Isolation and Characterization of Bacteriophages That Infect Citrobacter rodentium, a Model Pathogen for Intestinal Diseases

Enteropathogenic Escherichia coli (EPEC) is a major pathogen for diarrheal diseases among children. Antibiotics, when used appropriately, are effective; however, their overuse and misuse have led to the rise of antibiotic resistance worldwide. Thus, there are renewed efforts into the development of phage therapy as an alternative antibacterial therapy. Because EPEC in vivo models have shortcomings, a surrogate is used to study the mouse pathogen Citrobacter rodentium in animal models. In this study, two new phages CrRp3 and CrRp10, which infect C. rodentium, were isolated and characterized. CrRp3 was found to be a new species within the genus Vectrevirus, and CrRp10 is a new strain within the species Escherichia virus Ime09, in the genus Tequatrovirus. Both phages appear to have independently evolved from E. coli phages, rather than other Citrobacter spp. phages. Neither phage strain carries known genes associated with bacterial virulence, antibiotic resistance, or lysogeny. CrRp3 is more potent, having a 24-fold faster adsorption rate and shorter lytic cycle when compared to the same properties of CrRp10. However, a lysis curve analysis revealed that CrRp10 prevented growth of C. rodentium for 18 h, whereas resistance developed against CrRp3 within 9 h. We also show that hypoxic (5% oxygen) conditions decreased CrRp3 ability to control bacterial densities in culture. In contrast, low oxygen conditions did not affect CrRp10 ability to replicate on C. rodentium. Together, CrRp10 is likely to be the better candidate for future phage therapy investigations.


Introduction
Diarrheal diseases continue to be one of the foremost public health issues globally, responsible for more than 1.6 million deaths each year [1]. While mortality rates have reduced substantially (by 34% among children and by 21% among all people) over the last two decades, the incidence of diarrhea has not reduced nearly as much (10% reduction in children and 6% reduction overall) [1]. Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrhea morbidity and mortality among children < 5 years [1,2]. When used appropriately, antibiotics can reduce the severity and duration of diarrheal diseases; however, the overuse and misuse of antibiotics in the treatment of diarrhea has led to an alarming increase in antibiotic resistance (AMR) globally [1][2][3]. These observations illustrate that diarrhea mortality is largely avoidable and renewed efforts to reduce disease the burden with non-antibiotic strategies are urgently needed. (c) Genome alignment of CrRp3 and its closest taxonomic relative E. coli phage K1-5. Genes are colored according to nucleotide sequence homology, with genes exclusive to CrRp3 labeled red, genes exclusive to K1-5 labeled yellow, and homologous genes with <70% identity labeled blue. At the time of analysis, the closest taxonomic relative of CrRp3 was the E. coli phage K1-5 from the Vectrevirus genus in the recently created family Autographivirinae. The two phages share both gene content and genome organization with 90% (over 76% of the genome) and 77% (over 75% of the genome) pairwise nucleotide identity, respectively (Figure 1c). Nearly half of the CrRp3 gene products have the best hits with K1-5 genes, including the DNA and RNA polymerases, DNA ligase and the major capsid protein, as well as many hypothetical proteins (Table S3). Accordingly, CrRp3 is a tentative new species within the genus Vectrevirus in the family Autographiviridae [58]. For most phage genera, >95% DNA sequence identity is used by the International Committee on Taxonomy of Viruses (ICTV), as the species demarcation criterion [58]. Gene products unique to CrRp3 include the head-tail connector protein, endolysin, tailspike protein, lyase, minor structural protein, and several other proteins with unknown functions (Figure 1c and Table S1).

