Isolation and Characterization of a Myoviridae phage that lyses two Escherichia coli pathotypes

The emergence of pathogenic bacteria with multiple drug resistances (MDR) and the detrimental side effects to the microbiome from the overuse of antibiotics pose significant risks to human health. Bacteriophage (phage) can be an alternative or supplemental therapy to combat MDR infections. Phage can be isolated from environmental reservoirs, such as wastewater, and used to target desired hosts, including MDR bacterial pathogens. In this study, we isolate a Myoviridae phage from Portland, Oregon, wastewater named Escherichia virus PDX that lyses enteropathogenic (EPEC) and enteroaggregative (EAEC) E. coli strains. Host susceptibility assays, transmission electron microscopy, and genomic analysis confirmed the bacteriolytic activity of PDX against these E. coli pathogens, while time-kill assays demonstrate that PDX inhibited growth of both pathotypes in a dose-dependent manner. Metagenomic sequencing of cultured fecal bacteria treated with PDX revealed that alpha diversity of propagated fecal samples was not reduced the presence of the PDX phage.

for administration of ListShield™, EcoShield™ and SalmoFresh™ for treating ready-to-96 eat food to prevent foodborne illness (http://www.intralytix.com/index.php?page=faq). 97 Bacteriophage are ubiquitous in the environment, and sewage wastewater is a 98 rich resource for isolating potentially therapeutic phage. For phage isolation we used a 99 sequential scheme beginning with a laboratory strain of E. coli with sewage overlays 100 (see Figure 1). We show that the lytic PDX phage lyses pathogenic strains of EPEC and 101 a clinical isolate of EAEC. Our results indicate that the presence of PDX phage did not 102 correlate with reduction of the alpha diversity of cultured normal human feces by 16S 103 PFU/mL when propagated on EAEC. As a negative control, the phage was unable to 119 lyse a strain of Bacillus subtilis (ATCC #6051). To test how broadly PDX recognized 120 EPEC pathogens we found that the phage could lyse 9 and form plaques on 5 out of 20 121 clinical, diarrheal isolates from children living in the Seattle area (Table 1; (14)). Thus, 122 the PDX phage could be used to target some, but not a comprehensive set, of E. coli 123

