Pseudomonas aeruginosa PAO 1 In Vitro Time–Kill Kinetics Using Single Phages and Phage Formulations—Modulating Death, Adaptation, and Resistance

Pseudomonas aeruginosa is responsible for nosocomial and chronic infections in healthcare settings. The major challenge in treating P. aeruginosa-related diseases is its remarkable capacity for antibiotic resistance development. Bacteriophage (phage) therapy is regarded as a possible alternative that has, for years, attracted attention for fighting multidrug-resistant infections. In this work, we characterized five phages showing different lytic spectrums towards clinical isolates. Two of these phages were isolated from the Russian Microgen Sextaphage formulation and belong to the Phikmvviruses, while three Pbunaviruses were isolated from sewage. Different phage formulations for the treatment of P. aeruginosa PAO1 resulted in diversified time–kill outcomes. The best result was obtained with a formulation with all phages, prompting a lower frequency of resistant variants and considerable alterations in cell motility, resulting in a loss of 73.7% in swimming motility and a 79% change in swarming motility. These alterations diminished the virulence of the phage-resisting phenotypes but promoted their growth since most became insensitive to a single or even all phages. However, not all combinations drove to enhanced cell killings due to the competition and loss of receptors. This study highlights that more caution is needed when developing cocktail formulations to maximize phage therapy efficacy. Selecting phages for formulations should consider the emergence of phage-resistant bacteria and whether the formulations are intended for short-term or extended antibacterial application.


Introduction
Pseudomonas aeruginosa is an opportunistic pathogen and a leading cause of severe nosocomial infections [1,2]. P. aeruginosa infections in healthcare settings include pneumonia, surgical, wound, and urinary tract infections and are associated with risk factors such as mechanical ventilation, immunosuppression, catheterization [2][3][4][5]. Effective treatment of P. aeruginosa infections is challenging [6], and multidrug-resistant and extensively drug-resistant P. aeruginosa have increased in prevalence and augmented morbidity and mortality [2,7,8]. P. aeruginosa is listed since 2017 as one of the critical priority pathogens listed by the World Health Organization to encourage research and development into new antibacterials [9].
Bacteriophage (phage) therapy is in use under the umbrella of article §37 (Unproven Interventions in Clinical Practice) of the Helsinki Declaration of Ethical Principles for Medical Research Involving Human Subjects [10,11]. In addition, the compassionate treatment of patients using phages is set out by different regulatory agencies, such as the Food * M: male; F: female. Identification of P. aeruginosa clinical isolates using VITEK2, MicroScan WalkAway as well as MALDI-TOF (for strains isolated after 2017). Antibiograms performed using the Kirby-Bauer method using Müeller-Hinton agar according to CLSI rules (for strains isolated until January 2014) and according to EUCAST afterward using VITEK2 and MicroScan WalkAway. Metallo-beta-lactamases search was done using Etest (imipenem/imipenem + EDTA) and the Kirby-Bauer method using Müeller-Hinton agar. Antibiotics and concentrations tested: AK: amikacin ( Phages' lytic spectra v lowing tests in two urine i 2). In these clinical isolates the outer membrane of the release of progeny phages were utterly insensitive to antibiotic susceptibilities ch up to four antibiotic classe Isolates showing resistance to a particular antibiotic according to the MIC Breakpoint (EUCAST, http://www.eucast.org/clinical_breakpoints/, accessed 20 January 2020).
Phages' lytic spectra varied from 15 to 55%. Lysis from without was perceived following tests in two urine isolates (U572569, I97824) and one blood isolate (I60026) ( Table 2). In these clinical isolates, lysis occurred due to the interaction of multiple phages with the outer membrane of the isolates and not due to a lytic infection cycle and consequent  Four clinical isolates (I499131, I29074, I41151, and U14706) were utterly insensitive to all phages (Table 2). These four did not share similarities in antibiotic susceptibilities changing in resistance towards one antibiotic (aminoglycosides) up to four antibiotic classes (isolate I41151) ( Table 1). Table 2. Lytic spectra against P. aeruginosa PAO1 and different clinical isolates.
Phages' lytic spectra varied from 15 to 55%. Lysis from without was perceived following tests in two urine isolates (U572569, I97824) and one blood isolate (I60026) ( Table  2). In these clinical isolates, lysis occurred due to the interaction of multiple phages with the outer membrane of the isolates and not due to a lytic infection cycle and consequent release of progeny phages. Four clinical isolates (I499131, I29074, I41151, and U14706) were utterly insensitive to all phages (Table 2). These four did not share similarities in antibiotic susceptibilities changing in resistance towards one antibiotic (aminoglycosides) up to four antibiotic classes (isolate I41151) ( Table 1). showing resistance to a particular antibiotic according to the MIC Breakpoint (EUCAST, http://www.eucast.org/clini-cal_breakpoints/, accessed 20 January 2020).
Phages' lytic spectra varied from 15 to 55%. Lysis from without was perceived following tests in two urine isolates (U572569, I97824) and one blood isolate (I60026) ( Table  2). In these clinical isolates, lysis occurred due to the interaction of multiple phages with the outer membrane of the isolates and not due to a lytic infection cycle and consequent release of progeny phages. Four clinical isolates (I499131, I29074, I41151, and U14706) were utterly insensitive to all phages (Table 2). These four did not share similarities in antibiotic susceptibilities changing in resistance towards one antibiotic (aminoglycosides) up to four antibiotic classes (isolate I41151) ( Table 1). Phages were selected were based on differences in the spectrum of activity. Phages SPCB and SPCG, included in the Sextaphage formulation, lysed 50 and 55% of the tested isolates. The spectra showed SPCB's ability to infect C364224, I60026, and I60584, and SPCG's killing isolates I97824 and C80117. SMS12, SMS21, and SMS29 isolated from raw sewage, lysed between 30 and 45%. SMS12 infected U570696 that both Sextaphages did not lyse. SMS21 also killed this isolate and further lysed H73832; however, it could not infect I202628.