Morphology and Genome of Phage CrRp10
The second isolated phage, C. rodentium phage CrRp10 (formal name: vB_CroM_CrRp10) displays an elongated (prolate) icosahedral head connected to a long tail covered with a discernable sheath ( Figure 2a). These features are characteristic of T4-like myoviruses. Plaques produced by CrRp10 on ICC180 were of <1 mm 2 ( Figure 2b). This suggests that phage strain has a significant effect on the plaque size, when compared to plaques produced by CrRp3 (Figure 1b vs. Figure 2b).
Genome sequencing showed that CrRp10 has a large circularly permuted dsDNA genome of 171.5 kb with 267 CDSs, and harbors 10 tRNA genes ( Figure 2c and Table 1). Only 133 (~50%) of CDS have putative functions (Table S2). Similar to CrRp3, CrRp10 has a GC content of 35.5%, which is 20% lower than that of C. rodentium. The genome does not carry sequence homology to lysogeny-associated genes, which suggests that CrRp10 is a virulent phage. The genomes also exhibit no sequence homologies to known virulence-associated or antibiotic resistance genes.
At the time of analysis, the closest taxonomic relative of CrRp10 was the E. coli phage Ime09, which belongs to the Tequatrovirus genus in the subfamily Tevenvirinae ( Figure 2c and Table S3). They share significant synteny, with 98% nucleotide identity over the complete length, and thus, CrRp10 is considered a new strain. However, little is known about phage Ime09, with details restricted to genomic analysis [59]. CrRp10 has divergence from Ime09 within its tail fiber gene, with 3% amino acid dissimilarity over 80% of the corresponding protein. Another notable feature of the CrRp10 genome is what appears to be a recombination event, which resulted in the gain of dUTPase with high sequence similarity to that encoded by phage e11/2. Interestingly, phage e11/2 infects the enterohemorrhagic E. coli (EHEC); also an A/E pathogen [60]. Other recombination events appear to have added several putative endonucleases, with high similarity to homologs in other related Enterobacteriaceae phages (Figure 2c and Table S2).

Comparison of Citrobacter Phage Proteins
We performed amino acid (AA) sequence alignments to compare proteins among phages that infect C. rodentium and other Citrobacter species ( Figure S1). Surprisingly, CrRp3 has <40% AA homology to either C. rodentium phages (CR8 and CR44b) or C. freundii phages phiCFP-1 and Sh4, with the exception of homology between a putative lyase (68%) and minor structural protein (75%) of Citrobacter phage CR8 ( Figure S1a and Table S3). Several CrRp10 proteins exhibit low AA homology to the C. rodentium phage Moon, and even less homology with C. freundii phage CfP1 ( Figure S1b and Table S3).  (c) Genome alignment of CrRp10 and its closest taxonomic relative E. coli phage ime09. Genes are colored according to nucleotide sequence homology, with genes exclusive to CrRp10 labeled red, genes exclusive to ime09 labeled yellow, and homologous genes with <70% identity labeled blue.

CrRp3 Is Faster at Infecting a Host Cell, but Has a Smaller Burst Size
With an excess of host cells, the estimated adsorption rates for phages CrRp3 and CrRp10 are 3.5 ± 3.2 × 10 −10 and 8.52 ± 2.8 × 10 −11 mL −1 min −1 , respectively (Table 2). That is, when compared, CrRp3 would 'infect a host'~24x faster than CrRp10. This also suggests that these two phages have different cell surface binding receptors. In a well-mixed culture, the CrRp3 replication cycle (latent period) takes approximately 15 min (Table 2 and Figure S2). CrRp10 however, had a slightly longer latent period of about 17 min. The eclipse periods could not be determined because C. rodentium appears to be resistant to chloroform lysis ( Figure S2). The shorter replication cycle for CrRp3 correlated had a reduced burst size of 43 phage particles per cell, compared to CrRp10 that produced a burst of 85 phage particles (Table 2).

