bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
1 Genomic and functional characterization of Pseudomonas aeruginosa-targeting
2 bacteriophages isolated from hospital wastewater
3
4 Hayley R. Nordstrom1, Daniel R. Evans1, Amanda G. Finney1, Kevin J. Westbrook1, Paula F.
5 Zamora2, Alina Iovleva1, Mohamed H. Yassin1, Jennifer M. Bomberger2, Ryan K. Shields1, Yohei
6 Doi1, Daria Van Tyne1,*
7
8 1Division of Infectious Diseases, University of Pittsburgh School of Medicine
9 2Department of Microbiology and Molecular Genetics, University of Pittsburgh School of
10 Medicine
11
12 *Address correspondence to: [email protected]
13
14 Running head: Bacteriophages to target Pseudomonas aeruginosa
15
16 Abstract
17 Pseudomonas aeruginosa infections can be difficult to treat and new therapeutic approaches
18 are needed. Bacteriophage therapy is a promising alternative to traditional antibiotics, but large
19 numbers of isolated and characterized phages are lacking. We collected 23 genetically and
20 phenotypically diverse P. aeruginosa isolates from people with cystic fibrosis (CF) and clinical
21 infections, and characterized their genetic, phenotypic, and prophage diversity. We then used
22 these isolates to screen and isolate 14 new P. aeruginosa-targeting phages from hospital
23 wastewater. Phages were characterized with genome sequencing, comparative genomics, and
24 lytic activity screening against all 23 bacterial host isolates. For four different phages, we
25 evolved bacterial mutants that were resistant to phage infection. We then used genome
26 sequencing and functional analysis of the resistant mutants to study their mechanisms of phage bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
27 resistance as well as changes in virulence factor production and antibiotic resistance, which
28 differed from corresponding parent bacterial isolates. Finally, we tested two phages for their
29 ability to kill P. aeruginosa grown in biofilms in vitro, and observed that both phages reduced
30 viable bacteria in biofilms by least one order of magnitude. One of these phages also showed
31 activity against P. aeruginosa biofilms grown on CF airway epithelial cells. Overall, this study
32 demonstrates how systematic genomic and phenotypic characterization can be deployed to
33 develop bacteriophages as precision antibiotics.
34
35 Introduction
36 The evolution of multidrug-resistant bacteria continues to outpace the development of new
37 antimicrobials, posing a serious threat to public health. Rates of infection and mortality due to
38 antibiotic-resistant pathogens are continuing to grow in the United States and around the world,
39 despite efforts to curtail their spread (1, 2). Compounding the rise of multidrug-resistant bacterial
40 infections, antibiotic development pipelines at many pharmaceutical companies have slowed or
41 run dry (3). To help curtail this growing public health crisis, innovative approaches to
42 antimicrobial therapy are needed.
43
44 Pseudomonas aeruginosa is a Gram-negative bacterium that causes a variety of infections,
45 including bacteremia and pneumonia (4). P. aeruginosa chronically colonizes the airways of
46 people with cystic fibrosis (CF), and is associated with increased morbidity and mortality in CF
47 individuals (5). The P. aeruginosa species encompasses a wide breadth of genomic and
48 phenotypic diversity, and multidrug-resistant strains often evolve during the course of prolonged
49 antibiotic treatment (6). The success of P. aeruginosa as an opportunistic pathogen, its
50 propensity for developing drug resistance, and the major threat it poses to CF patients, are
51 compelling reasons to develop new and more effective therapies to treat P. aeruginosa
52 infections. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
53
54 Modern medicine is quickly approaching a “post-antibiotic” era, in which current antibiotics may
55 no longer be effective treatments for bacterial infections due to the rampant spread of drug
56 resistance (7). The urgent need for alternative therapies has prompted clinicians and scientists
57 to reconsider the use of bacteriophage therapy (8), particularly for treating infections that cannot
58 be resolved with antibiotics alone (9). Recent advances in genomics and genetic engineering
59 have facilitated the development of phage-based therapies that have proven successful in
60 clinical settings (9), including P. aeruginosa infections in CF (10). Here, we used a genetically
61 diverse panel of 23 P. aeruginosa clinical isolates, collected mostly from CF patients, to isolate
62 over a dozen distinct bacteriophages from hospital wastewater. We characterized the genomic
63 and phenotypic diversity of the bacterial isolates and phages, including a subset of evolved
64 phage-resistant bacterial mutants. We also tested the ability of some of the isolated phages to
65 clear bacterial biofilms in vitro and ex vivo. These data can aid in the rational design of tailored,
66 phage-based therapies for the treatment of P. aeruginosa infections.
67
68 Materials and Methods
69 Bacterial isolate collection
70 P. aeruginosa bacterial isolates were collected from patients treated at the University of
71 Pittsburgh Medical Center (UPMC) (n=21), or were purchased from the American Type Culture
72 Collection (ATCC) (n=2). Collection of UPMC isolates was conducted with Institutional Review
73 Board Approval (protocol #PRO12060302). Of the UPMC isolates, 20 were collected from
74 people with CF and one was a clinical isolate from sputum collected from a non-CF patient.
75 Both ATCC isolates were of clinical origin. All isolates were cryopreserved in brain heart
76 infusion (BHI) media with 16.7% glycerol and stored at -80°C.
77
78 Hospital wastewater collection and processing bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
79 Wastewater effluent was sampled from the main sewer outflow of a Pittsburgh area hospital, at
80 a point before the outflow joined with the municipal sewer system. A total of four samples were
81 collected over a six-month period. Each effluent sample was centrifuged at 4,000rpm for 20
82 minutes to pellet solid debris, the supernatant was filtered through a 0.22-µm filter, and the
83 sample was then concentrated by centrifugation using an Amicon 100kDa filter unit
84 (MilliporeSigma, Burlington, MA) at 4,000rpm for 15 minutes.