pathogens. 124
In order to obtain a presumptive identification of PDX, we employed transmission 125 electron microscopy (TEM). Imagining revealed the phage belonged to the family 126 phages (FastQC Galaxy). We identified 206 putative coding domain sequences (CDS) 188 (PROKKA, ORFfinder), but no toxin-encoding or lysogenic genes, which are 189 characterized as any genes which contained the terms "integrase", "excisionase", 190 "recombinase", and "repressor" were identified in the genome when compared to the 191 NCBI BLAST database. Additionally, in a 20-standard amino acid (aa) search, a total of 192 6 putative tRNAs were predicted with open reading frames (ORFs) ranging from 72-88 193 bp in length (tRNAscan-SE). Virfam software uses the translated amino acid (aa) of 194 CDS from a phage genome as input and searches for homology to other structural 195 phage protein sequences deposited in the Aclame database. Based on the results of 196 the Virfam analysis, the head-neck-tail structure genome organization in PDX belongs 197 to the Neck Type One -Cluster 7 category (Fig. 7A). Neck Type One phage genomes 198 contain the following proteins: portal protein, Adaptor of type 1 (Ad1), Head-closure of 199 type 1(Hc1), Neck protein of type 1(Ne1), and tail-completion of type 1 (Tc1) (Fig. 7B). 200 Cluster 7 phage genomes are reported in Virfam to be strictly myophages with small 201 genome sizes and gene content (61-150 genes). However, it is noteworthy that the 202 genome of PDX contains 206 CDS, which is more than the other phage in this cluster in 203 the current Aclame database (Table 2). Cluster 7 myophages all infect Proteobacteria. 204 Cluster 7 phage genomes are organized such that there is a different gene order 205 compared to the canonical order that is observed in most clusters of the type 1 family 206 (Fig. 7B). 207 By BLAST analysis, the three genomes with the highest whole-genome 208 nucleotide identity match were Escherichia coli O157 typing phage 4 (98%), Escherichia 209 All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint (which . http://dx.doi.org/10.1101/385104 doi: bioRxiv preprint first posted online Aug. 4, 2018; that shares a 97% nucleotide identity with PDX, is strictly lytic against enterotoxigenic 211 (ETEC), and is being researched as a potential treatment of contaminated food in lieu of 212 was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/385104 doi: bioRxiv preprint first posted online Aug. 4, 2018; EPEC and EAEC infections in humans are endemic to underdeveloped regions of the 230 world and are commonly isolated from stool samples of diarrheal patients living in these 231 countries ((1)). The rise in MDR strains of EPEC and EAEC demand an alternative 232 treatment (3), and in this study we identified and characterized a bacteriophage, PDX, 233 and investigated its potential as a therapeutic agent to treat EPEC and EAEC disease. 234 Our findings revealed that PDX is a good candidate for phage therapy based on its 235 efficiency and specificity in killing pathogenic E. coli. Furthermore, the presence of PDX 236 did not correlate with reduced alpha diversity of the microbiota when cultured with 237 normal human feces (Fig. 5B). This is in stark contrast to the up to 40% reduction in gut 238 microbiome alpha diversity observed in patients given ciprofloxacin, a fluoroquinolone 239 commonly prescribed for E. coli infections (23). 240 Phage are generally thought to be present in the same environment as their 241 bacterial hosts. However, we isolated the PDX phage, able to infect two pathotypes, 242 from the wastewater treatment plant located in Portland, Oregon even though infections 243 by EPEC and EAEC bacteria are rare in the Pacific Northwest of the United States. The 244 phage isolated might also replicate in related E. coli host bacteria via the recognition of 245 conserved receptors. However, to the initial point, these pathogens may be more 246 common in the Pacific Northwest than initially thought because PDX was able to form 247 plaques on 5 of 20 EPEC strains, clinical samples from children living in the Seattle 248 area (see Table 1; (14)). 249 In order for phage to be used as an efficacious therapeutic agent they must be 250 strictly lytic. Lysogenic phage, such as phage lambda, transfer bacterial genetic material 251 All rights reserved. No reuse allowed without permission.
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/385104 doi: bioRxiv preprint first posted online Aug. 4, 2018; from one population to another upon infection and integration into the host genome. 252 This can lead to the transfer of antibiotic resistance genes and toxin encoding genes 253 between bacterial hosts (24). Several lines of evidence indicate that PDX is a lytic 254 phage. PDX was classified as a member of the strictly lytic (25) Myoviridae 255 bacteriophage family based on morphological characteristics from TEM imaging (Fig. 2). 256 Bioinformatic analysis of the head-neck structure CDS in the Virfam suite identified PDX 257 as belonging to the Neck Type One -Cluster 7 comprised only of members of the 258 myophage family (Fig. 7). Neither CI nor CI-associated genes that are responsible for 259 lysogeny in phage lambda were identified in the genome of PDX. Lastly, phage 260 induction, by treatment with Mitomycin C, did not induce PDX plaques in mutant 261 derivatives of EPEC and EAEC resistant to PDX infection (data not shown). Thus, we 262 concluded that the PDX phage was a member of the Myoviridae family having a strictly 263 lytic lifestyle. 264 The PDX genome is a midsized with 138,828 bp, compared to the 11,000 to 265 271,000 bp range found within the 547 complete Myoviridae genomes currently in the 266 NCBI database. Additionally, it is noteworthy that PDX has a higher putative CDS count 267 than any of the other members of Cluster 7 category in the Virfam and Aclame 268

databases. 269
One of the dangerous side effects of antibiotics is the drastic disruption of the 270 microbiome, or dysbiosis, due to the conserved nature of the drug targets. The 271 metagenomic analysis of fecal cultures treated with phage suggested that PDX 272 treatment did not significantly alter the community structure of the commensal 273 microbiome in feces cultured with PDX, in contrast to what occurs with antibiotic 274 All rights reserved. No reuse allowed without permission.
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/385104 doi: bioRxiv preprint first posted online Aug. 4, 2018; Enterbacteriaceae family was reduced when comparing the feces and feces + PDX 276 samples in Figure 5. This suggested that some commensal bacteria belonging to this 277 family were targeted by PDX. Even so, bacteria in this family make up a small fraction of 278 human gut microbiomes, in most cases less than 1% of the population, and the overall 279 impact on the microbiota from treatment with PDX is predicted to be minimal. Phage 280 with a limited host range avoid the consequences of non-specific microbiome disruption, 281 and thus offer an alternative to antibiotic treatment against MDR, or even antibiotic 282