Hypoxia Reduces Lysis at Low MOIs but Not Resistance
Healthy gastrointestinal luminal oxygen levels decreased from 7% (58 mmHg) in the stomach to 0.5% in the colon, and mucosa oxygen levels ranged between 2-6% oxygen [61]. Both CrRp3 and CrRp10 were able to reduce C. rodentium population densities compared to untreated controls at different MOIs under both normoxic (~21% O 2 ) and hypoxic (5% O 2 ) culturing conditions ( Figure 3). Under normoxia, CrRp3 took less time than CrRp10 to reverse bacterial population growth (~2.5 h post treatment), and reduced bacterial density below OD limits of detection (LOD) within 3 h (Figure 3a). For CrRp10, the same results took 3.5 and 5.5 h, respectively ( Figure 3b). Of these two phages, CrRp3 appears to be more 'potent'. Under hypoxia, however, CrRp3 at MOIs <0.01 failed to reduce bacterial density below the LOD, whereas CrRp10 could ( Figure 3). Indeed, hypoxia also dampened exponential bacterial growth ( Figure 3) in the absence of phages, but final cell densities were similar after 18 h. In addition, the regrowth of C. rodentium occurred between 8-9 h after inoculation with CrRp3. In contrast, no observable bacterial regrowth occurred for CrRp10 after 18 h.

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, along with other representative phages against several bacterial strains (Table 3). In addition to their isolation strain of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broader host range, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strain CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, it cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strains tested, were resistant to both CrRp3 and CrRp10. Growth curves of C. rodentium ICC180 infected with CrRp3 (a-e) or CrRp10 (f-j) at MOI ranging between 10-0.001 (solid line) or no phage control (dashed line). Bacteria were growth under either 21% (blue line) or 5% (red line) oxygen and 5% CO 2 conditions. N = 6 per condition.  ange , we tested the host range of the virulent phages CrRp3 and CrRp10, along with other ative phages against several bacterial strains (Table 3). In addition to their isolation strain tium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broader e, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strain . Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, it fect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strains re resistant to both CrRp3 and CrRp10.  Growth curves of C. rodentium ICC180 infected with CrRp3 (a-e) or CrRp10 (f-j) at MOI ranging between 10-0.001 (solid line) or no phage control (dashed line). Bacteria were growth under either 21% (blue line) or 5% (red line) oxygen and 5% CO2 conditions. N = 6 per condition.

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, along with other representative phages against several bacterial strains (Table 3). In addition to their isolation strain of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broader host range, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strain CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, it cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strains tested, were resistant to both CrRp3 and CrRp10.

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, alon representative phages against several bacterial strains (Table 3). In addition to their iso of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a m host range, including K-12 and several pathotypes of E. coli, as well as the E. caro CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad h cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marce tested, were resistant to both CrRp3 and CrRp10.  Figure 3. CrRp3 or CrRp10 lysis curves of C. rodentium under normoxic and hypoxic c Growth curves of C. rodentium ICC180 infected with CrRp3 (a-e) or CrRp10 (f-j) at MO between 10-0.001 (solid line) or no phage control (dashed line). Bacteria were growth un 21% (blue line) or 5% (red line) oxygen and 5% CO2 conditions. N = 6 per condition.

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, alo representative phages against several bacterial strains (Table 3). In addition to their i of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a host range, including K-12 and several pathotypes of E. coli, as well as the E. ca CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia mar tested, were resistant to both CrRp3 and CrRp10. Citrobacter rodentium CrRp3 or CrRp10 lysis curves of C. rodentium under normoxic and hypoxic conditions. th curves of C. rodentium ICC180 infected with CrRp3 (a-e) or CrRp10 (f-j) at MOI ranging een 10-0.001 (solid line) or no phage control (dashed line). Bacteria were growth under either blue line) or 5% (red line) oxygen and 5% CO2 conditions. N = 6 per condition.
ange , we tested the host range of the virulent phages CrRp3 and CrRp10, along with other ative phages against several bacterial strains (Table 3). In addition to their isolation strain tium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broader e, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strain . Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, it fect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strains re resistant to both CrRp3 and CrRp10. Citrobacter rodentium Figure 3. CrRp3 or CrRp10 lysis curves of C. rodentium under normoxic and hypoxic conditions. Growth curves of C. rodentium ICC180 infected with CrRp3 (a-e) or CrRp10 (f-j) at MOI ranging between 10-0.001 (solid line) or no phage control (dashed line). Bacteria were growth under either 21% (blue line) or 5% (red line) oxygen and 5% CO2 conditions. N = 6 per condition.

