Bacteriophages To Sensitize A Pathogenic New Delhi Metallo Î²

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Bacteriophages To Sensitize A Pathogenic New Delhi Metallo #-lactamase-positive Escherichia coli To Solar Disinfection Nada Al-Jassim, David Mantilla-Calderon, Giantommaso Scarascia, and Peiying Hong Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b04501 • Publication Date (Web): 20 Nov 2018 Downloaded from http://pubs.acs.org on November 21, 2018

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Environmental Science & Technology

Inactivation curve with bacteriophages

Log10 E. coli

Log10 E. coli

Inactivation curve without bacteriophages

time

time Pathogenic & antibiotic- resistant E. coli

Pathogenic & antibioticresistant E. coli + Bacteriophages

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Log10 reduction in CFU numbers Log10(C/C0)

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Fluence (J/cm2) 150 200

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-1.00 R² = 0.9733

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Figure 1.


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Log10 reduction in PFU numbers Log10(C/C0)

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Time (h) Dark control

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Bacteriophages to Sensitize a Pathogenic New Delhi Metallo β-lactamase-positive

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Escherichia coli to Solar Disinfection

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Nada Al-Jassim 1, David Mantilla-Calderon 1, Giantommaso Scarascia 1, Pei-Ying Hong * 1

4 5 6 7

1. King Abdullah University of Science and Technology (KAUST), Water Desalination and Reuse Center (WDRC), Biological and Environmental Sciences & Engineering Division (BESE), Thuwal, 23955-6900, Saudi Arabia

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

* Corresponding author: Pei-Ying Hong Email: [email protected] Phone: +966-12-8082218

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Keywords: Ultraviolet disinfection; decay kinetics; antibiotic-resistant bacteria; transcriptomics

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Running title: Bacteriophage-supplemented disinfection of E. coli

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Abstract

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Bacteriophages active against a New Delhi metallo beta lactamase (NDM)-positive E. coli PI-7

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were isolated from municipal wastewater and tested for their lytic effect against the bacterial

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host. Bacteriophages were highly specific to E. coli PI-7 when tested for host-range. After

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determining host-specificity, bacteriophages were tested for their ability to sensitize E. coli PI-

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7 to solar irradiation. Solar irradiation coupled with bacteriophages successfully reduced the

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length of the lag-phase for E. coli PI-7 from 4.11 ± 0.78 h to 1.82 ± 0.60 h in buffer solution.

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The reduction of lag-phase length was also observed in filtered wastewater effluent and

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chlorinated effluent. Previously, we found through gene expression analysis that cell wall,

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oxidative stress and DNA repair functions played a large role in protecting E. coli PI-7 against

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solar damage. Here, gene expression analysis of bacteriophage-supplemented solar-irradiated

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E. coli PI-7 revealed downregulation of cell wall functions. Downregulation of functions

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implicated in scavenging and detoxifying reactive oxygen species, as well as DNA repair genes

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was also observed in bacteriophage-supplemented solar-irradiated E. coli PI-7. Moreover, solar

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irradiation activates recA which can induce lytic activity of bacteriophages. Overall, the

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combined treatment led to gene responses that appeared to make E. coli PI-7 more susceptible

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to solar disinfection and bacteriophage infection. Our findings suggest that bacteriophages

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show good potential to be used as a biocontrol tool to complement solar irradiation in

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mitigating the persistence of antibiotic-resistant bacteria in reuse waters.

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1. Introduction

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Antibiotic-resistant bacteria (ARB) are among emerging microbial contaminants of

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increasing concern, and are challenging our current antimicrobial strategies. In fact, several

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reports have raised concerns that the development of new drugs alone will not adequately

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address the threat posed by the spread of antibiotic-resistant pathogens1-3. A major hotspot

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implicated in the creation of superbugs, and the dissemination of these emerging contaminants

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is wastewater and its treatment facilities as abundance of nutrients, high bacterial numbers and

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sub-lethal levels of antibiotics are considered favorable for both the survival ARB and the

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horizontal transfer of bacterial resistance genes4, 5.

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In particular, an earlier study has isolated an E. coli strain PI-7 from local wastewater

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influent in Jeddah, Saudi Arabia, and found it to be pathogenic, highly antibiotic-resistant and,

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more alarmingly, carrying the New Delhi metallo-β-lactamase (NDM) antibiotic resistance

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gene, blaNDM6. Other studies have also independently reported the presence of blaNDM genes

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and NDM-harboring bacteria in urban wastewater6-8, and in the final chlorinated secondary

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effluent despite having received treatment through a municipal wastewater treatment process7.