85
86 Isolation of bacteriophages and phage-resistant mutants
87 Lytic bacteriophages were identified with a soft agar overlay assay. Briefly, bottom agar plates
88 were prepared containing BHI media with 1.5% agar, 1mM CaCl2 and 1mM MgCl2. Bacterial
89 isolates were inoculated into BHI media and grown overnight at 37ºC. 100µL of bacterial culture
90 was added to a tube containing 100µL of filtered concentrated wastewater for 5-10 minutes at
91 room temperature, and then 10mL of top agarose (BHI with 0.5% agarose, 1mM CaCl2, and
92 1mM MgCl2) cooled to 55ºC was added and the mixture was plated onto two bottom agar
93 plates. Plates were incubated overnight at 37ºC and were examined the following day to identify
94 lytic phage plaques. Phages were passaged by sequential picking and plating of individual
95 plaques grown on the same bacterial isolate. Phages were picked from individual plaques using
96 a pipette tip and were placed into 100µL of SM buffer (50mM Tris-HCl pH 7.5, 100mM NaCl,
97 8mM MgSO4) and incubated overnight at 37ºC. The following day, serial 10-fold dilutions were
98 made in SM buffer, and 3µL of each dilution was spotted onto a plate containing 5mL of top
99 agarose mixed with 50µL of bacterial culture and layered on top of a bottom agar plate. After
100 overnight incubation at 37ºC, an individual plaque was picked and passaged again. Each phage
101 was passaged at least twice before the generation of high-titer stocks.
102
103 To generate high-titer stocks, a single plaque was picked into 100µL of SM buffer and incubated
104 overnight. Then, 100µL of overnight bacterial culture was added and the mixture was incubated bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
105 for 5-10 minutes at room temperature, followed by addition of 10mL of top agarose and plating
106 onto two bottom agar plates. Plates were incubated overnight at 37ºC, and then plates with high
107 plaque density were flooded with 5mL of SM buffer and incubated at 37ºC for at least 1 hour to
108 elute phages from the top agar. SM buffer was removed from each plate, pooled, spun down at
109 4,000rpm for 20 minutes, and filtered through a 0.22µm filter. Phage-containing lysates were
110 extracted with 0.1 volumes of chloroform followed by 0.4 volumes of 1-octanol, and were stored
111 at 4ºC.
112
113 When phage-resistant mutants were observed in the course of preparing high-titer phage
114 lysates, they were saved for additional characterization. Individual bacterial colonies were
115 picked, restreaked onto BHI agar, and tested by plaque assay to confirm their resistance to the
116 isolated phage, as well by spotting onto a lawn of the parent bacterial isolate to confirm that they
117 were not lysogens.
118
119 Phenotypic characterization of bacterial hosts and phage-resistant mutants
120 Biofilm assays were performed following a previously published protocol (11). Briefly, bacteria
121 were first inoculated into LB media and incubated overnight at 37ºC. Cultures were then diluted
122 1:100 into M63 media. Diluted cultures were aliquoted into vinyl 96-well plates (100µl per well)
123 sealed, and incubated at 37ºC for 24 hours. After incubation, planktonic cells were removed by
124 inverting the plates and shaking liquid out into a sterilization tub. Plates were then submerged in
125 water and rinsed twice to remove unattached cells. Wells were stained with 0.1% crystal violet
126 and incubated at room temperature for 15 minutes. Plates were rinsed three times with water
127 and shaken out vigorously, then allowed to dry completely. Crystal violet stain was solubilized
128 with 30% acetic acid. Absorbance was read in each well at 550nm using a BioTech Synergy H1
129 microplate reader with GenMark software (BioTech, Winooski, VT). Two biological replicates,
130 each containing 24 technical replicates, were run for each isolate. To test phage activity against bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
131 bacteria grown in biofilms, biofilms were inoculated into 96-well plates as above and incubated
132 for 24 hours at 37ºC. Planktonic cells were removed and biofilms were washed with sterile
133 1xPBS using a multichannel pipettor, and either fresh M63 media or phage at 1x1012 PFU/mL in
134 M63 media was added to each well. Plates were incubated for 24 hours at 37ºC, then bacteria
135 in each well were resuspended and serial 10-fold dilutions were tracked onto BHI agar plates to
136 determine the number of colony-forming units per mL (CFU/mL) in each condition.
137
138 Extracellular protease production was measured by spotting 2.5µL of an overnight culture of
139 each isolate grown in BHI media onto a BHI agar plate containing 10% milk. Plates were
140 incubated overnight at 30ºC and read the following day. Protease activity was detected as a
141 clear halo surrounding the bacterial spot. Swimming motility was measured by spotting 2.5µL
142 from an overnight bacterial culture grown in BHI media onto plates containing LB + 0.3% agar.
143 Plates were incubated overnight at 37ºC and read the following day. Swimming motility was
144 detected as bacterial growth away from the central spot. Twitching motility was assessed by
145 inserting a pipet tip coated in overnight bacterial culture completely through a BHI agar plate to
146 create a small hole in the agar, and then incubating for 48 hours at 37ºC. Mucoidy was
147 measured by assessing the morphology of each isolate when grown on both LB agar and
148 Pseudomonas isolation agar (PIA) plates. Each isolate was struck onto each agar type, then
149 incubated at 37ºC overnight, followed by a 48-hour incubation at room temperature.
150
151 Antibiotic susceptibility testing was performed by broth microdilution in Mueller-Hinton Broth
152 according to the standard protocol established by the Clinical Laboratory Standards Institute
153 (CLSI) (12). Serial two-fold dilutions of ceftazidime were tested and the minimum inhibitory
154 concentration (MIC) was recorded as the lowest antibiotic concentration that inhibited bacterial
155 growth by visual inspection. Pyoverdine production was measured by first growing isolates to
156 stationary phase in LB media and then inoculating bacteria into M9 media supplemented with bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
157 20mM sodium succinate and 0.5% iron-depleted casamino acids (produced by pre-treating a
158 10% stock solution with 0.05g/mL Chelex-100 for one hour). Bacteria were grown overnight,
159 then pyoverdine fluorescence was measured at excitation=400nm and emission=447nm
160 wavelengths on a BioTech Synergy H1 microplate reader with GenMark software (BioTech,
161 Winooski, VT). Raw fluorescence unit values were collected, background fluorescence was
162 subtracted, and fluorescence units were normalized by OD600. Three biological replicates, each
163 consisting of four technical replicates, were tested.