Virion Particle and Plaque Morphologies
The characteristics of two phages previously isolated [15] and three additional phages were studied in terms of virion and plaque morphologies ( Figure 1). Phages SPCB and SPCG had short tails resembling members of the Autographiviridae family. On the other hand, phages SMS12, SMS21, and SMS29 had long contractile tails resembling phages of the Myoviridae family.

Virion Particle and Plaque Morphologies
The characteristics of two phages previously isolated [15] and three additional phages were studied in terms of virion and plaque morphologies ( Figure 1). Phages SPCB and SPCG had short tails resembling members of the Autographiviridae family. On the other hand, phages SMS12, SMS21, and SMS29 had long contractile tails resembling phages of the Myoviridae family. Plaque morphologies varied from tiny (1 mm) to big (5 mm), and the plaque + halo (p + h) diameters ranged between 3 and 35 mm ( Figure 2). SPCB and SPCG had considerably larger plaques (p) which did not alter with time, and haloes that increased, since the start (Figure 2A), at average speeds of 0.41 ± 0.24 and 0.43 ± 0.18 cm/day until 168 h, respectively ( Figure 2C). In addition, the lysis zones of SPCB and SPCG had significant numbers of colonies already after 24 h. The haloes of SMS12, SMS21, and SMS29 were smaller (p + h, Figure 2B) and started to increase after 96 h at average speeds of 0.51 ± 0.33, 0.51 ± 0.48, and 0.69 ± 0.35 cm/day ( Figure 2D), respectively. Plaque morphologies varied from tiny (1 mm) to big (5 mm), and the plaque + halo (p + h) diameters ranged between 3 and 35 mm ( Figure 2). SPCB and SPCG had considerably larger plaques (p) which did not alter with time, and haloes that increased, since the start (Figure 2A), at average speeds of 0.41 ± 0.24 and 0.43 ± 0.18 cm/day until 168 h, respectively ( Figure 2C). In addition, the lysis zones of SPCB and SPCG had significant numbers of colonies already after 24 h. The haloes of SMS12, SMS21, and SMS29 were smaller (p + h, Figure 2B) and started to increase after 96 h at average speeds of 0.51 ± 0.33, 0.51 ± 0.48, and 0.69 ± 0.35 cm/day ( Figure 2D), respectively.

One-Step Growth Characteristics
One-step growth experiments with the five phages were performed ( Figure 3).

One-Step Growth Characteristics
One-step growth experiments with the five phages were performed ( Figure 3).

One-Step Growth Characteristics
One-step growth experiments with the five phages were performed ( Figure 3).