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, along with othe resentative phages against several bacterial strains (

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, along with oth representative phages against several bacterial strains (Table 3). In addition to their isolation str of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broad host range, including K-12 and several pathotypes of E. coli, as well as the E. carotovora str CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens stra tested, were resistant to both CrRp3 and CrRp10. Table 3. Phage strain host range.

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, along with othe resentative phages against several bacterial strains (Table 3). In addition to their isolation strai . rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broade t range, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strai P2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, not infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strain ed, were resistant to both CrRp3 and CrRp10. Table 3. Phage strain host range.

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, alon representative phages against several bacterial strains (Table 3). In addition to their iso of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a m host range, including K-12 and several pathotypes of E. coli, as well as the E. caro CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad h cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marce tested, were resistant to both CrRp3 and CrRp10. Table 3. Phage strain host range.

Bacteria
Strain CrRp3 or CrRp10 lysis curves of C. rodentium under normoxic and hypoxic conditions. th curves of C. rodentium ICC180 infected with CrRp3 (a-e) or CrRp10 (f-j) at MOI ranging een 10-0.001 (solid line) or no phage control (dashed line). Bacteria were growth under either blue line) or 5% (red line) oxygen and 5% CO2 conditions. N = 6 per condition.
ange , we tested the host range of the virulent phages CrRp3 and CrRp10, along with other ative phages against several bacterial strains (Table 3). In addition to their isolation strain tium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broader e, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strain . Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, it fect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strains re resistant to both CrRp3 and CrRp10.

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, alo representative phages against several bacterial strains (Table 3). In addition to their i of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a host range, including K-12 and several pathotypes of E. coli, as well as the E. ca CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia mar tested, were resistant to both CrRp3 and CrRp10.

Host Range
Next, we tested the host range of the virulent phages CrRp3 and representative phages against several bacterial strains (Table 3). In addit of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp1 host range, including K-12 and several pathotypes of E. coli, as well CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a rela cannot infect C. rodentium. Moreover, most of the E. coli, as well as the tested, were resistant to both CrRp3 and CrRp10. Citrobacter rodentium CrRp3 or CrRp10 lysis curves of C. rodentium under normoxic and hypoxic conditions. th curves of C. rodentium ICC180 infected with CrRp3 (a-e) or CrRp10 (f-j) at MOI ranging een 10-0.001 (solid line) or no phage control (dashed line). Bacteria were growth under either blue line) or 5% (red line) oxygen and 5% CO2 conditions. N = 6 per condition.
ange , we tested the host range of the virulent phages CrRp3 and CrRp10, along with other ative phages against several bacterial strains (Table 3). In addition to their isolation strain tium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broader e, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strain . Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, it fect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strains re resistant to both CrRp3 and CrRp10.  Growth curves of C. rodentium ICC180 infected with CrRp3 (a-e) or CrRp10 (f-j) at MOI ranging between 10-0.001 (solid line) or no phage control (dashed line). Bacteria were growth under either 21% (blue line) or 5% (red line) oxygen and 5% CO2 conditions. N = 6 per condition.
Host Range Next, we tested the host range of the virulent phages CrRp3 and CrRp10, along with othe resentative phages against several bacterial strains (

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp1 representative phages against several bacterial strains (Table 3). In addition to of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 disp host range, including K-12 and several pathotypes of E. coli, as well as the CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serrat tested, were resistant to both CrRp3 and CrRp10. Citrobacter rodentium

Host Range
Next, we tested the host range of the virulent phages CrRp3 and representative phages against several bacterial strains (Table 3). In addit of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp1 host range, including K-12 and several pathotypes of E. coli, as well CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a rela cannot infect C. rodentium. Moreover, most of the E. coli, as well as the tested, were resistant to both CrRp3 and CrRp10. Citrobacter rodentium

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, along wi representative phages against several bacterial strains (Table 3). In addition to their isolatio of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much host range, including K-12 and several pathotypes of E. coli, as well as the E. carotovor CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host r cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens tested, were resistant to both CrRp3 and CrRp10. Citrobacter rodentium

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, alo representative phages against several bacterial strains (Table 3). In addition to their i of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a host range, including K-12 and several pathotypes of E. coli, as well as the E. ca CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia mar tested, were resistant to both CrRp3 and CrRp10. Citrobacter rodentium