164
165 Genome sequencing and analysis
166 Bacterial genomic DNA was extracted from 1mL overnight cultures grown in BHI media using a
167 Qiagen DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD) following the manufacturer’s
168 protocol. Illumina sequencing libraries were prepared with a Nextera XT or Nextera kit (Illumina,
169 San Diego, CA), and libraries were sequenced on a MiSeq using 300-bp paired-end reads, or
170 on a NextSeq using 150-bp paired-end reads. Genomic DNA was also used to construct long-
171 read sequencing libraries using a rapid barcoding kit (SQK-RBK004, Oxford Nanopore
172 Technologies, Oxford, UK), and libraries were sequenced on a MinION device. Base-calling of
173 nanopore reads was performed with Guppy. Genomes were hybrid assembled with unicycler
174 (13), annotated with prokka (14), and were compared to one another with Roary (15). A core
175 genome phylogenetic tree was generated using RAxML with the GTRCAT substitution model
176 and 1000 iterations (16). Prophages were identified in each bacterial genome using PHASTER
177 (17). Prophages of any length that were predicted to be intact or questionable by PHASTER
178 were included. Prophage sequences were compared to one another with nucleotide BLAST,
179 and clusters of similar prophage sequences were identified as those sharing >90% sequence
180 coverage and >90% sequence identity.
181 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
182 Phage genomic DNA was extracted from 500µL of phage lysate using phenol chloroform,
183 followed by ethanol precipitation. Briefly, 500µL phenol:chloroform:isoamyl alcohol (25:24:1)
184 was added to each lysate, samples were vortexed and then centrifuged at 16,000 x g for 1
185 minute. The upper aqueous phase was transferred to a new tube and 500µL of chloroform was
186 added. Samples were vortexed and centrifuged again at 16,000 x g for 1 minute, and the upper
187 aqueous phase was again transferred to a new tube. Then 1µL glycogen, 0.1x volume 3M
188 sodium acetate, and 2.5x volume 100% ethanol were added and samples were incubated
189 overnight at -20ºC. The next day samples were centrifuged at 16,000 x g for 30 minutes at 4ºC,
190 then the supernatant was removed and the DNA pellet was washed with 150µL 70% ethanol.
191 DNA pellets were resuspended in 100µL nuclease-free water, and DNA was quantified with a
192 Qubit fluorimeter (Thermo Fisher Scientific, Waltham, MA). Illumina sequencing libraries were
193 prepared with a Nextera XT or Nextera kit (Illumina, San Diego, CA), and libraries were
194 sequenced on a MiSeq using 300-bp paired-end reads, or on a NextSeq using 150-bp paired-
195 end reads. Phage genomes were assembled with SPAdes v3.13.0 (18), and were annotated
196 with prokka (14). Phage genomes were compared to one another and to other available phage
197 genomes using BLAST (19), PHASTER (17), and Mauve (20).
198
199 Ex vivo biofilm assay
200 Immortalized human bronchial epithelial cells isolated from a ΔF508/ΔF508 CF patient
201 (CFBE41o- cells) (21) were cultured on Transwell inserts (Corning, Corning, NY) and grown for
202 7-10 days at the air-liquid interface, as described (22). Basolateral medium was replaced with
203 minimum essential medium (MEM) supplemented with 2mM L-glutamine 24h before bacterial
204 inoculation. P. aeruginosa isolate 427 was inoculated into the apical compartment as described
205 previously (23), with the following modifications: bacteria were inoculated at a multiplicity of
206 infection of 0.015 CFU/cell in MEM and were incubated at 37ºC for 1h, followed by inoculum
207 removal and addition of MEM supplemented with 2mM L-glutamine and 23mM L-arginine. P. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
208 aeruginosa phage PSA07/PB1 was added to the apical compartment 8h post-bacterial
209 inoculation at a concentration of 8x106 PFUs/mL and incubated at 37ºC for 16h. Following
210 washing of the apical and basolateral media, biofilms were collected by adding 0.1% Triton X-
211 100 apically and centrifuged to remove soluble phages. CFUs were quantified by dilution
212 plating onto LB agar.
213
214 Statistical Analyses
215 Two-tailed t-tests were used to assess the significance of prophage differences between
216 CRISPR+ and CRISPR- isolates, differences in pyoverdine production between parent and
217 phage-resistant mutant isolates, and differences in bacterial cell density in biofilm killing
218 experiments.
219
220 Data Availability
221 Hybrid assembled bacterial host genomes were submitted to NCBI under BioProject
222 PRJNA610040. Bacteriophage genomes were submitted to NCBI under BioProject
223 PRJNA721956.
224
225 Results
226 P. aeruginosa clinical isolates used for phage screening are genetically and
227 phenotypically diverse
228 To isolate bacteriophages that could be maximally useful for the treatment of P. aeruginosa
229 infections, we assembled a genetically and phenotypically diverse panel of 23 P. aeruginosa
230 isolates collected from clinical sources (Table S1). Two isolates were purchased from the
231 American Type Culture Collection (ATCC), 20 isolates were collected from adults with cystic
232 fibrosis (CF), and one isolate was collected from the sputum of a hospitalized non-CF patient.
233 All isolates were collected from different patients. The genome of each isolate was sequenced bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
234 on both the Illumina and Oxford Nanopore MinION platforms, and the resulting sequencing data
235 were hybrid assembled (13). Over half (14/23) of the genomes were closed to a single
236 chromosome, and the remaining nine assemblies all contained 10 or fewer contigs (Table S1).
237 Among the 23 isolates, a total of 19 different multi-locus sequence types (STs) were identified
238 (Table S1). A core genome phylogeny of all 23 isolates confirmed that they were highly
239 genetically diverse (Fig. 1A). All isolates were tested for their ability to form biofilms, produce
240 extracellular protease, exhibit swimming motility, and display a mucoid phenotype when grown
241 on LB and Pseudomonas isolation agars. These phenotypes were found to be variable among
242 the collected isolates (Fig. 1A), demonstrating that the assembled isolate panel was both
243 genetically and phenotypically diverse.
244
245 We assessed the abundance and diversity of prophage sequences in the 23 P. aeruginosa
246 clinical isolates we collected. The genome of each isolate was mined for prophage sequences
247 using the PHASTER online tool (17). Between 0 and 6 prophages were found in each isolate
248 genome (Fig. 1A, Table S2). Prophages varied in length from 5.5-74.5 Kb and in GC-content
249 from 52.5%-66.0%. A total of 54 prophage sequences were extracted, and were compared to
250 one another using nucleotide BLAST to assess both the nucleotide identity and coverage across
251 all pairwise comparisons (Fig. 1B). A total of six different prophages were found to be present in
252 more than one isolate; these included three distinct phiCTX-like phages and two Pf1-like
253 filamentous phages. Finally, we assessed the number of prophages in isolates that were
254 predicted to have either functional or non-functional Clustered Regularly Interspaced Short
255 Palindromic Repeats (CRISPR) loci, based on the presence or absence of Cas enzymes in the
256 genome of each isolate (Table S1). We found that the seven isolates predicted to have non-
257 functional CRISPR-Cas systems had more prophages compared to isolates with intact CRISPR-
258 Cas loci (Fig. 1C, P=0.03).
259 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
260 P. aeruginosa-targeting bacteriophages isolated from hospital wastewater
261 We used the 23 P. aeruginosa isolates we collected to screen for lytic bacteriophages in
262 wastewater effluent collected from a Pittsburgh area hospital. A total of 14 phages were isolated
263 on 10 different P. aeruginosa isolates (Table 1). One additional phage, PB1, was purchased
264 from ATCC and was propagated and characterized alongside the newly isolated phages.