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The blaNDM gene codes for a carbapenemase, a class of enzymes that confer resistance to

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carbapenems and other β-lactam antibiotics that are normally reserved as a last line of defense

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against gram-negative bacterial infections. Moreover, NDM-harboring bacteria typically carry

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resistance determinants against multiple other antibiotic classes, leaving very few treatment

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options available 8-11. These findings highlight the risks of disseminating ARB and their genes

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through wastewater reuse12, 13, with potential implications to public health. This is particularly

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relevant in places like Saudi Arabia, where pressure on water resources is high, and treated

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wastewater is being considered to alleviate considerable portions of this water-scarcity pressure

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14.

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Solar irradiation can serve as a biocidal barrier to mitigate this problem. Many studies

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found rapid inactivation of fecal indicator organisms within a few hours of exposure to natural

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sunlight15-19. However, it was found that the NDM-positive E. coli PI-7 underwent a

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significantly longer lag-phase prior to decay, and exhibited a longer inactivation half-life

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compared to another non-pathogenic and less antibiotic-resistant E. coli DSM1103 upon solar

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irradiation20. This suggests that a higher solar fluence or longer exposure time would be

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required to achieve the same log reduction for the NDM-positive E. coli compared to a non-

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resistant strain. Furthermore, E. coli PI-7 was observed to upregulate a larger suite of genes

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related to cell repair, including upregulation of cell wall synthesis and extracellular polymeric 3 ACS Paragon Plus Environment

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substance genes that may have served as a physical barrier to solar damage, upregulation of

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oxidative stress response and ROS (reactive oxygen species) scavenger genes which may have

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protected the cell from indirect mechanisms of solar damage, as well as upregulation of DNA

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synthesis and repair genes that may have enabled longer survival. Moreover, the same study

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found upregulation of genes involved in persister cell formation, along with genes related to

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antibiotic resistance and virulence functions. These observations suggest that pathogenic

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antibiotic-resistant bacteria like E. coli PI-7 remain viable for longer period compared to an

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antibiotic-susceptible strain despite solar irradiation. There exists a need for an additional

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mitigation strategy besides solar irradiation to enhance biocidal effects against the E. coli PI-

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7.

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A complementary treatment method to explore would be the use of lytic

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bacteriophages. Lytic bacteriophages are viruses that infect the bacterium host, replicate within

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and kill bacteria by lysing the host. In wastewater related applications, bacteriophages have

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been applied to control biofilm formation on filtration membranes and to restore flux to fouled

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membranes1, 21. Bacteriophage therapy has also been demonstrated to solve the problem of

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sludge bulking caused by filamentous bacteria22-24. However, those studies did not demonstrate

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the complementary use of bacteriophages along with solar irradiation to inactivate ARB like

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E. coli PI-7.

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It is hypothesized that bacteriophages can aid in achieving further reduction of E. coli

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PI-7 numbers during disinfection by solar irradiation. This is inferred from the earlier study

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where downregulation of phage-shock functions as well as upregulation of phage integrase and

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DNA transfer genes were observed in E. coli PI-7 in response to solar irradiation20. The aim of

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this study is to investigate the use of bacteriophages as a biocidal tool in combination with solar

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irradiation for inactivating NDM-carrying E. coli PI-7. In this study, seven bacteriophages

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targeting E. coli PI-7 were isolated from wastewater. They were then tested for host-specificity

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and stability in controlling E. coli PI-7’s growth under various conditions. Three bacteriophage

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candidates were used in combination with solar irradiation to test the possibility of hastening

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E. coli PI-7’s inactivation. The response was assessed in terms cell survival via quantification

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of culturable cell counts, and gene expression changes to reveal survival and damage responses

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at the molecular level.