Host Range
Next, we tested the host range of the virulent phages CrRp3 and representative phages against several bacterial strains (Table 3). In addit of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp1 host range, including K-12 and several pathotypes of E. coli, as well CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a rela cannot infect C. rodentium. Moreover, most of the E. coli, as well as the tested, were resistant to both CrRp3 and CrRp10. Citrobacter rodentium Viruses 2020, 12, x FOR PEER REVIEW 9 of 18

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, along with other representative phages against several bacterial strains (Table 3). In addition to their isolation strain of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broader host range, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strain CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, it cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strains tested, were resistant to both CrRp3 and CrRp10.

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, along with other representative phages against several bacterial strains (Table 3). In addition to their isolation strain of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broader host range, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strain CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, it cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strains tested, were resistant to both CrRp3 and CrRp10.

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, along with other representative phages against several bacterial strains (Table 3). In addition to their isolation strain of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broader host range, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strain CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, it cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strains tested, were resistant to both CrRp3 and CrRp10.  Host Range Next, we tested the host range of the virulent phages CrRp3 and CrRp10, along with othe resentative phages against several bacterial strains (Table 3). In addition to their isolation strai . rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broade t range, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strai P2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, not infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strain ed, were resistant to both CrRp3 and CrRp10.

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, along with other representative phages against several bacterial strains (Table 3). In addition to their isolation strain of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broader host range, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strain CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, it cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strains tested, were resistant to both CrRp3 and CrRp10.

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp1 representative phages against several bacterial strains (Table 3). In addition to of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 disp host range, including K-12 and several pathotypes of E. coli, as well as the CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serrat tested, were resistant to both CrRp3 and CrRp10. Citrobacter rodentium

Host Range
Next, we tested the host range of the virulent phages CrRp3 and representative phages against several bacterial strains (Table 3). In addit of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp1 host range, including K-12 and several pathotypes of E. coli, as well CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a rela cannot infect C. rodentium. Moreover, most of the E. coli, as well as the tested, were resistant to both CrRp3 and CrRp10. Citrobacter rodentium ange , we tested the host range of the virulent phages CrRp3 and CrRp10, along with other ative phages against several bacterial strains (Table 3). In addition to their isolation strain tium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broader e, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strain . Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, it fect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strains re resistant to both CrRp3 and CrRp10. Citrobacter rodentium Host Range Next, we tested the host range of the virulent phages CrRp3 and CrRp10, along with othe resentative phages against several bacterial strains (Table 3). In addition to their isolation strai . rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broade t range, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strai P2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, not infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strain ed, were resistant to both CrRp3 and CrRp10. Citrobacter rodentium

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, along with other representative phages against several bacterial strains (Table 3). In addition to their isolation strain of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broader host range, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strain CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, it cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strains tested, were resistant to both CrRp3 and CrRp10. Table 3. Phage strain host range.

Bacteria
Strain Host Range Next, we tested the host range of the virulent phages CrRp3 and CrRp10, along with othe resentative phages against several bacterial strains (Table 3). In addition to their isolation strai . rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broade t range, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strai P2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, not infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strain ed, were resistant to both CrRp3 and CrRp10.  ange , we tested the host range of the virulent phages CrRp3 and CrRp10, along with other ative phages against several bacterial strains (Table 3). In addition to their isolation strain tium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broader e, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strain . Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, it fect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strains re resistant to both CrRp3 and CrRp10.

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, alo representative phages against several bacterial strains (Table 3). In addition to their i of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a host range, including K-12 and several pathotypes of E. coli, as well as the E. ca CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia mar tested, were resistant to both CrRp3 and CrRp10. Table 3. Phage strain host range.