265 Because sequencing the genome of this phage revealed multiple mutations when compared to
266 the PB1 sequence deposited in NCBI, we refer to it here as PSA07/PB1. Phages were picked
267 and repeatedly passaged as single plaques, and were then amplified to generate high-titer
268 stocks. Genomic DNA was extracted from each phage stock, and was sequenced on the
269 Illumina platform. Phage genomes were found to be between 43.7-65.9 Kb in length and had
270 GC-content ranging from 44.9%-64.5% (Table 1). Phages were compared to publicly available
271 genomes using PHASTER and NCBI BLAST, and the predicted family and genus of each
272 phage were determined based on similarity to previously described phages. Despite appearing
273 to be lytic on the isolates used to propagate them, three phages (PSA04, PSA20, and PSA21)
274 were predicted to have a lysogenic lifestyle due to the presence of annotated phage integrases.
275 The PSA04 genome was most similar to the JBD44 lysogenic phage (24), however the
276 homology between these two phages was not particularly high (Table 1). The PSA20 and
277 PSA21 phage genomes showed moderate sequence similarity to the Yuavirus phages AN14
278 and LKO4, in which the putative integrase is instead believed to be a DNA primase (25). The
279 lack of previously described lysogenic activity among Yuavirus phages is consistent with our
280 observations of lytic behavior for phages PSA04, PSA20 and PSA21.
281
282 Next, we compared the genomes of the isolated phages to one another, and to the publicly
283 available phage genomes that were most similar to them, using nucleotide BLAST (Table 1, Fig.
284 2). Phages within the same genus showed varying degrees of genomic similarity with one
285 another, and no similarity was observed across different families or genera. Six of the phages bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
286 we isolated belonged to the Bruynoghevirus genus within the Podoviridae family; three of these
287 phages (PSA31, PSA37, and PSA40) were highly similar to one another, despite having been
288 isolated on three different P. aeruginosa isolates and from three different wastewater samples
289 (Fig. S1). These data suggest that Bruynoghevirus phages might have been particularly
290 abundant in the wastewater that we sampled, and that they are able to infect genetically diverse
291 P. aeruginosa isolates.
292
293 Phage susceptibility of P. aeruginosa isolates and bacteriophage infectivity
294 To examine the phage susceptibilities of our P. aeruginosa isolates as well as the infectivity
295 profile of each phage, we performed a lytic activity screen of the 15 bacteriophages studied here
296 against all 23 bacterial isolates (Fig. 3). Serial dilutions of each phage were spotted onto top
297 agar lawns of each bacterial isolate, and individual plaques were counted to determine the titer
298 of each phage against each isolate. Three of the P. aeruginosa isolates we tested (413, 414,
299 and 729) were resistant to all phages tested, however the other 20 isolates (87% of all isolates
300 tested) were susceptible to multiple phages belonging to different families (Fig. 3). Phage
301 susceptibility profiles of the isolates were highly variable, with the exception of isolate pairs
302 418/423 and 427/466; these pairs contained isolates belonging to the same ST, which were
303 more genetically similar to one another than to the other isolates in the study. While phages
304 were found to infect between 9 and 19 different isolates, activity of the same phage was often
305 variable against different isolates. For example, phage PSA07/PB1 displayed titers varying from
306 102 to 1010 PFU/mL against different P. aeruginosa isolates (Fig. 3). Finally, compared to
307 Myoviridae and Siphoviridae phages, the Podoviridae phages we isolated were able to infect
308 more isolates and had higher average infectivity against the isolates tested here.
309
310 Genomic and phenotypic differences of phage-resistant mutants bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
311 During the course of phage propagation, we isolated single colonies of phage-resistant mutants
312 for four phages: PSA09, PSA11, PSA20, and PSA34 (Fig. 4). Phage-resistant mutant isolates
313 were tested to confirm their resistance, and were then subjected to whole-genome sequencing.
314 Sequencing reads were mapped to the hybrid assembled genome of the corresponding phage-
315 susceptible parent isolate, and protein-altering mutations in each resistant mutant were
316 identified (Table 2). Each phage-resistant mutant genome encoded 2-3 protein-altering
317 mutations. Based on the annotation of each mutated gene, we were able to identify putative
318 phage resistance-conferring mutations in each mutant isolate genome. A phage-resistant
319 mutant in the 639 isolate background that was resistant to phage PSA20 was found to have a
320 Thr278Pro mutation in the Type IV pilus protein PilB (Table 2). Because Type IV pili are
321 involved in twitching motility, we compared the twitching motility of the 639 P. aeruginosa parent
322 isolate and the PSA20-resistant mutant, and found that the resistant mutant showed diminished
323 twitching motility (Fig. 4A). Two other phage-resistant mutants raised in different P. aeruginosa
324 parent isolates against different phages both encoded mutations in genes predicted to impact
325 LPS biosynthesis, including a RfaB-like glycosyltransferase and the dTDP‑4‑dehydrorhamnose
326 reductase RfbD (Table 2). We compared the susceptibilities of both phage-resistant mutants
327 and their corresponding parent isolates to ceftazidime, an antibiotic that is used to treat P.
328 aeruginosa infections (26). The mutants showed either four-fold or eight-fold sensitization to
329 ceftazidime compared to their parents (Fig. 4B), indicating that the phage resistance-conferring
330 alterations to the LPS in these mutants also increased their susceptibility to a cell wall-targeting
331 antibiotic. A final mutant was found to carry a frameshift mutation that disrupted the coding
332 sequence of the quorum-sensing master regulator LasR (Table 2). Because LasR is known to
333 regulate the production of P. aeruginosa virulence factors, we measured the production of
334 extracellular protease and pyoverdine in both the parent and phage-resistant mutant isolates
335 (Fig. 4C and D). Extracellular protease production was absent and pyoverdine production was
336 greatly diminished in the phage-resistant mutant compared to the parent isolate. Overall these bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
337 data demonstrate the variability of genetic mechanisms underlying phage resistance, as well as
338 the collateral phenotypic effects of resistance.