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2. Materials and Methods

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2.1. Bacteriophage isolation

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Bacteriophages specific to a NDM-positive pathogenic E. coli strain PI-76 were isolated

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from wastewater as follows. 50 ml of wastewater influent was centrifuged at 10,000 x g for 15

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min, and the supernatant was filtered through a 0.2 µm filter. Then 50 ml of 2X LB broth was

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added to the filtrate, as well as 50 ml of overnight PI-7 culture with 8 µg/ml meropenem. The

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mixture was incubated at 37 °C and 200 rpm for 24 h. Chloroform was added in to a final

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concentration of 1% and the mix was incubated for 1 h to lyse bacterial cells. Next, the mixture

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was centrifuged at 10,000 x g for 30 min at 4 °C, and the supernatant was filtered through a

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0.2 µm filter. The phage lysate filtrate was diluted in SM buffer (100 mM NaCl, 8 mM

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MgSO4•7H2O, 50 mM Tris buffer at pH 7.5). To isolate individual phages, 10 µl of phage

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lysate and 100 µL of overnight PI-7 culture were mixed together in soft LB agar supplemented

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with 2 mM CaCl2, then poured over CaCl2-supplemented LB (1.5 % w/v) agar. After 24 h

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incubation at 37 °C, isolated plaques were individually picked, re-suspended in SM buffer and

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filtered through a 0.2 µm filter. Each plaque then went through seven rounds of plating and re-

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isolation to isolate a total of seven individual bacteriophages.

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2.2. High-titer bacteriophage stock preparation

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Bacteriophage stocks containing high phage titer were prepared by first mixing 50 µl

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of original phage stock with 100 µl of fresh E. coli PI-7 in 3 ml of soft LB agar (0.3%)

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supplemented with 2 mM CaCl2. The mixture was plated onto 1.5% w/v LB agar plates

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supplemented with 2 mM CaCl2, and incubated overnight at 37 °C. Then, 3 ml of SM buffer

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was added onto each plate, and a sterile spreader was used to scrape off the top agar layer

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containing bacteriophages and bacterial cells. This mixture was then centrifuged at 10,000 g

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for 15 min, and the supernatant was filtered through 0.2 µm filters (GE Healthcare Life

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Sciences, Little Chalfont, Buckinghamshire, UK). Afterwards, polyethylene glycol 8000 was

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added to the filtrate to form 15% w/v, and the mixture was incubated overnight with stirring at

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4 °C. Subsequently, the mixture was centrifuged at 10,000 g for 20 mins, and the cell pellet

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containing the bacteriophage particles was resuspended in 1 ml of 1X PBS. Stocks were

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enumerated for titer concentration by plating serial dilutions of the stock with fresh E. coli PI-7

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using the soft agar method described above, and quantifying PFUs (or plaque-forming units)

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after overnight incubation. Bacteriophages were characterized for their morphology and

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genome sizes as described in Supplementary Information 1. 5 ACS Paragon Plus Environment

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2.3. Bacteriophage lytic activity

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To check the lytic activity of the bacteriophage isolates against different bacteria,

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cultures of various bacteria from clinical and environmental origins (Table 1) were prepared

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overnight. 100 µl of bacteria with were plated in soft agar, mixed with the bacteriophages to

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perform a plaque assay, or followed by spotting of serial dilutions of different bacteriophages

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for spot test evaluation25.

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Based on the lytic activity tests, bacteriophages were specific against E. coli PI-7 and

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further determination was done to monitor the impact on growth curve of E. coli PI-7 in

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presence of bacteriophages. A fresh culture of E. coli PI-7 was prepared overnight, then 100 µl

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was inoculated into 10 ml of 1:1 SM buffer mixed with LB broth in sterile transparent glass

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tubes. The OD600 of each tube was periodically measured to monitor E. coli PI-7’s growth in

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presence or absence of bacteriophages at different temperatures (40, 37, 32 and 20 °C) and pH

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(6, 7 and 8).

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2.4. Solar inactivation and decay kinetics

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Solar inactivation trials were conducted using an Atlas Suntest ® XLS+ photosimulator

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(Chicago, IL) equipped with a xenon arc lamp as described in our previous work20. The solar

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simulator’s irradiance is described in Supplementary Information 2 and shown in Figure S1.