Phage Genomes and Comparative Analysis
SPCB and SPCG resembled Phikmvviruses from the Krylovirinae sub-family of the Autographiviridae family. Pairwise identity at the nucleotide level was 97.1%, with minor differences in the regions between ORFs 4-6 of SPCG and ORF 25 of SPCB ( Figure 4A, Table  S1). In addition, both SPCB and SPCG showed homology to phages PT5 (EU056923) and vB_Pae_TbilisiM32 (NC_017865) but at different percentages (Supplementary Tables S2  and S3 and Supplementary Figures S1 and S2).
No transmembrane domains and no tRNAs were present in the genomes, indicating their sole dependence on the host tRNA molecules. Promoter numbers varied between 1 and 3. Rho-independent terminators found no terminators for SPCB, one for SPCG, eight for SMS12, seven for SMS21, and six for SMS29, respectively.

Time-Kill of Single Phage and Phage Cocktail Formulations
Time-kill experiments were performed with single phages or phage formulations ( Figure 5A,B). Time-kill experiments showed that Pbunaviruses (SMS12, SMS21, and SMS29) produced the best antibacterial effect (reducing approximately 4 log10 CFU/mL after 3 h). The Phikmvviruses SPCB and SPCG led to 3 log10 CFU/mL reductions ( Figure 5A). After 3 h post-infection, a growth of P. aeruginosa cells was observed, which was faster for Phikmvviruses than Pbunaviruses. All phages, except SPCB, exhibited after 24 h post-infection significantly lower (p ≤ 0.05) viable cell counts compared to the controls.
Due to the increase in P. aeruginosa after being challenged following a single and a multi-phage approach, surviving cells were recovered post-infection. Their susceptibility to all phages and possible changes in motility were evaluated to understand this growth phenomenon.

Assessment of the Survivor's Susceptibility and Motility
Survivors were isolated following 24 h post-infection and assessed for their suscep- Time-kill experiments showed that Pbunaviruses (SMS12, SMS21, and SMS29) produced the best antibacterial effect (reducing approximately 4 log10 CFU/mL after 3 h). The Phikmvviruses SPCB and SPCG led to 3 log10 CFU/mL reductions ( Figure 5A). After 3 h post-infection, a growth of P. aeruginosa cells was observed, which was faster for Phikmvviruses than Pbunaviruses. All phages, except SPCB, exhibited after 24 h post-infection significantly lower (p ≤ 0.05) viable cell counts compared to the controls.
Due to the increase in P. aeruginosa after being challenged following a single and a multi-phage approach, surviving cells were recovered post-infection. Their suscepti-bility to all phages and possible changes in motility were evaluated to understand this growth phenomenon.

Assessment of the Survivor's Susceptibility and Motility
Survivors were isolated following 24 h post-infection and assessed for their susceptibility to all phages used in this study (Table 3). Surviving cells were challenged with all phages and grouped into 18 specific resistance patterns, where R stands for resistance and S for susceptible to a given phage (Table 3). Even though we registered different susceptibility patterns following phage treatment, some recovered survivors continued to show susceptibility to the phage used but had acquired resistance towards other phages (Table 3). These susceptible survivors are highlighted in green, and an example of this is what was observed with phage SMS12. For example, survivors of SMS12 treatment showing patterns 7, 8, 10, 12, and 15, continued to be susceptible to this phage but had become resistant to one (patterns 12 and 15), two (patterns 8 and 10), or three (pattern 7) phages, respectively. It is also worth mentioning that P. aeruginosa PAO1 is susceptible to all phages, and these replicate to produce progeny. However, after 24 h, colonies obtained from the control samples (not challenged with any phage) presented resistance towards phages SPCB and SPCG (patterns 15-SRSSS (30%), and 8-RRSSS (30%)).  Phages' lytic spectra varied from 15 to 55%. Lysis from without was perceived foling tests in two urine isolates (U572569, I97824) and one blood isolate (I60026) ( Table  In these clinical isolates, lysis occurred due to the interaction of multiple phages with e outer membrane of the isolates and not due to a lytic infection cycle and consequent lease of progeny phages. Four clinical isolates (I499131, I29074, I41151, and U14706) re utterly insensitive to all phages (Table 2). These four did not share similarities in tibiotic susceptibilities changing in resistance towards one antibiotic (aminoglycosides) to four antibiotic classes (isolate I41151) ( Table 1).