Bacteria
Strain

Host Range
Next, we tested the host range of the virulent phages CrRp3 and CrRp10, along wi representative phages against several bacterial strains (Table 3). In addition to their isolatio of C. rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much host range, including K-12 and several pathotypes of E. coli, as well as the E. carotovor CFBP2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host r cannot infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens tested, were resistant to both CrRp3 and CrRp10.  ange , we tested the host range of the virulent phages CrRp3 and CrRp10, along with other ative phages against several bacterial strains (Table 3). In addition to their isolation strain tium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broader e, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strain . Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, it fect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strains re resistant to both CrRp3 and CrRp10.  Host Range Next, we tested the host range of the virulent phages CrRp3 and CrRp10, along with othe resentative phages against several bacterial strains (Table 3). In addition to their isolation strai . rodentium, CrRp3 could infect only the E. coli strain K-12, while CrRp10 displays a much broade t range, including K-12 and several pathotypes of E. coli, as well as the E. carotovora strai P2141. Although the E. coli phage LF82_P10 [46] also exhibits a relatively broad host range, not infect C. rodentium. Moreover, most of the E. coli, as well as the Serratia marcescens strain ed, were resistant to both CrRp3 and CrRp10.  virulent phages CrRp3 and CrRp10, along with other rial strains (Table 3). In addition to their isolation strain . coli strain K-12, while CrRp10 displays a much broader thotypes of E. coli, as well as the E. carotovora strain _P10 [46] also exhibits a relatively broad host range, it of the E. coli, as well as the Serratia marcescens strains p10.
hage strain host range.

Bacteriophages b
Type a CrRp3 CrRp10 LF82_P10 LM33_P1 536_P7 AL505_P2 CLB_P2  (Table 3). In addition to their isolation strain infect only the E. coli strain K-12, while CrRp10 displays a much broader and several pathotypes of E. coli, as well as the E. carotovora strain coli phage LF82_P10 [46] also exhibits a relatively broad host range, it Moreover, most of the E. coli, as well as the Serratia marcescens strains CrRp3 and CrRp10.

Phage Phylogenetic Relationships
To investigate phylogenetic relationships among Citrobacter and Escherichia podoviruses including CrRp3, we constructed a genome-blast distance phylogeny tree using 37 complete genome nucleotide sequences from GenBank ( Figure 5 and Table S4). We found that these phages display the heterogeneous clustering of closely related Autographiviridae into three distinct clades with maximal bootstrap support (Figure 4a). CrRp3 has closer relationships with podoviruses infecting Escherichia (Figure 4b) than those also infecting C. rodentium (CR8 and CR44b) (Figure 4a; see C1 vs. C3). Rather, CR8 and CR44b clusters with some phages that infect the human pathogen C. freundii (SH3 and SH4). Other C. freundii podoviruses (phiCFP1, SH1, and SH2) cluster in clade 2 (Figure 4c), and phage CVT2 branches separately. The latter was not unexpected, because CVT2 was isolated from the gut of termites with an uncharacterized Citrobacter species [51]. To determine the relationship of CrRp10 to other Citrobacter and Escherichia Myoviridae, we constructed a genome-blast distance tree using 69 complete genome sequences from GenBank ( Figure 5 and Table S4), also showing heterogeneous clustering (Figure 5a and Table S3). CrRp10 clusters in a clade with several E. coli myoviruses ( Figure 5). Other myoviruses that infect C. freundii cluster in clades 2-4. Within clade 2 (C2), C. freundii phages Merlin and Moon are further distantly related to 8 Escherichia phages (Figure 5c). In contrast, clade 3 (C3) is almost exclusively composed of C. freundii phages (IME CF2, Miller, CfP1, and Margaery).

