-348-5903. Fax: 713-348-5203. E-mail: alvarez@ rice.edu.

ACS Paragon Plus Environment


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ABSTRACT

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Bacteriophages are widely recognized for their importance in microbial ecology and bacterial

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control. However, little is known about how phage polyvalence (i.e., broad host range) affects

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bacterial suppression and interspecies competition in environments harboring enteric pathogens

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and soil bacteria. Here we compare the efficacy of polyvalent phage PEf1 versus coliphage T4 in

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suppressing a model enteric bacterium (E. coli K-12) in mixtures with soil bacteria

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(Pseudomonas putida F1 and Bacillus subtilis 168). Although T4 was more effective than PEf1

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in infecting E. coli K-12 in pure cultures, PEf1 was 20-fold more effective in suppressing E. coli

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under simulated multispecies biofilm conditions because polyvalence enhanced PEf1

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propagation in P. putida. In contrast, soil bacteria do not propagate coliphages and hindered T4

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diffusion through the biofilm. Similar tests were also conducted under planktonic conditions to

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discern how interspecies competition contributes to E. coli suppression without the confounding

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effects of restricted phage diffusion. Significant synergistic suppression was observed by the

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combined effects of phages plus competing bacteria. T4 was slightly more effective in

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suppressing E. coli in these planktonic mixed cultures, even though PEf1 reached higher

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concentrations by reproducing also in P. putida (7.2±0.4 vs. 6.0±1.0 log10PFU/mL). Apparently,

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enhanced suppression by higher PEf1 propagation was offset by P. putida lysis, which decreased

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stress from interspecies competition relative to incubations with T4. In similar planktonic tests

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with more competing soil bacteria species, P. putida lysis was less critical in mitigating

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interspecies competition and PEf1 eliminated E. coli faster than T4 (36 vs. 42 h). Overall, this

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study shows that polyvalent phages can propagate in soil bacteria and significantly enhance

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suppression of co-occurring enteric species.

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1 ACS Paragon Plus Environment

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INTRODUCTION Bacteriophages, which are viruses that infect and replicate within bacteria, are the most

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abundant and diverse living entities in the biosphere.1 Estimates place their numbers in the range

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of 1031, with a common environmental virus-to-prokaryote ratio of about 10:1.2, 3 As antibacterial

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agents and important vectors for horizontal gene transfer4, phages are significant drivers of

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bacterial evolution and community structure. Consequently, phages can also significantly

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influence biogeochemical cycles and energy flows in various ecosystems.5 Given their ecological

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importance and potential applications for controlling problematic bacteria6, there is a need to

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advance fundamental understanding of phage-host interactions in complex microbial

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communities. This includes discerning how phage host range affects their propagation dynamics

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and microbial community structure in both planktonic (i.e., suspended) and biofilm systems.

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Phages alter their hosts’ metabolic repertoire, fitness and competitive capability, or directly

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eliminate the most susceptible bacteria.7, 8 Interspecific bacterial competition in the presence of

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phages has been studied theoretically8, 9 and empirically10, 11. Depending on the nature of phage-

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host associations and environmental conditions, phages can facilitate competitive exclusion9, 12

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or enhance the bacterial hosts’ coexistence11, 13. However, most these studies have only

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considered phages that typically infect only one species and cannot reproduce by using hosts

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from other genera in the microbial community. No previous publications have considered

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how (broad host range) polyvalent phages (i.e., phages capable of intergeneric infection14)

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affect bacterial suppression and interspecific competition. This is a critical knowledge gap

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given the recent recognition of the prevalence of polyvalent phages in the environment15-17 and

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their potential to exhibit more complex propagation dynamics. Specifically, whereas polyvalent

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phages use a wider variety of bacteria to reproduce, they generally experience lower efficiency



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of infection (i.e., the ratio between the infectious titer for a given host and the maximum

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observed titer18) and slower growth17, 19, which may offset their ability to influence microbial

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community structure.