Bacterial Isolates
Phages' lytic spectra varied from 15 to 55%. Lysis lowing tests in two urine isolates (U572569, I97824) and 2). In these clinical isolates, lysis occurred due to the int the outer membrane of the isolates and not due to a lyt release of progeny phages. Four clinical isolates (I499 were utterly insensitive to all phages (Table 2). These antibiotic susceptibilities changing in resistance towards up to four antibiotic classes (isolate I41151) ( Table 1). surviving cells that remained susceptible to the phage used in the treatment. When cocktail formulations were used, the value is highlighted in green only if the survivors remained susceptible to all phages present in the specific formulation. * R and S refer to the percentage of survivors showing resistance (R) or susceptibility (S) to a particular phage. 5PCF (phages SPCB + SPCG + SMS12 + SMS21 + SMS29); 4PCF (SPCG + SMS12 + SMS21 + SMS29); 3PCF (SMS12 + SMS21 + SMS29).
In addition to the susceptibilities of surviving cells towards the different phages, survivors were also evaluated for possible changes in motility (Table 4). Several motility differences were registered, particularly considering the predominant swimming and swarming motilities (blue and grey highlighted values). Although the control cells (nonphage challenged) showed changes in susceptibility to phages, 100% of the survivors maintained excellent swimming characteristics and dendritic swarming motility. Additionally, survivors from single SMS21 and SMS29 phage experiments remained mostly good swimmers (87.5 and 79%, respectively), while those following treatment with phages SPCB, SPCG, and SMS12 changed their swimming predominance to nonswimmers. Compared to the non-phage-treated survivors, changes in swarming were only perceived for single phage treatments with SMS12 and SMS29. surviving cells that remained susceptible to the phage used in the treatment. When cocktail formulations were used, the value is highlighted in green only if the survivors remained susceptible to all phages present in the specific formulation. * R and S refer to the percentage of survivors showing resistance (R) or susceptibility (S) to a particular phage. 5PCF (phages SPCB + SPCG + SMS12 + SMS21 + SMS29); 4PCF (SPCG + SMS12 + SMS21 + SMS29); 3PCF (SMS12 + SMS21 + SMS29).
In addition to the susceptibilities of surviving cells towards the different phages, survivors were also evaluated for possible changes in motility (Table 4). Several motility differences were registered, particularly considering the predominant swimming and swarming motilities (blue and grey highlighted values). Although the control cells (nonphage challenged) showed changes in susceptibility to phages, 100% of the survivors maintained excellent swimming characteristics and dendritic swarming motility. Additionally, survivors from single SMS21 and SMS29 phage experiments remained mostly good swimmers (87.5 and 79%, respectively), while those following treatment with phages SPCB, SPCG, and SMS12 changed their swimming predominance to nonswimmers. Compared to the non-phage-treated survivors, changes in swarming were only perceived for single phage treatments with SMS12 and SMS29. The use of phage cocktails also resulted in motility shifts compared to non-phage exposed P. aeruginosa PAO1 (Table 4). Only survivors from SPCB + SPCG remained good swimmers (50%), although in a fairly similar amount to nonswimmers (45.5%). Therefore, the other cocktail formulations will be compared, regarding swimming characteristics, only in terms of the percentage changes in nonswimming survivors. The 5PCF treatment resulted in 73.7% of nonswimmers and a predominant smooth edge swarming (79.0%). The removal of phage SPCB from the 5PCF decreased nonswimmers to 38.4% (4PCF). However, this removal increased nonswarmers from 15.8% (5PCF) to 57.1% (4PCF) ( Table   predominant swimming pattern; Antibiotics 2021, 10, x FOR PEER REVIEW 9 of 17 R surviving P. aeruginosa cells that became resistant; surviving cells that remained susceptible to the phage used in the treatment. When cocktail formulations were used, the value is highlighted in green only if the survivors remained susceptible to all phages present in the specific formulation. * R and S refer to the percentage of survivors showing resistance (R) or susceptibility (S) to a particular phage. 5PCF (phages SPCB + SPCG + SMS12 + SMS21 + SMS29); 4PCF (SPCG + SMS12 + SMS21 + SMS29); 3PCF (SMS12 + SMS21 + SMS29).