Discussion
EPEC, EHEC, and the mouse pathogen C. rodentium, are all attaching/effacing pathogens and causative agents of diarrheal disease. In this study, we isolated and characterized two new phages, CrRp3 and CrRp10, which infect C. rodentium. We show that the podovirus CrRp3 is a tentative new species within the genus Vectrevirus in the family Autographiviridae, while the myovirus CrRp10 is a new strain within the Tequatrovirus genus in the family Myoviridae. Between CrRp3 and CrRp10, only 48% of their combined gene repertoire have assigned putative functions. Of the assignments, we found that neither phage harbor known genes associated with bacterial virulence or antibiotic resistance. The latter is consistent with previous findings that antibiotic resistance genes are rarely carried in phage genomes [62]. In addition, we show that CrRp3 and CrRp10 genomes lack identifiable integrase genes. This implies that both phages carry out strictly lytic replication cycles. Indeed, with more than 50% of genes having an unknown function, these findings emphasize the critical need for significant gene functional studies before any certainty that CrRp3 and CrRp10 do not carry harmful genes.
While these phages expand our knowledge of viral biodiversity, we found that neither CrRp3 nor CrRp10 genomes exhibit nucleotide sequence homology with other C. rodentium phages, including CR8 and CR44b from the genus Caroctavirus [21]. In addition, CrRp3 and CrRp10 are distantly related to phages that infect C. freundii, including members of the genera Moonvirus (Merlin, Miller, Moon) [52][53][54], Mooglevirus (Moogle, Michonne, Mordin) [55,56,63], Tlsvirus (Stevie) [57], and Teetrevirus (phiCFP-1, SH1, SH2, SH3, SH4, SH5) [64,65]. C. freundii can cause a variety of nosocomial acquired extraintestinal human diseases, such as the urinary tract, respiratory tract, and wound infections [66]. Thus, we show that CrRp3 and CrRp10 appear to have independently evolved from closely related E. coli phages, presumably because it was advantageous to expand the host range to infect C. rodentium, and as a result, occupy new niches. Petty et al. showed that the genome of C. rodentium exhibits several features typical of recently passing through an evolutionary bottleneck, including several large-scale genomic rearrangements and functional gene loss in the core genomic regions [67][68][69]. This led the authors to hypothesize that C. rodentium evolved from a human E. coli strain [69]. Our results strengthen this hypothesis by showing that phages that infect C. rodentium appear to have also evolved from phages that infect E. coli. Nonetheless, CrRp3 carries genes that have diverged significantly from presumably ancestral genes of E. coli K1-5-like phage, in particular, gene products responsible for receptor recognition (tail fibers) and cell lysis (endolysin). Interestingly, phage K1-5 exhibits two tail fiber genes, which allow it to be promiscuous between E. coli strains with different capsule compositions [70]. CrRp3 endolysin gene has homology to other Citrobacter phage endolysin genes. This suggests a mosaic genome structure driven by recombination events from diverse viruses. This is consistent with other autographiviruses that exhibit a high genetic identity, structure, and specific RNA polymerase, with the modest differences observed in genes implicated in adaptation to host constraints [71].
Lysis of bacterial cells provides insight into the dynamics between individual phages and their host bacteria. We found that CrRp3 appears to be the more 'potent' virus of the two. First, it was determined that CrRp3 would be 24x faster at infecting a host cell based on adsorption rates compared to CrRp10. Either this could be due to cell surface receptors for CrRp3 outnumbering receptors for CrRp10, or CrRp3 tail fibers have a higher affinity to a shared receptor. Second, in well-mixed cultures, CrRp3 took approximately 2 min less than CrRp10 to complete a single lytic replication cycle. This short cycle time correlated to CrRp3 reversing C. rodentium exponential growth sooner than CrRp10 (Figure 3). CrRp3 also exhibited, on average, a 51% reduced progeny burst compared to CrRp10 s burst size (Table 2). This raises the question of whether shorter replication cycles are favorable to higher phage progeny production to eliminate bacterial infections.