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This study addresses how phage host range affects interspecific bacterial competition and

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enteric bacteria suppression. We compare the effect of coliphage T4 versus polyvalent phage

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PEf1 on three competing bacteria: (1) Escherichia coli, representing an enteric bacterium

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commonly associated with fecal pollution and (for some E. coli strains) infectious disease20, (2)

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Pseudomonas putida (alternative host to PEf1 but not to T4) and (3) non-host Bacillus subtilis,

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which cannot be infected by these phages. The latter two represent indigenous soil bacteria

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commonly associated with biodegradation of pollutants.21 E. coli suppression is quantified under

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both planktonic conditions and model biofilms, which offer potential resistance to phage

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diffusion and to propagation of phage-resistant mutants. We discern the contributions to E. coli

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suppression from phages versus competition by soil bacteria (demonstrating synergism), and

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highlight the importance of polyvalence in enhancing phage propagation in alternative hosts (e.g.,

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soil bacteria) and significantly boosting suppression of potential enteric pathogens.

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MATERIALS AND METHODS

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Bacteria, bacteriophage and culture conditions. The bacterial strains in this study included

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E. coli K-12 (ATCC 700926), P. putida F1 (ATCC 700007), and B. subtilis 168 (ATCC 23857).

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Culturing was conducted in M63 glucose medium at 30°C [12 g of KH2PO4, 28 g of K2HPO4, 8

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g of (NH4)2SO4 per liter water supplemented with 1 mM of MgSO4, 0.2% glucose and 0.5%

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Casamino Acids].22 Coliphage T4 (Carolina Biological 12-4330), which was reported to infect

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only E. coli and closely related Shigella species23, infected E. coli K-12 but none of other tested

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bacterial strains. The polyvalent phage PEf1, which can infect both E. coli and P. putida (Fig.


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S1) but not B. subtilis, was previously isolated by a sequential multi-host approach.19 Bacteria in

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exponential phase were used for phage treatment, phage enumeration and phage characterization.

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Double-layer plaque assays24 used tryptone agar plates containing 0.7% agar for the soft agar and

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1.1% for the base layer. Phage titration was performed using E. coli K-12 as the host and

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expressed as plaque forming units (PFU) per ml. The samples were centrifuged to obtain

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supernatant for extracellular phage titration. Phages were enumerated after direct dilution using

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the double-layer plaque assay. Quantitative phage abundance analysis was performed by

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observation of virus-like-particles (VLP) via fluorescence microscopy after staining with SYBR

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Green I according to the standard protocol.25

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Bacteriophage growth parameters characterization. The fraction of free phages was

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measured as function of time in adsorption tests, and phage adsorption rate constants were

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determined based on the first-order kinetics: ݇‫(= ܤ‬ln(ܲ0) − ln (ܲ‫))ݐ‬/‫ݐ‬, where k is the adsorption

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rate constant (mL/min), B is bacterial density (CFU/mL), P0 is number of free phages at time 0

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(PFU/mL), and Pt is number of free phages at time t (PFU/mL).26 One-step growth curve

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experiments were conducted to measure the burst size and latent time of each phage.27 Efficiency

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of plating (EOP) was quantified by calculating the ratio of phage plaque titers obtained with

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a given host to the phage plaque titers obtained with a reference strain18 (i.e., E. coli K-12 in

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this case). All parameters were determined with bacteria in exponential phase in M63 medium at

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30 °C (Table S1).

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Bacterial challenge test in planktonic state. To compare the inhibitory efficiency of phage

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T4 and PEf1 in planktonic states, 10 ml 105 CFU/ml E. coli K-12 were subjected to the following

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treatments: (i) 106 PFU/ml phage T4, (ii) 106 PFU/ml phage PEf1, (iii) soil bacteria (1 P. putida

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and 1 B. subtilis per E. coli), (iv) soil bacteria phage mixture I (1 P. putida, 1 B. subtilis and 10



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T4 per E. coli), and (v) soil bacteria phage mixture II (1 P. putida, 1 B. subtilis and 10 PEf1 per

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E. coli). During the 72-h experiment, to avoid nutrient depletion and the accumulation of phage

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particles (which confounds analysis), samples were collected at 12h intervals and 0.1 ml culture

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were transferred into 9.9 ml fresh medium. The samples were centrifuged to obtain supernatant

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for extracellular phage titration and cell pellets for bacterial DNA extraction. Since cultures were

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diluted 100-fold each step, bacteria were considered completely eliminated when densities were

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below 100 CFU/ml.