sensitive, EPEC and EAEC infections. 283
Future studies should investigate the efficacy of this phage, both in isolation and 284 in combination with other phage (a phage cocktail), in attenuating bacterial infections. 285 Though certainly not exhaustive, we did observe that PDX could form plaques, 286 indicating replication, in 5 out of 20 clinical isolates of EPEC from Northwest children 287 (Table 1). The use of phage cocktails would then allow for the broadening of serotypes 288 and pathotypes that can be targeted by phage therapy. Further, an in vivo approach 289 could utilize mice whose microbiomes have been "humanized", inoculating subjects with announced that UC San Diego will launch The Center for Innovative Phage Applications 297 All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint (which . http://dx.doi.org/10.1101/385104 doi: bioRxiv preprint first posted online Aug. 4, 2018; and Therapeutics (IPATH) to refine treatments and to bring them to market 298 (http://www.sciencemag.org/news/2018/06/can-bacteria-slaying-viruses-defeat-299 antibiotic-resistant-infections-new-us-clinical). They hope to generate libraries of phages 300 and use cocktails for individual patients to treat MDR infections in order to meet FDA 301 approval challenges. Thus, our efforts and those of other researchers will be essential 302 for isolating therapeutic phages to be used against the increasing number of untreatable 303 bacterial infections. 304 305 306 All rights reserved. No reuse allowed without permission.
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. October 1, 2015. The raw sewage was filtered through a 0.2 µm filter (Corning, Corning 315 NY) to remove bacteria. The filtered sewage was then added to host bacteria in liquid 316 culture (Fig. 1A). Phage-host solutions were combined with 0.5% LB soft agar and 317 overlaid on LB agar plates. Plates were then observed for the presence of plaques after 318 overnight growth at 37 o C. Clear, defined plaques of interest were isolated and 319 transferred using a sterilized inoculating needle and agitation in 1 mL solution of lambda 320 diluent (10mM Tris, pH 7.5; 10 mM MgSO4). To obtain a high titer phage preparation, a 321 crude lysate was generated. A clear PDX plaque was isolated and suspended in 1 mL 322 of sterile lambda diluent. The phage suspension was combined with overnight culture of 323 the bacterial host and incubated at 37 o C with shaking at 225 RPM overnight. Next, 324 chloroform was added to kill any remaining bacteria. This solution was centrifuged at 325 4 o C at 4000 x g for 10 minutes to remove bacterial cells. Finally, the supernatant was 326 recovered and treated with chloroform again to kill any remaining bacterial cells. 327 Several rounds of overlay purification were used to ensure lysate purity after 328 sequential host isolation. From the final overlay of phage sequentially isolated from 329 All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint (which . http://dx.doi.org/10.1101/385104 doi: bioRxiv preprint first posted online Aug. 4, 2018; Several clearly defined plaques from the final overlay plate were picked using a sterile 331 inoculating needle and suspended in microcentrifuge tubes containing SM buffer. The 332 phage suspension was incubated at room temperature for 15 minutes to allow the 333 phage to diffuse through the medium, then filtered through a sterile 0.2-μm filter to 334 remove any bacteria that may have been transferred from the overlay plate. A host 335 susceptibility assay was performed as described below to test the ability of each 336 selected plaque to lyse MC4100, EPEC, and EAEC. This process was performed four 337 more times until uniform plaques were observed indicating pure phage suspension. 338 The final phage suspension was then propagated to make a purified high titer 339 lysate. Exponential EAEC with undiluted phage suspension was incubated in LB 340 overnight at 37°C. Bacteria were removed from the culture by centrifugation at 6,055 x 341 g, then the supernatant was sterile filtered with a 0.2 μm filter. The titer of the lysate was 342 determined by plating dilutions (10 -2 -10 -16 ) using the EAEC overlay procedure described 343 previously. After overnight incubation, the plates were observed and the number of 344 plaque forming units per mL (PFU/mL) was calculated. The purified high titer lysate in 345 SM buffer was stored at 4°C. 346 Electron Microscopy. PDX was analyzed using transmission electron microscopy 347 (TEM). Purified high titer lysate was precipitated in 25% PEG 6000-8000 in 2.5M NaCl 348 and stored overnight at 4 o C. Precipitated phage solutions were centrifuged at 4 o C, 4000 349 x g for 10 minutes. The supernatant was removed and the phage pellet was suspended 350 in 40μL of 50 mM Tris, pH 7.4, 10 mM MgSO4. Phage suspension was placed on 351 copper coated formvar grids and negatively stained with 1% uranyl acetate. Samples 352 All rights reserved. No reuse allowed without permission.
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/385104 doi: bioRxiv preprint first posted online Aug. 4, 2018; and the PFU/mL was calculated. The titer over time (120 minutes) was plotted to obtain 376 a one-step growth curve. This experiment was repeated using EPEC and EAEC strains. and 100 µL of purified lysate dilutions in SM buffer. Bacterial cultures were grown to late 382 exponential phase and diluted to 10 6 CFU/mL. PDX lysate was diluted to 10 5 , 10 6 , 10 7 383 or 10 8 PFU/mL, corresponding to MOIs of 0.1, 1, 10, and 100, respectively. Each 384 phage-host combination at specific MOIs was performed in triplicate wells, and each 385 experiment was performed three times. Controls for plate sterility, phage suspension 386 sterility, and bacterial growth without phage addition were also included. To prevent 387 State University Center for Genome Research and Biocomputing for multiplex 423 sequencing analysis using the Illumina MiSeq sequencing platform. The V3-V4 region of 424 the 16S rRNA gene was amplified and used to prepare a dual index library using the 425 Nextera XT library preparation kit. The analysis produced forward and reverse paired 426 end reads of 150 bp at a sequencing depth of 1 million reads. Sequence quality was 427 quantified using FastQC and demultiplexed. was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which . http://dx.doi.org/10.1101/385104 doi: bioRxiv preprint first posted online Aug. 4, 2018; was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
All rights reserved. No reuse allowed without permission.
was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.