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Briefly, prior to the experiments, overnight cultures of E. coli PI-7 were thoroughly washed in

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1 mM NaHCO3, then re-suspended in 80 ml of the final matrix of either 1X PBS, filtered

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tertiary-treated wastewater effluent (collected after treatment in an anoxic-oxic activated

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sludge tank followed by microfiltration) or filtered chlorinated effluent collected after

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microfiltration. All wastewater samples were filtered through 0.2 µm polycarbonate membrane

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filters (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK) and stored at 4°C

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in sterile glass bottles within 4 h of collection. No additional step was taken to quench residual

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chlorine in chlorinated effluent samples prior to inactivation experiment as no residual chlorine

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were detected by DPD method (Hach, Loveland, CO). Starting bacterial numbers for solar

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inactivation experiments were always approximately 1-9x108 CFU/ml. For bacteriophage-

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supplemented experiments (SI+phage), 1 ml of high titer phage solution was added to the

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microcosms to a final PFU concentration similar to the bacterial concentration (i.e., MOI = 1).

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Microcosms were then either covered with foil (dark controls) or glass filters permitting

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wavelength 280 nm and above (Newport Corporation, Irvine, CA). A set of non-bacteriophage-

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supplemented experiments (SI-only), both dark and irradiated were always included in parallel 6 ACS Paragon Plus Environment


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to bacteriophage-supplemented solar irradiation (SI+phage) trials, but SI-only microcosms

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were inoculated with sterile buffer solution instead of phage solution. After inoculation with

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bacteria and bacteriophages where applicable, the microcosms were placed inside the Atlas

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Suntest and exposed to solar irradiation. SI+phage and SI-only inactivation kinetics data

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presented in this study was obtained from four biological replicates.

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A set of solar inactivation experiments with seven bacteriophages individually

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inoculated into buffer solution in separate microcosms were also performed without replication

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in order to denote the stability of these bacteriophages upon exposure to sunlight. This

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observation, along with that obtained from lytic activity tests and growth curve impediment

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experiments (described in section 2.3), were used to select for the most suitable bacteriophage

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isolates to form a bacteriophage mixture. Upon selection of the bacteriophage mix (i.e.,

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bacteriophage P1, P4 and P5a), solar inactivation was performed on this mixture inoculated in

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sterile buffer solution and in presence of E. coli PI-7. Three biological replicates were

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performed for this experiment to determine the bacteriophage mixture’s decay kinetics.

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The number of colony forming units (CFU/ml) and/or plaque forming unites (PFU/ml)

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in the irradiated and dark experimental microcosms was quantified at the starting point and

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over irradiation time. Inactivation results were converted to log10 and natural log (ln) curves of

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concentration values normalized as CFUt/CFUt=0 for E. coli PI-7 or PFU/PFUt=0 for

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bacteriophages, and plotted against time to obtain the inactivation curves. The decay rate

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constants (k) of dark control and irradiated sample inactivation curves were calculated as the

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negative slopes of natural log inactivation curves, with the following caveat: in cases where

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there was a detectable lag-phase, the curve was considered bi-modal and the lag-phase data

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points were excluded from the slope calculation. Further detail on lag-phase can be found in

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Supplementary Information 3 and Figure S2. The decay rate constants were mathematically

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corrected for attenuation of light penetration caused by light screening (due to variable light

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absorbance) but not light scattering (due to turbidity) in the samples. The corrected decay rate

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constants were then used in statistical comparisons or half-life calculations. Because

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differences in light absorbance and screening can also occur due to sample to sample variation

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or different water matrices (Figure S3), this adjustment was hence performed for each

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experimental microcosm by measuring each individual sample’s absorbance at the start of the

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experiment as described previously,

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inactivation by full spectrum irradiation (280-700 nm) as is shown in this study, correction

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factor for the decay rate constants was based on the concept that different wavelengths differ

26, 27

and shown in Supplementary Information 2. For


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in their inactivation efficiencies. UV portions of the spectrum are generally the most important

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wavelengths that are most lethal to the microorganism compared to the visible range

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wavelengths. Mathematical calculations of correction factors therefore also weigh in the

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difference in contribution of UV and visible range to the decay27. The half-lives for each

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experiment, or the durations needed to reduce the bacterial or bacteriophage concentration by

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half (after and excluding lag-phase), were calculated using the first-order decay kinetics

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equation:

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ln(CFUt/CFU0) = -k*t

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where k* is the decay rate constant, corrected for light screening, of the natural log inactivation

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curve, and t is time.