339
340 Phage-mediated killing of bacterial biofilms in vitro and ex vivo
341 Because P. aeruginosa causing infections frequently grows in biofilms (27), we tested whether
342 phages that were active against bacteria in our top agar lawn-based activity assays could also
343 kill bacteria grown in biofilms (Fig. 5). We first tested the ability of the PSA07/PB1 and PSA34
344 phages to kill the 427 P. aeruginosa isolate grown in biofilms in vitro. Biofilms were grown for 24
345 hours, planktonic cells were removed and biofilms were washed, and then phages were applied
346 and plates were incubated for an additional 24 hours. PSA07/PB1 treatment resulted in >100-
347 fold bacterial killing, and PSA34 treatment resulted in >10-fold bacterial killing (Fig. 5A). Next,
348 we tested the ability of the PSA07/PB1 phage to kill the 427 isolate grown in biofilms in
349 association with human CF airway epithelial cells. Bacteria were incubated with epithelial cells
350 for 8 hours, then phage was added and incubated for additional 16 hours before cell-associated
351 bacteria were collected and quantified for viability. We found that similar to the in vitro assay,
352 PSA07/PB1 treatment resulted in >100-fold bacterial killing (Fig. 5B), suggesting that phages
353 can also kill P. aeruginosa grown in biofilms under conditions that more closely mimic infection
354 in humans.
355
356 Discussion
357 The objective of this study was to isolate and characterize lytic bacteriophages from hospital
358 wastewater with activity against clinically relevant P. aeruginosa isolates. By screening
359 wastewater samples against a genetically and phenotypically diverse panel of P. aeruginosa
360 bacterial isolates, we were able to isolate a diverse group of P. aeruginosa-targeting phages
361 representing three families: Myoviridae, Siphoviridae and Podoviridae. In testing our panel of P.
362 aeruginosa isolates for susceptibility to the isolated phages, we found a broad range of phage bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
363 activities. Additionally, our analysis of select phage-resistant mutants showed that evolving
364 phage resistance often conferred an increase in antibiotic susceptibility or a reduction in
365 bacterial virulence. Finally, two of the phages we isolated were able to kill P. aeruginosa grown
366 in biofilms in vitro and ex vivo, suggesting that they have therapeutic utility for the treatment of
367 P. aeruginosa infections.
368
369 The bacteriophages we isolated in this study were similar in terms of phage family, genus, and
370 other genome characteristics to P. aeruginosa-targeting phages isolated previously (28-31).
371 This could be due to the fact that these prior studies also isolated phages from sewage, similar
372 to what we did in this study. Our findings, however, are in keeping with the idea that phages
373 active against P. aeruginosa mirror the abundant genetic and phenotypic diversity of their hosts.
374 While it has been noted that newly isolated phages do not often represent novel phylogenetic
375 lineages (30), sampling and screening from more diverse sources could potentially uncover a
376 broader range of phage genetic diversity.
377
378 While the vast majority of P. aeruginosa isolates we screened were susceptible to one or more
379 of the phages we isolated, three bacterial isolates were resistant to all phages studied here.
380 These three isolates (413, 414, and 729) were genetically distinct from one another, and no
381 clear trends emerged to explain their resistance to phage infection. For example, they did not all
382 have functional CRISPR-Cas systems or a higher relative abundance of prophages compared
383 to phage-susceptible isolates. We did note that the P. aeruginosa 729 isolate grew very poorly,
384 and was predicted to be a hypermutator due to a frameshift mutation in the DNA mismatch
385 repair gene mutS. It is unknown whether hypermutators in P. aeruginosa are more resistant to
386 phage infection; this would be a worthwhile avenue of future investigation. Nonetheless, the
387 specific mechanism(s) conferring phage resistance in the clinical isolates we studied here
388 remain unclear. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
389
390 During the course of phage propagation, we isolated four phage-resistant P. aeruginosa
391 mutants and studied them further. From whole-genome sequencing of these mutants, we
392 identified three kinds of mutations that lead to measurable phenotypic changes. First, in the
393 phage-resistant mutant of isolate 639, disruption of the Type IV pilus protein PilB appears to
394 have also caused a reduction in twitching motility. Because the Type IV pilus has been
395 previously described as a surface receptor used by P. aeruginosa phages for infection (32), we
396 suspect that the phage PSA20, and also perhaps the other Yuavirus phages we isolated, use
397 the Type IV pilus as a receptor for infection. Second, we identified mutations in genes affecting
398 LPS biosynthesis in phage-resistant mutants of isolates 410 and 427. Bacterial LPS is also a
399 well-known surface receptor used for phage infection in P. aeruginosa (33). Notably, we
400 observed that the resulting phage-resistant mutants showed increased susceptibility to
401 ceftazidime, a cell-wall targeting antibiotic. Finally, in the phage-resistant mutant of ATCC
402 14210, a disruption in the quorum-sensing master regulator LasR resulted in a decrease in the
403 production of extracellular protease as well as pyoverdine, which are two prominent virulence
404 factors in P. aeruginosa (34). Taken together, these findings are consistent with the notion that
405 the development of phage resistance is often coupled with collateral effects like decreased
406 bacterial virulence or increased antibiotic susceptibility (35). This has potentially promising
407 implications for treatment of P. aeruginosa infections using phages, where a tradeoff between
408 phage resistance and antibacterial resistance could be exploited.
409
410 When we tested whether two different P. aeruginosa phages could kill bacteria grown in
411 biofilms, we observed reductions in viable bacteria upon phage treatment of biofilms both in
412 vitro and ex vivo. While application of phage did not completely eradicate bacteria growing in
413 the biofilms, it did substantially decrease the bacterial loads measured in both assays to similar
414 levels to the ones obtained after antibiotic treatment (36). This finding is consistent with other bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
415 studies that have also documented phage-mediated reductions in P. aeruginosa biofilm density
416 in vitro (31, 37, 38). Here we have extended these in vitro findings to test the ability of phages to
417 infect bacteria grown on human CF airway epithelial cells, a setting that more closely mimics
418 bacterial growth in the CF airway (39, 40). Testing of phage efficacy in a context that includes
419 eukaryotic cells is an important feature of this study. Whether and how bacteriophages interact
420 with eukaryotic cells, and how this interaction may impact phage activity, is a focus on ongoing
421 work by us and others (41).