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To avoid interference by dead cells, ethidium bromide monoazide (EMA) was used to bind

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the DNA from dead cells 20. Briefly, cell suspensions were stained with 20 µg/ml EMA (Sigma-

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Aldrich) in the dark for 5 min, placed on ice for 1 min, and exposed to bright visible light for 10

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min prior to DNA extraction. Bacterial genomic DNA was then extracted using an UltraClean

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Microbial DNA Isolation Kit (MoBio, Carlsbad, CA) according to the manufacturer’s

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instructions.

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Challenge tests with bacteria embedded in soft agar. The two-species mixture (105 CFU

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E. coli K-12 and B. subtilis 168, or 105 CFU E. coli K-12 and P. putida F1) and three-species

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mixture (105 CFU E. coli K-12, B. subtilis 168 and P. putida F1) were inoculated with coliphage

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T4 or polyvalent phage PEf1 (105 PFU) in the soft agar of double-layer plates. The plates were

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incubated for 12h at 30°C and then three disk-shaped samples (2cm in diameter) were randomly

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taken from the soft agar of each plate. The samples were physically ground and bacterial DNA

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was extracted using a PowerSoil DNA Isolation Kit (MoBio, Carlsbad, CA). Phage λ DNA was

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used as an internal standard to calculate the recovery rate of all DNA extractions 28.

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Isolation and characterization of BIMs. Bacteriophage-insensitive mutants (BIMs) resistant to phage PEf1 and T4 were isolated as follows 29. The soft agar of double-layer plates


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was seeded with 107 CFU hosts and 109 PFU phage (multiplicity of infection, MOI = 100) and

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then incubated for 24 h at 30°C. Surviving colonies were picked from the confluent lysis assay

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plate and streaked onto a nutrient agar. Bacterial contamination was excluded by using selective

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agar for plating (EMB agar for E. coli and Pseudomonas selective agar for P. putida), and

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verifying purity by colony PCR with specific biomarkers (E. coli-specific universal stress protein

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gene A30 and P. putida-specific toluene dioxygenase gene todC131). The ratio between the

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number of surviving colonies and the number of initially inoculated bacteria (107 CFU) was

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calculated as the BIM frequency.

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BIMs were streaked once to avoid re-sensitizing and remove potentially co-evolving

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phages.32 Spot tests were conducted to confirm the phage resistance of new isolated BIMs.18 To

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determine potential fitness costs associated with phage resistance development, growth kinetics

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and biofilm formation were measured. Bacterial specific growth rates were determined based on

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OD600 measurements during the 12-h exponential growth phase (all tested bacteria reached

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stationary phase after 12 h). The biofilm formed on the surface of 96-well tissue culture plates

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after a 24h incubation was measured according to a microtiter dish biofilm formation

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assay.33

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Inhibition of E. coli K-12 in newly-established biofilm. E. coli K-12, P. putida F1 and B.

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subtilis 168 (105 CFU/ml for each bacterium) were incubated in glass vials (KimbleTM 20 mL

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Disposable Scintillation Vials) containing 5 ml of medium and 1.0 g of quartz sand (Fig. S3).

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The microcosms were shaken at 240 rpm and 30 °C using a Multi-ThermTM shaker (Benchmark

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Scientific Inc. Edison, NJ). After 24 h incubation, the bulk solution was replaced with 5 ml fresh

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medium inoculated with bacteria (105 CFU/ml for each strain) and phages (T4 or PEf1 at 106

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PFU/ml) to adjust the initial MOI in the bulk solution to the same value used in the prior liquid



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culture experiments in order to facilitate comparison. The time point when phages were added

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was considered as time zero. No phages were added in the control group. During the 5 day

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experiment, 4.9 ml of liquid culture was replaced with an equal volume of fresh medium at 24 h

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intervals.

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The bulk solution was collected and centrifuged to obtain supernatant for phage titration and

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cell pellets for bacteria enumeration. The sand was gently rinsed three times with 0.2 ml of PBS.

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Sand sampled were suspended in 0.2 ml of PBS supplemented with 0.05% (v/v) Tween-20, and

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gently sonicated at 40 kHz for 20 min in a 4°C bath sonicator (Branson, Danbury, CT) to disrupt

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the biofilm matrix and disperse the bacterial cells and entrapped phages.34 The 0.2 ml suspension

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was centrifuged to obtain the supernatant for phage titration and the cell pellet for bacteria

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quantification.