In addition to the susceptibilities of surviving cells towards the different phages, survivors were also evaluated for possible changes in motility (Table 4). Several motility differences were registered, particularly considering the predominant swimming and swarming motilities (blue and grey highlighted values). Although the control cells (nonphage challenged) showed changes in susceptibility to phages, 100% of the survivors maintained excellent swimming characteristics and dendritic swarming motility. Additionally, survivors from single SMS21 and SMS29 phage experiments remained mostly good swimmers (87.5 and 79%, respectively), while those following treatment with phages SPCB, SPCG, and SMS12 changed their swimming predominance to nonswimmers. Compared to the non-phage-treated survivors, changes in swarming were only perceived for single phage treatments with SMS12 and SMS29. The use of phage cocktails also resulted in motility shifts compared to non-phage exposed P. aeruginosa PAO1 (Table 4). Only survivors from SPCB + SPCG remained good swimmers (50%), although in a fairly similar amount to nonswimmers (45.5%). Therefore, the other cocktail formulations will be compared, regarding swimming characteristics, only in terms of the percentage changes in nonswimming survivors. The 5PCF treatment resulted in 73.7% of nonswimmers and a predominant smooth edge swarming (79.0%). The removal of phage SPCB from the 5PCF decreased nonswimmers to 38 The use of phage cocktails also resulted in motility shifts compared to non-phage exposed P. aeruginosa PAO1 (Table 4). Only survivors from SPCB + SPCG remained good swimmers (50%), although in a fairly similar amount to nonswimmers (45.5%). Therefore, the other cocktail formulations will be compared, regarding swimming characteristics, only in terms of the percentage changes in nonswimming survivors. The 5PCF treatment resulted in 73.7% of nonswimmers and a predominant smooth edge swarming (79.0%). The removal of phage SPCB from the 5PCF decreased nonswimmers to 38.4% (4PCF). However, this removal increased nonswarmers from 15.8% (5PCF) to 57.1% (4PCF) ( Table 4). The removal of both SPCB and SPCG from the 5PCF caused a decrease in nonswimmers (42.9%, 3PCF) and increased nonswarmers (87.5%, 3PCF).
A few examples of swimming and swarming profiles are present in Figures 6 and 7. In terms of swimming, some survivors had no motility ( Figure 6A) while others presented reduced ( Figure 6B,C), moderate ( Figure 6D-F), and excellent swimming competencies ( Figure 6G-I). A few survivors also produced flares that swam beyond the uniform swimming zone ( Figure 6J,K), and a minor fraction presented, after 48 h, a red/brown pigmented area indicative of pyorubrin production ( Figure 6I). 4). The removal of both SPCB and SPCG from the 5PCF caused a decrease in nonswimmers (42.9%, 3PCF) and increased nonswarmers (87.5%, 3PCF).
A few examples of swimming and swarming profiles are present in Figures 6 and 7. In terms of swimming, some survivors had no motility ( Figure 6A) while others presented reduced ( Figure 6B,C), moderate ( Figure 6D-F), and excellent swimming competencies ( Figure 6G-I). A few survivors also produced flares that swam beyond the uniform swimming zone ( Figure 6J,K), and a minor fraction presented, after 48 h, a red/brown pigmented area indicative of pyorubrin production ( Figure 6I).  Differences in swarming behaviors were perceived, particularly in the swarm zone coverage, tendril formation, and alterations related to the swarm zone edges (Figure 7). 4). The removal of both SPCB and SPCG from the 5PCF caused a decrease in nonswimmers (42.9%, 3PCF) and increased nonswarmers (87.5%, 3PCF).
A few examples of swimming and swarming profiles are present in Figures 6 and 7. In terms of swimming, some survivors had no motility ( Figure 6A) while others presented reduced ( Figure 6B,C), moderate ( Figure 6D-F), and excellent swimming competencies ( Figure 6G-I). A few survivors also produced flares that swam beyond the uniform swimming zone ( Figure 6J,K), and a minor fraction presented, after 48 h, a red/brown pigmented area indicative of pyorubrin production ( Figure 6I).  Differences in swarming behaviors were perceived, particularly in the swarm zone coverage, tendril formation, and alterations related to the swarm zone edges (Figure 7). Differences in swarming behaviors were perceived, particularly in the swarm zone coverage, tendril formation, and alterations related to the swarm zone edges (Figure 7). Some survivors lost their swarming motility ( Figure 7A). Furthermore, most cells presented smooth edges with wandering colonies (Figure 7D-F) or multiple fronts radiating outwards that swarmed fast due to the developed tendrils ( Figure 7I,J). In addition, some survivors showed deep creases connected to the central swarm zone (Figure 7C,H), while others showed suppressors emerging from the central colony as motility flares ( Figure 7B,G).