422
423 This study had several limitations. Many of the phages we isolated showed variable activity, and
424 their activity was generally diminished against isolates other than the host isolate used for their
425 initial isolation and propagation. While we attempted to be unbiased in our phage isolation
426 methods, we observed some redundancy in isolated phages within the Podoviridae family,
427 suggesting a possible enrichment of our wastewater source with Podoviridae phages.
428 Additionally, we only isolated and studied four different phage-resistant mutants, and we did not
429 confirm that any of the resistance-associated mutations identified were indeed the cause of
430 phage resistance, for example through genetic complementation. Finally, all work performed in
431 this study was conducted in vitro or ex vivo, thus we are unable to conclude that any of the
432 phages we isolated would be useful therapeutic candidates without additional testing, for
433 example in relevant animal models of P. aeruginosa infection.
434
435 Taken together, the genotypic and phenotypic data presented here have promising implications
436 for the therapeutic potential of P. aeruginosa-targeting bacteriophages. This study provides a
437 valuable addition to the growing literature documenting the abundance and diversity of P.
438 aeruginosa phages, and demonstrates how systematic characterization can aid in the
439 development of phages for clinical use as precision antibiotics.
440 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
441 Acknowledgements
442 We gratefully acknowledge all members of the Van Tyne lab, and in particular Shu-Ting Cho for
443 helpful input during the preparation of this manuscript. We also thank Carlos Guerrero-
444 Bustamante and Catherine Armbruster for helpful contributions to the study. Research reported
445 in this publication was supported in part by the National Institute of Allergy and Infectious
446 Diseases of the National Institutes of Health under Award Number UM1AI104681. The content
447 is solely the responsibility of the authors and does not necessarily represent the official views of
448 the National Institutes of Health. This work was also supported by grants BOMBER19R0,
449 BOMBER21P0, and ZAMORA20F0 from the Cystic Fibrosis Foundation, and by the Department
450 of Medicine at the University of Pittsburgh School of Medicine. The funders had no role in study
451 design, data collection and analysis, decision to publish, or preparation of the manuscript.
452
453 Conflicts of Interest
454 J.B. is a consultant for BiomX, Inc. The other authors have no relevant conflicts of interest.
455
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571 41. Van Belleghem JD, Dabrowska K, Vaneechoutte M, Barr JJ, Bollyky PL. 2018. 572 Interactions between Bacteriophage, Bacteria, and the Mammalian Immune System. 573 Viruses 11. 574 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Length Predicted Predicted NCBI Similar Phage Coverage/ Phage Source1 Host %GC BioSample (bp) Family Genus (Accession)2 Identity PSA04 HWW 418 48544 59.2 Siphoviridae - JBD44 (NC_030929) 68%/98% SAMN18741767 PSA07/PB1 ATCC ATCC 15692 65891 54.9 Myoviridae Pbunavirus PB1 (NC_011810) 100%/100% SAMN18741768 PSA09 HWW 410 62015 55.5 Myoviridae Pbunavirus Pa193 (NC_050148) 99%/93% SAMN18741769 PSA11 HWW ATCC 14210 48815 44.9 Podoviridae - PA11 (NC_007808) 97%/99% SAMN18741770 PSA13 HWW 427 45731 52.6 Podoviridae Bruynoghevirus Pa222 (MK837011) 99%/93% SAMN18741771 PSA16 HWW 466 45623 52.5 Podoviridae Bruynoghevirus Pa222 (MK837011) 98%/98% SAMN18741772 PSA20 HWW 639 62296 64.4 Siphoviridae Yuavirus AN14 (KX198613) 95%/98% SAMN18741773 PSA21 HWW 639 62243 64.5 Siphoviridae Yuavirus LKO4 (NC_041934) 96%/97% SAMN18741774 PSA25 HWW 426 64290 55.5 Myoviridae Pbunavirus LBL3 (NC_011165) 99%/95% SAMN18741775 PSA28 HWW 428 48440 58.3 Siphoviridae - PMBT28 (MG641885) 96%/86% SAMN18741776 PSA31 HWW 411 45505 52.5 Podoviridae Bruynoghevirus Pa222 (MK837011) 98%/97% SAMN18741777 PSA34 HWW 427 43749 52.3 Podoviridae Bruynoghevirus Pa222 (MK837011) 98%/98% SAMN18741778 PSA37 HWW 639 45506 52.5 Podoviridae Bruynoghevirus Pa222 (MK837011) 98%/97% SAMN18741779 PSA39 HWW 423 47030 64.2 Siphoviridae Yuavirus LKO4 (NC_041934) 95%/98% SAMN18741780 PSA40 HWW 466 45506 52.5 Podoviridae Bruynoghevirus Pa222 (MK837011) 98%/97% SAMN18741781 575 Table 1. Genome characteristics of P. aeruginosa-targeting bacteriophages.
576 1HWW = Hospital wastewater; 2Most similar phage based on BLAST to the NCBI nr database bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
577 Table 2. Protein-altering mutations identified in phage-resistant P. aeruginosa mutants
Isolate Phage Location1 Mutation Description 639⏀R PSA20 3,133,682 N401I Hypothetical protein 5,758,428 T278P Type IV pilus protein PilB
410⏀R PSA09 2,674,126 V671A 16S rRNA endonuclease CdiA 2,688,288 L5392F 16S rRNA endonuclease CdiA
5,582,688 D279G RfaB-like glycosyltransferase
427⏀R PSA34 2,909,283 E643G Linear gramicidin synthase subunit D lgrD 5,953,386 frameshift +TG dTDP-4-dehydrorhamnose reductase RfbD
ATCC 14210⏀R PSA11 3,871,727 D304S Cbb3-type cytochrome c oxidase subunit CcoN1 4,104,560 frameshift +A Transcriptional activator protein LasR
578 1Genome coordinates in parent P. aeruginosa genome. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
579 Figure Legends
580 Figure 1. Diverse P. aeruginosa clinical isolates used for bacteriophage isolation and screening.
581 (A) Core genome phylogeny of 23 P. aeruginosa isolates used for phage isolation. Isolates were
582 typed for biofilm formation (measured as crystal violet staining intensity), protease production,
583 swimming motility, mucoidy, and prophage abundance. Black squares show the presence of
584 binary phenotypes. (B) Clusters of similar prophages found in the genomes of different P.