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Statistical analyses was performed using Minitab version 1.4.0. One-way ANOVA was

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performed to compare the lengths of lag-phases, the corrected decay rate constants and the

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half-life values of the different experimental conditions and isolates. Linear regression analysis

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was performed to compare the slope of each experiments inactivation curve to 0 (α = 0.05) to

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determine if significant change/decay is observed (Supplementary Information 3). Errors

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presented at various points in the text and tables, and as error bars in some charts, are standard

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deviation from the mean values reported. No error was propagated on C0 in (C/C0)-based plots.

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This is because each sample had its own C0. After samples were enumerated for C, Log10(C/C0)

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or Ln(C/C0) values were generated for each microcosm. Afterwards the average Log10(C/C0)

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or Ln(C/C0) – including all technical and biological replicates - for one experimental condition

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was calculated, and then the standard deviation error for that average was calculated. Lag-

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phase length and half-life were not analyzed for dark control samples due to the lack of

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significant decay in these samples, as determined by single linear regression analysis.

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2.5. RNA-seq sampling, extraction and sequencing

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RNA-seq was performed to provide comparative analysis of gene expression between

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samples with and without bacteriophage addition upon solar irradiation. Samples were taken

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at various time points (at 0, 1, 2, 4, 6, 8 and 10 h) to capture the gene expression at different

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phases of the bacterial CFU inactivation curves. For bacteriophage-supplemented experiments

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(SI+phage), three solar inactivation experiments were conducted for RNA sampling to obtain

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three biological replicates, each with two dark and two irradiated technical replicate

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microcosms at every sampling point. For non-bacteriophage-supplemented (SI-only)



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experiments, two solar inactivation experiments for RNA sampling were performed, also with

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two dark and two irradiated technical replicate microcosms at every sampling point.

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RNA preservation, extraction and sequencing were performed as described

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previously20. Briefly, sample volumes varying between 1 to 4 mL were taken from the

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experimental microcosms, and an equal volume of RNAProtect reagent (Qiagen, Valencia,

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CA) was added in. Samples were stored at 4 °C for less than a week until RNA extraction.

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Samples were centrifuged at 5,000 g for 10 mins, and the pellets used for total RNA extraction

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using the RNeasy Mini kit (Qiagen, Hilden, Germany) with the on-column DNase treatment.

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The RNA was eluted with RNase-free water and RNA quality was determined using 2200

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Tapestation Bioanalyzer (Agilent Technologies, Santa Clara, CA). All samples that passed the

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quality control were submitted to KAUST Genomics Core lab for RNA-Seq on Illumina HiSeq

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platform. Prior to sequencing, the bacterial ribosomal RNA was removed from 1 µg of total

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RNA using the Ribo Zero rRNA removal kit (Illumina, San Diego, CA, USA). The enriched

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mRNA fraction was converted to RNA-seq libraries compatible for runs on Illumina HiSeq

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platforms based on protocols described by manufacturer. Fastq files were generated and

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demultiplexed by KAUST Bioinformatics core lab. All fastq files associated with E. coli PI-7

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with and without bacteriophages are available on the ENA SRA public depository under

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accession numbers PRJEB25911 and 25812.

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2.6. RNA-seq data analysis

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The RNA-seq data was analyzed using CLC Genomics Workbench version 8.0.1 from CLC

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Bio (Cambridge, MA). For the reference genome sequence, and to assign genes to specific

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categories, genomic DNA sequences annotated using RAST were used for E. coli PI-7 6. RNA-

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seq reads were mapped onto the reference sequences using CLC. Reads were only assembled

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if the fraction of reads that aligned to the reference genome was greater than 0.9 and if the read

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matched other regions of the reference genome at fewer than 10 nucleotide positions.

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Summarized RNA-seq mapping results are shown in Tables S1 and S2 and Supplementary

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Information 4.

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CLC was used to generate normalized mean expression values by applying a scaling

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correction to samples to facilitate cross-sample comparisons within and across biological

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batches. The gene expression results were then analyzed for statistical significance, separately

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for each of the biological experimental replicate batches. The statistical comparison was based 9 ACS Paragon Plus Environment

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on the proportion-based test described by Baggerly et al. 28. Mean gene expression values as

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normalized RPKM (reads per kilobase million) in irradiated samples were compared with the

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mean normalized RPKM values of their respective dark controls at each time point for each

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experimental batch. Then, for each condition, the fold-change in expression level was taken

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into consideration if it was statistically significant (p-value

Bacteriophages To Sensitize A Pathogenic New Delhi Metallo Î²

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