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Primer design and real-time quantitative PCR analysis. The E. coli-specific universal

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stress protein gene A (uspA) and toluene dioxygenase gene todC1 were chosen as specific

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biomarkers to assess the abundance of E.coli K-12 and P. putida F1, respectively. Both genes

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were quantified by qPCR using SYBR Green. The bacterial 16S rRNA gene, quantified by

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Taqman qPCR, was used for the total bacteria enumeration 35. The extracted genomic DNA from

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E.coli K-12 and P. putida F1 with different bacterial densities were used to establish the standard

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curve between threshold cycle (Ct) value and log10 CFU for each bacteria individually. Detailed

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information of SYBR Green qPCR and Taqman qPCR on primers and probes, reaction reagents

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and temperature programs is included in the supplementary material (Table S2).

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Scanning electron microscopy of biofilm on sand surface. The washed sand samples were

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fixed in 4% glutaraldehyde 0.1M phosphate buffer solution (PBS, pH=7.4) overnight at 4°C. The

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samples were dehydrated in a series of ethanol-water solutions (50, 60, 70, 80, 90, 95, 100%,


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vol/vol) for 30 min at 4°C. The sand was placed on the carbon tape pasted stub and then sputter-

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coated with ~7 nm gold film under vacuum using Denton Desk V Sputter System. The

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microstructure of biofilm on the sand surface was observed using JEOL 6500 scanning electron

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microscopy. Duplicates were performed for samples from both control and phage treatment

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microcosms and more than 10 representative images were captured for each sample.

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Statistical analysis. Student's t-test (two-tailed) was used to determine the significance of the

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differences between treatments. Differences were considered to be significant at the 95% level (p

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< 0.05). Synergy quantification of combination treatments was performed using the Bliss

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independence model 36. Combination indices (CI) were calculated as follows:

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CI = (ECompetitors + EPhages - ECompetitors × EPhages)/ ECompetitors+Phages

Eqn. (1)

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where ECompetitors, EPhages and ECompetitors+Phages represent suppression factors for E. coli caused by

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interspecific competition, phage infection and combination of interspecific competition and

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phage infection, respectively 37. The Bliss model was selected because the modes of action for

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bacterial inhibition by interspecies competition and phage infection are independent. Each

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suppression factor was calculated as the ratio of E. coli density in the presence of a specific

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stressor (phages or competitors) to the density of unexposed E. coli.

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RESULTS

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Coliphage T4 suppressed E. coli monocultures more effectively than polyvalent phage PEf1.

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E. coli growth in suspended pure culture was inhibited to a greater extent by T4 than by PEf1

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(Fig. 1A). Relative to uninfected controls, E. coli viability (i.e., the ratio between E. coli

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abundance in the phage-treated group to that in the phage-free control) after 12-h incubation in

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the presence of T4 at MOI = 1 (i.e., one phage initially present per E. coli cell) was 12±4%,

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which is significantly lower (p < 0.05) than 28±4% in the presence of PEf1 at the same MOI.



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Similar results were observed for biofilm formation, T4 was more effective than PEf1 (10±3%

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versus 18±4% E. coli viability after 24 h exposure, n = 3, p < 0.05) at inhibiting E. coli growth in

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microtiter dish biofilm formation assays (Fig. 1B). These results are consistent with the higher

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adsorption rate constant (2.3×10-9 vs. 0.6×10-9 ml/min), shorter latent time (35 vs. 50 min), and

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larger burst size (117±8 vs. 99±5 PFU/cell) for T4 than PEf1 (Table S1), which results in faster

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propagation and higher probability of infecting E. coli in monoculture.

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E. coli resistance to T4 results in higher fitness costs than resistance to PEf1.