Discussion
The clinical use of phages has witnessed significant advances after several compassionate reports and the successful healing of antibiotic-resistant infections. However, despite the enthusiasm for phage therapy, phage treatments cause the emergence of phage-insensitive phenotypes, which can compromise the therapeutic outcome. A recent review showed that phage-insensitive variants occurred in 80% of studies targeting the intestinal milieu, 50% of studies using sepsis models, and 75% of humans [15].
This study focuses on the impact of different phage formulations on the killing, survival, and resistance of P. aeruginosa. P. aeruginosa is a member of the challenging ESKAPE pathogens group, which shows an excellent ability to "escape" killing by antibiotics [16][17][18]. The virulence of P. aeruginosa has been attributed to several cell-associated factors such as LPS, flagellum, as well as pilus and non-pilus adhesins, and to exoenzymes or different secretory virulence factors [19].
Single phage application significantly reduced the living cell population, and the best antibacterial efficacy was perceived 3 h post-treatment with the Pbunaviruses ( Figure 5A). Despite Phikmvviruses' (SPCB and SPCG) shorter latent period, this factor did not significantly outweigh the antibacterial performance of other phages. In fact, SPCB and SPCG caused the lowest viable cell reductions and the most rapid increase in survivors. On the other hand, Myoviruses use in the experiments maintained the viable cell counts low until 7 h of treatment.
In terms of combinatory phage experiments, only six phage combinations were tested ( Figure 5B). The selection of the combinations tested relied on the limited antibacterial action of the dual combination of SPCB + SPCG. Different phage formulations gave rise to startling results at the population level. Cell death was much faster after one hour and may hypothetically be due: (i) to lysis-from-without phenomena; or (ii) cooperation between Autographiviridae and Myoviridae, which use different host cell receptors for adsorption (discussed below). Lysis was not further enhanced between 1 and 3 h of treatment ( Figure 5B), as had previously occurred in single-phage treatments ( Figure 5A). The loss of further activity may be due to competition between the access of the phages to the host receptors. Although the multiplicity of infection remains the same, taking into account each phage and number of bacteria present in the culture, the combination of phages in a cocktail can double the number of phages targeting a specific receptor when the two Phikmvviruses or two of the three Pbunaviruses are combined or even triple the phages available for adsorption to a receptor when SMS12, SMS21, and SMS29 are combined. Competition for receptors in the cell wall delays adsorption and concomitantly delays phage lysis [24,25], as recently demonstrated using fluorescently labeled phages [24]. In that work, the authors showed observed that the lysis only derived due to one phage relative to the mixed lysis fluorescence, suggesting a direct or indirect suppression of one of the phages at some point. Phage dominance was due to the blocking of DNA replication through resource sequestration, and the impotence of phage was due to an incomplete ejection of the DNA into the cell.
The selective pressure from phages on their host population can result in potential alterations in phage receptors that may hinder the phage adsorption step [26,27] and change the virulence of the emerged variants [28]. Many have reported lower frequencies of phageresistant mutants using cocktail formulations rather than monotherapy against Klebsiella pneumoniae [29] and Escherichia coli [30,31]. The results presented herein are in agreement with these articles. The 4PCF and 3PCF gave higher percentages of phage-resistant bacteria compared to 5PCF. Nevertheless, this was not universal, since this hypothesis did not apply to all two-phage formulations that were prepared using the same phages used to produce the 3PCF.
Survivors from control experiments became insensitive to two phages, but this can be due to the phenotypic variations of P. aeruginosa PAO1 itself that are known to occur at a relatively high frequency [32].
P. aeruginosa possesses two surface structures, a single polar flagellum (flagella) and a polar type IV pili (TFP), that facilitate its motility [40], and both can serve as phage receptors. Flagella are mostly virulence factors for the establishment, persistence, and inflammatory profile and a common cause of acute and chronic of P. aeruginosa infections [23]. Flagella are not permanent cellular structures; instead, the cell's probability of having a flagellum differs across different growth phases [41]. Bacteria without flagella generally cause less inflammation and mortality [42,43]. The swimming motility of the survivors varied according to the phage(s) used, and higher losses in motility were achieved in treatments with the 5PCF and the SMS12 + SMS21 cocktails, presenting 73.7 and 71.4% of nonswimmers, respectively. In theory, these nonswimmers may be defective in flagella, and, as a positive consequence, these cells will have a decline in virulence and be less prone to form biofilms on surfaces and tissues.
The swarming of P. aeruginosa PAO1 is typically characterized by a dendritic colonial appearance. However, when changes in flagellar quantity and placement or both occur, swarming motility can be compromised. Individual Myoviruses, the 3PCF, and SMS12 + SMS21 revealed the highest losses in swarming motility. Diminished or absent swarming motility can result from a loss of the signal recognition particle-like protein FlhF, resulting in the assembly of flagella at nonpolar locations on the cell resulting in defective swimming and swarming motilities in P. aeruginosa [44]. Additionally, mutations in LPS can influence the exposition of flagella and pili on the bacterial surface, such as that observed with PAO1 ∆waaL mutants which encoded a functional O-antigen ligase, which showed drastic alterations in swimming and twitching motilities [45,46]. This may justify the loss of infectivity of phages in some of these experiments performed with the Myoviruses.