585 aeruginosa isolates. Bacterial isolate names are listed inside the nodes of each cluster, and
586 lines connect prophages that share >90% sequence coverage and >90% sequence identity. (C)
587 Prophage abundance in isolates that do (CRISPR+) or do not (CRISPR-) encode functional
588 CRISPR-Cas systems. P-value is from a two-tailed t-test.
589 Figure 2. Genomic similarity among isolated P. aeruginosa bacteriophages and publicly
590 available phage genomes. Phages are organized by family and genus, which are labeled at the
591 top of the figure. Phage genomes were compared with one another using nucleotide BLAST to
592 determine sequence coverage and nucleotide identity for each pairwise comparison. Coverage
593 and identity values were multiplied to calculate the “sequence similarity” for each comparison.
594 Similarity values range from 0-100%, and are shown with red shading (0% = white, 100% =
595 red). Dendrograms at top were generated by Pearson correlation clustering of sequence
596 similarity values across all pairwise comparisons.
597 Figure 3. Infectivity of isolated phages against genetically diverse P. aeruginosa isolates.
598 Bacterial isolates are ordered according to the core genome phylogeny in Figure 1. Infectivity is
599 shown as the log10 titer (PFU/mL) of each phage against each isolate. Boxed white values
600 indicate the P. aeruginosa isolate that each phage was isolated and propagated on. Blue
601 shading corresponds to phage titer, with darker shading indicating higher titer. White shading
602 indicates no phage activity.
603 Figure 4. Phenotypic consequences of phage resistance. (A) Twitching motility differences
604 between isolate 639 and 639⏀R, a phage-resistant mutant raised against phage PSA20 that bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
605 harbors a mutation in the Type IV pilus protein PilB. (B) Ceftazidime (CAZ) susceptibilities of
606 two pairs of wild type parent isolates and corresponding phage-resistant mutants, both of which
607 harbor mutations in genes impacting LPS biosynthesis. (C) Protease production differences
608 between isolate ATCC 14210 and ATCC 14210⏀R, a phage-resistant mutant raised against
609 phage PSA11 that harbors a mutation in the quorum-sensing transcriptional regulator LasR. (D)
610 Pyoverdine production quantified in ATCC 14210 and ATCC 14210⏀R. P-value is from a two-
611 tailed t-test.
612 Figure 5. Phage-mediated killing of P. aeruginosa grown in biofilms in vitro and ex vivo. (A)
613 Bacterial viability measured after P. aeruginosa isolate 427 biofilms were grown in vitro and then
614 treated with either fresh media (Untreated), or with phages PSA07/PB1 or PSA34. (B) Viability
615 after phage PSA07/PB1 treatment of P. aeruginosa isolate 427 biofilms grown on human-
616 derived CF airway epithelial cells. Viable bacteria were quantified as CFU/mL, and phage-
617 treated conditions were compared to the untreated condition using two-tailed t-tests. **P < 0.01,
618 ****P < 0.0001.
619 Figure S1. Genome sequence alignment of similar Bruynoghevirus phages. The genomes of
620 phages PSA31, PSA37, and PSA40 were aligned to one another using Mauve. Grey arrows
621 indicate coding sequences, and pink arrowheads show the location of tRNA genes. Vertical
622 black lines show differences in nucleotide sequence between phage genomes, and horizontal
623 black lines show differences due to nucleotide insertions or deletions. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made A available under aCC-BY-NC-ND 4.0 International license. Isolate Prophages Biofilm Protease Motility Mucoidy 639639 413413 422422 419419 426426 ATCC 15692 Tree scale: 0.001 402 412412 428428 Biofilm Prophages 415415 0.0040 0 423423 0.0570.05 1 418418 2 0.110.10 411 3 41 0.1630.15 4 425425 0.2160.20 5 729729 0.2690.25 6 429429 0.3220.30 410410 0.3750.35 417417 0.4280.40 414414 0.4810.45 401ATCC 14210 0.0.53450 421421 779779 466466 427427
B C 8 421 ATCC_15692 415 410 414 418 * P = 0.03 426 429 428 423 417 779 6 425
419 ATCC_14210
Cluster 1 Cluster 2 Cluster 3 4 phiCTX-like phiCTX-like phiCTX-like
413 639 418 2 Number of prophages 422
639 414 415 ATCC_14210 Cluster 4 Cluster 5 Cluster 6 0 Pf1-like Pf1-like JBD25-like CRISPR+ CRISPR-
Figure 1. Diverse P. aeruginosa clinical isolates used for bacteriophage isolation and screening. (A) Core genome phylogeny of 23 P. aeruginosa isolates used for phage isolation. Isolates were typed for biofilm formation (measured as crystal violet staining intensity), protease production, swimming motility, mucoidy, and prophage abundance. Black squares show the presence of binary phenotypes. (B) Clusters of similar prophages found in the genomes of different P. aeruginosa isolates. Bacterial isolate names are listed inside the nodes of each cluster, and lines connect prophages that share >90% sequence coverage and >90% sequence identity. (C) Prophage abundance in isolates that do (CRISPR+) or do not (CRISPR-) encode functional CRISPR-Cas systems. P-value is from a two-tailed t-test. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Myoviridaeavailable- Siphoviridaeunder aCC-BY-NC-ND- 4.0 International license. Podoviridae- Pbunavirus Yuavirus Siphoviridae Podoviridae Bruynoghevirus
Sequence similarity PSA07PB1 0.000% 100%1.00 id id