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Development of resistance is a common bacterial response to phage infection, but a given

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host may experience different susceptibility and fitness costs for different phages. We assessed

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the BIM frequency of E. coli exposed to T4 or PEf1 (MOI = 100) and compared the resulting

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fitness costs associated with phage resistance. The BIM frequency of E. coli with T4 was

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6.2±1.1×10-7, which is significantly lower p < 0.05) than that of E. coli toward PEf1

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(3.8±0.5×10-6). Resistance to T4 decreased the (exponential phase) specific growth rate of E. coli

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by 28.8±3.6% (from 0.52±0.03 to 0.37±0.02 h-1), compared to a 19.3±1.6% decrease (from

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0.52±0.03 to 0.42±0.02 h-1) associated with resistance to PEf1 (Fig. 2A). Similarly, the 24 h

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biofilm formation of E. coli resistant to T4 decreased by 22.4±2.1% (from 0.69±0.04 to

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0.53±0.04 OD595), while that of E. coli resistant to PEf1 decreased by only 14.3±2.9% (from

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0.69±0.04 to 0.60±0.05 OD595) (Fig. 2B). The higher fitness cost associated with resistance to T4

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than to PEf1 is consistent with the more effective suppression of E. coli pure cultures by T4.

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Polyvalent phage PEf1 proliferates to a greater extent and is more effective than T4 at

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suppressing E. coli in mixed cultures under simulated biofilm and sandy slurry conditions.

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Although PEf1 was less effective than T4 in suppressing E. coli in mono-culture (Fig. 1), it was significantly more effective than T4 under more realistic mixed cultures in both sandy slurry


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microcosms (Table 1, Fig. 3 and Fig. 4A) and in soft agar microtiter formation assays (Table S3

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and Fig. 4B). In these mixed cultures, polyvalence enhanced PEf1 propagation in other hosts (i.e.,

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P. putida F1 serving as alternative host for PEf119) and reached significantly higher

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concentrations than T4 in the bulk solution (7.88±0.16 vs 6.83±0.17 log10PFU/ml, p < 0.05),

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which enhanced E. coli suppression.

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In sandy slurry microcosms, PEf1 could reproduce in both E. coli and P. putida and reached

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higher (10-fold) concentrations than T4, which could reproduce only in E. coli (Fig. 3 and Fig.

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S5). Higher PEf1 concentrations in mixed cultures facilitated E. coli infection and suppression

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(Fig. 3 and Fig. 4A). E. coli concentrations in the bulk liquid stabilized after 3 days at 4.7±0.1

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log10CFU/ml in the presence of PEf1, which is 1.3 orders of magnitude lower than in

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microcosms with T4 (Fig. 3). PEf1 also suppressed E. coli to a greater extent and for a longer

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duration than T4 in the mixed-culture biofilm attached on the sand surface (Fig. 4A). Prior to

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phage amendment, the initial densities of E. coli in newly formed mixed-species biofilm were

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5.64±0.15 log10 CFU/mg sand (Fig. 4A). After PEf1 amendment, the attached 5-day E. coli

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density decreased by 93±2% to 4.51±0.21 log10 CFU/mg sand, while that in microcosms

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amended with T4 it increased by 44±26% to 5.80±0.23 log10 CFU/mg sand (Fig. 4A). Compared

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to controls without phage, PEf1 reduced attached E. coli viability by 2.4 orders of magnitude,

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while T4 achieved only 1.1 order of magnitude suppression. Furthermore, scanning electron

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microscopy (SEM) of the sand biofilm revealed significantly greater disruption in microcosms

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treated with PEf1 than those treated with T4 (Fig. 5).

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Additional experiments were conducted with bacteria embedded in soft agar plates to assess

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the effect of phages under biofilm-like conditions without confounding effects such as phage

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reproduction in suspended hosts or recolonization from the bulk solution. Whereas soft agar tests



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do not represent the complexity of biofilms commonly encountered in natural and engineered

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systems, they offer a simple system to investigate factors that influence bacterial attachment and

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biofilm formation in reductionist experiments that exclude such confounding factors.38, 39 When

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E. coli and B. subtilis were co-cultured without phages, the E. coli density reached 8.57±0.17

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log10 CFU/cm2 after 12 h (Fig. 4B). E. coli densities decreased dramatically when the bacteria

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were challenged with either PEf1 (6.60±0.18 log10 CFU/cm2) or T4 (6.77±0.18 log10 CFU/cm2)

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(Fig. 4B, p

Suppression of Enteric Bacteria by Bacteriophages ... - ACS Publications

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