Phage Host Range Determination
Phages were tested against a panel of isolates using the standard spot test, with phages being serially diluted in SM buffer (5.8 g/L NaCl, 2 g/L MgSO4.7H2O, and 50 mL/L of 50 mM Tris/HCl (pH 7.5)) to investigate lysis from within and from without phenomena [49].

Phage Propagation and Titration
Phage amplification was performed using the plate lysis and elution method [50], and phage titrations were performed according to a previously described method [51].

Phage Plaque Morphology and Replication Characteristics
Ten different plaques were analyzed in terms of plaque and halo widths using a high-performance imaging apparatus (Chemi XT4, GBOX-CHEMI-XT4-E, AlphaMetrix Biotech, Rödermark, Germany), coupled with a 4.2 MP imaging 16-bit CCD camera. Phage growth parameters were determined, as previously described [48]. In brief, for single-step experiments, 10 mL of a mid-exponential-phase culture was harvested by centrifugation (7000× g, 5 min, 4 • C) and the pellet resuspended in 5 mL fresh TSB (OD 600 of 1.0). Phages (5 mL) were added at a MOI of 0.001, homogenized and allowed to adsorb for 5 min at room temperature. The samples were centrifuged (7000× g, 5 min, 4 • C) and the pellet resuspended in 10 mL of fresh TSB. Samples were taken during a period of 70 min. The samples were serially 10-fold diluted and plated immediately. Plaque forming units (PFU) were determined following 16 h incubation at 37 • C.

Time-Kill Experiments with Different Formulations
The M26-A document of the Clinical & Laboratory Standards Institute was adopted to carry the time-kill experiments [64]. In brief, P. aeruginosa (1 mL, 5 × 10 8 CFU/mL) was diluted in 9 mL of TSB, and 100 µL of phage (5 × 10 9 PFU/mL) or 100 µL of SM buffer (control) were added. The mixtures were incubated at 37 • C (120 rpm), and samples were taken at 0, 1, 3, 5, 7, and 24 h post-infection. Serial 10-fold dilutions of P. aeruginosa cells were performed in saline containing 10 mM ferrous ammonium sulphate. Three independent experiments conducted in triplicate were performed. After 24 h of each independent time-kill experiment, 25 surviving colonies were isolated, and tested for their susceptibility not only to the phage they had been challenged with but also all other phages. The susceptibility assay was performed according to a previously described protocol [65].

Characterization of the Motility Properties of Survivor Cells
The swimming and swarming motilities of survivors were analyzed as previously described [66,67], and Petri dishes observed using Chemi XT4 coupled with a 4.2 MP imaging 16-bit CCD camera.

Statistical Analysis
Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparison statistical tests, using GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA). At least three independent experiments were performed, and the results are presented as mean ± standard deviation (SD). Differences were considered as statistically different if p ≤ 0.05 (95% confidence interval).

Conclusions
The results of this study show that universal assumptions regarding the decrease of phage-resistant variants using cocktails are not valid for any given phage combination. Many studies found in literature isolate phages, briefly characterize them, and combine a few in cocktails failing to properly identify the host receptors which they target. Therefore, caution is necessary when combining phages from the same genus due to potentially adverse outcomes. Ideally, the phage formulations should combine different genera, more than just the two used in this study, to better understand the phage-phage and phage-bacteria interactions and produce better antibacterial solutions than the commonly available ones.