id PSA09 Pa193 PSA07PB1 PSA25 LBL3 PSA39 PSA20 PSA21 AN14 LKO4 PSA28 PMBT28 PSA04 JBD44 PSA11 PA11 PSA13 PSA34 PSA16 PSA31 PSA37 PSA40 Pa222 PSA09am_cut PSA09 Pa193 Pa193 PSA07/PB1 PSA07/PB1 PSA25 PSA25 LBL3 LBL3 PSA39 PSA39 PSA20 PSA20 PSA21 PSA21 AN14 AN14 LKO4 LK PSA28 PSA28 PMBT28 PMBT28 PSA04 PSA04 JBD44 JBD44 PSA11 PSA11 PA11 PA11 PSA13 PSA13 PSA34 PSA34 PSA16 PSA16 PSA31 PSA31 PSA37 PSA37 PSA40 PSA40 Pa222 Pa222
Figure. 2 Genomic similarity among isolated P. aeruginosa bacteriophages and publicly available phage genomes. Phages are organized by family and genus, which are labeled at the top of the figure. Phage genomes were compared with one another using nucleotide BLAST to determine sequence coverage and nucleotide identity for each pairwise comparison. Coverage and identity values were multiplied to calculate the “sequence similarity” for each comparison. Similarity values range from 0-100%, and are shown with red shading (0% = white, 100% = red). Dendrograms at top were generated by Pearson correlation clustering of sequence similarity values across all pairwise comparisons. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Myoviridaeavailable under aCC-BY-NC-NDS iphoviridae4.0 International license. Podoviridae PSA07/ PSA09 PB1 PSA25 PSA39 PSA20 PSA21 PSA28 PSA04 PSA11 PSA13 PSA34 PSA16 PSA31 PSA37 PSA40 1 639639 5.8 4.6 6.5 3.2 8.5 2.8 2 413413 3 422422 8.6 7.1 4.0 3.8 3.3 4 419419 2.5 2.8 6.8 7.0 8.5 7.7 7.3 6 426426 5.0 9.2 7.5 3.5 3.0 3.0 5.3 5.8 5.5 4.1 4.8 18 402ATCC 15692 5.0 10.5 7.6 12.5 6.1 4.0 4.3 3.4 5.9 8.4 5.8 4.4 9.8 9.0 16 412412 4.1 10.5 3.3 2.5 2.5 5.0 7.3 5.1 6.5 4.2 4.3 6.8 12 428428 2.8 4.1 3.5 3.8 2.8 3.5 8.8 2.8 8.6 2.5 4.0 5.3 4.1 3.5 2.5 Biofilm 21 415415 6.2 0.004 22 423423 4.3 8.4 9.2 4.5 6.5 3.5 7.5 5.1 7.6 4.8 6.8 4.5 4.5 0.057 23 418418 3.8 7.3 8.4 4.8 5.3 3.2 7.5 5.2 7.3 4.8 5.8 5.0 3.5 2.5 0.11 13 41411 4.0 10.2 3.2 4.3 3.6 3.5 5.0 9.2 8.7 8.8 10.2 8.8 8.1 0.163 19 425425 3.5 5.1 3.8 2.8 3.5 3.2 6.8 5.4 7.1 5.5 0.216 20 729729 0.269 17 429429 3.8 6.5 3.8 4.5 4.5 4.6 4.5 4.8 6.5 7.5 8.8 9.8 6.5 8.1 0.322 5 410410 5.8 6.0 7.2 7.5 7.4 4.3 0.375 8 417417 4.5 9.0 3.0 4.0 3.3 5.5 5.4 5.0 7.2 7.1 4.5 6.2 0.428 7 414414 0.481 11 401ATCC 14210 7.6 3.5 3.1 3.1 8.9 8.4 3.5 3.5 5.5 3.0 2.8 0.534 10 421421 5.4 3.5 6.2 5.8 5.8 9 779779 3.1 6.0 3.4 3.5 7.0 7.5 7.8 8.2 2.5 7.8 14 466466 4.0 4.8 3.5 3.8 3.5 4.1 4.8 4.8 7.8 7.5 8.2 8.4 5.5 7.3 15 427427 4.0 7.5 5.0 4.5 4.5 5.1 4.8 6.7 8.4 8.8 8.5 10.5 7.8 8.1
Figure 3. Infectivity of isolated phages against genetically diverse P. aeruginosa isolates. Bacterial isolates are ordered according to the core genome phylogeny in Figure 1. Infectivity is shown as the log10 titer (PFU/mL) of each phage against each isolate. Boxed white values indicate the P. aeruginosa isolate that each phage was isolated and propagated on. Blue shading corresponds to phage titer, with darker shading indicating higher titer. White shading indicates no phage activity. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made A available under aCC-BY-NC-ND 4.0 InternationalC license.
639 639⏀R (PSA20) ATCC 14210 ATCC 14210 ⏀R (PSA11)
B 512 D
256
128 g/mL)
µ 64
32
CAZ MIC ( 16
8
4 410 410 ϕR 427 427 ϕR (PSA09) (PSA34) Isolate
Figure 4. Phenotypic consequences of phage resistance. (A) Twitching motility differences between isolate 639 and 639⏀R, a phage-resistant mutant raised against phage PSA20 that harbors a mutation in the Type IV pilus protein PilB. (B) Ceftazidime (CAZ) susceptibilities of two pairs of wild type parent isolates and corresponding phage-resistant mutants, both of which harbor mutations in genes impacting LPS biosynthesis. (C) Protease production differences between isolate ATCC 14210 and ATCC 14210⏀R, a phage-resistant mutant raised against phage PSA11 that harbors a mutation in the quorum-sensing transcriptional regulator LasR. (D) Pyoverdine production quantified in ATCC 14210 and ATCC 14210⏀R. P- value is from a two-tailed t-test. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made A 10 available under aCC-BY-NC-NDB 4.010 International license.
8 8
**** 6 6 **** ** (CFU/mL) (CFU/mL) 10 4 10 4 Log Log
2 2
0 0
+PSA34 Untreated Untreated +PSA07/PB1 +PSA07/PB1
Figure 5. Phage-mediated killing of P. aeruginosa grown in biofilms in vitro and ex vivo. (A) Bacterial viability measured after P. aeruginosa isolate 427 biofilms were grown in vitro and then treated with either fresh media (Untreated), or with phages PSA07/PB1 or PSA34. (B) Viability after phage PSA07/PB1 treatment of P. aeruginosa isolate 427 biofilms grown on human-derived CF airway epithelial cells. Viable bacteria were quantified as CFU/mL, and phage-treated conditions were compared to the untreated condition using two-tailed t-tests. **P < 0.01, ****P < 0.0001. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.08.451722; this version posted July 9, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
Figure S1. Genome sequence alignment of similar Bruynoghevirus phages. The genomes of phages PSA31, PSA37, and PSA40 were aligned to one another using Mauve. Grey arrows indicate coding sequences, and pink arrowheads show the location of tRNA genes. Vertical black lines show differences in nucleotide sequence between phage genomes, and horizontal black lines show differences due to nucleotide insertions or deletions.