However, CrRp10 was more resilient to resistance. We found that CrRp10 did not allow the population growth of C. rodentium after 18 h (end of study), even at an initial MOI of 0.001 (i.e., 1 virion to 1000 cells) (Figure 3b). In contrast, C. rodentium exhibited regrowth after 9 h of co-incubation with CrRp3. Bacteria thwart phage attack through an arsenal of antiviral mechanisms targeting most steps of the phage replication cycle. The transition from phage-sensitive to phage resistant is often due to spontaneous chromosomal mutations that modify cell surface receptors for the phages [72]. Resistance mutations may be expected to impart a fitness cost because they target important biological functions in the cell [73]. This suggests that the mutation of the specific receptors used by CrRp10 to bind to bacterial hosts imposes much higher fitness costs than resistance to CrRp3 s receptors. Furthermore, second-site compensatory mutations did not lessen or alleviate the fitness costs associated with resistance to CrRp10. In relation to phage therapy, El Haddad et al. found that 7 out of 12 clinical studies confirmed that resistance had emerged during phage treatment [74]. Although the consequences of phage resistance in the clinic are under-studied, they could be the root cause of phage therapy clinical trial failures, for example [75]. Thus, selecting therapeutic agents like phage CrRp10, which was resilient to resistance in vitro, may lead to improvements in phage therapy efficacy.
To the best of our knowledge, this is the first investigation of phage infection under a controlled low-oxygen atmosphere (<10 mmHg); herein described as "physiologic hypoxia". Hypoxia is a common feature during inflammation associated with bacterial infection and the intestinal luminal environment [61,76]. In addition, host intestinal epithelial cells maintain physiologic hypoxia by counter-current blood flow [77]. Enteropathogenic A/E pathogens secrete the virulence protein (effector) NleB, which alters the function of a master regulator of cellular O 2 homeostasis, HIF-1α, thereby increasing O 2 levels between 2-5% for glycolysis [77]. We show that 5% oxygen caused a marked delay in the ability of CrRp3 to create a bacterial population decline compared to infections under normoxia. With the higher concentration of oxygen, the physiologic conditions of the host bacterium may have been at a more favorable metabolic state for a higher productive phage burst or faster lytic cycle completions [78,79]. By contrast, enteric pathogens adapt to oxygen limitations by entering into a metabolically reduced state [80], which was observed as a slightly slower growth rate (Figure 3). The reduced growth rate may have affected phage-bacteria interactions and/or reduced the expression of phage receptors. This contrasts with our knowledge that other phages do not replicate in slow-growing bacteria, for example [81,82]. Our results warrant further investigations into the effects of hypoxia on phage-bacteria interactions.
Another criterion for the selection of therapeutic phages is the spectrum of bacterial species or strains lysed. We found that CrRp3 and CrRp10 exhibit polyvalence in hosts across genera within the Enterobacteriaceae. We show that, while CrRp3 produced plaques on lawns of C. rodentium and the non-pathogenic E. coli strain K12, CrRp10 also produced plaques on eight other E. coli strains (pathogenic and non-pathogenic) and the plant pathogen E. carotovora. In contrast, most phages are confined to a single host species and often to a subset of strains [47,83]. For example, the C. rodentium phage phiCR1 was unable to produce plaques on E. coli [19]. Considering the well-documented, collateral effects of broad-spectrum antibiotics, which are notorious for secondary outcomes such as antibiotic-associated diarrhea [84], a narrow phage spectrum may be advantageous during therapy. However, species specificity comes with inherent constraints. By selecting a phage that is limited to a single species or limited number of strains, treatment is likely to be less effective against multispecies or polystrain infections [85].
The overuse and misuse of antibiotics in the treatment of diarrhea have led to an alarming increase of AMR in diarrheagenic bacteria [1][2][3]. Phage therapy has been effective at reducing E. coli burden in the murine gut with antibiotic pretreatment [5][6][7][8]. Although C. rodentium is widely used as an exemplary in vivo model system for gastrointestinal bacterial diseases [9,86], there are no reports using this species for phage therapy development. Prior to animal modeling, the careful selection of phages for therapeutic applications is especially important. Indeed, it is important to select phages that do not undergo lysogeny and do not carry toxin and antibiotic resistance genes. The selection of phage strains that are resilient to resistance has received little attention, which could be due to the unproven premise that phage cocktails will prevent resistance development [23,87]. Among 12 phage therapy human clinical studies that implemented phage cocktails, 7 cases confirmed the emergence of phage resistance during treatment [74]. For example, Acinetobacter baumannii developed resistance to all eight phages used to treat bacteremia after just 1 week of treatment [88]. Another criterion for phage selection that is lacking exploration is phage infection performance under human physiologic hypoxia conditions. Together, the features of CrRp10 suggest that it could be a promising therapeutic agent in mouse models of diarrheal diseases.