Synergistic action of phage and antibiotics: parameters to enhance the killing efficacy against mono and dual-species biofilms

antibiotics Article Synergistic Action of Phage and Antibiotics: Parameters to Enhance the Killing Efficacy Against Mono and Dual-Species Biofilms Ergun Akturk 1, Hugo Oliveira 1, Sílvio B. Santos 1, Susana Costa 1, Suleyman Kuyumcu 2, Luís D. R. Melo 1,* and Joana Azeredo 1,* 1LIBRO-Laboratório de Investigação em Biofilmes Rosário Oliveira, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4700-057 Braga, Portugal 2Department of Medical Genetics, Medical Faculty, Sifa University, 35535 Izmir, Turkey *Correspondence: [email protected] (L.D.R.M.); [email protected] (J.A.) Received: 13 June 2019; Accepted: 22 July 2019; Published: 25 July 2019   Abstract: Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens and are commonly found in polymicrobial biofilm-associated diseases, namely chronic wounds. Their co-existence in a biofilm contributes to an increased tolerance of the biofilm to antibiotics. Combined treatments of bacteriophages and antibiotics have shown a promising antibiofilm activity, due to the profound differences in their mechanisms of action. In this study, 48 h old mono and dual-species biofilms were treated with a newly isolated P. aeruginosa infecting phage (EPA1) and seven different antibiotics (gentamicin, kanamycin, tetracycline, chloramphenicol, erythromycin, ciprofloxacin, and meropenem), alone and in simultaneous or sequential combinations. The therapeutic efficacy of the tested antimicrobials was determined. Phage or antibiotics alone had a modest effect in reducing biofilm bacteria. However, when applied simultaneously, a profound improvement in the killing effect was observed. Moreover, an impressive biofilm reduction (below the detection limit) was observed when gentamicin or ciprofloxacin were added sequentially after 6 h of phage treatment. The effect observed does not depend on the type of antibiotic but is influenced by its concentration. Moreover, in dual-species biofilms it was necessary to increase gentamicin concentration to obtain a similar killing effect as occurs in mono-species. Overall, combining phages with antibiotics can be synergistic in reducing the bacterial density in biofilms. However, the concentration of antibiotic and the time of antibiotic application are essential factors that need to be considered in the combined treatments. Keywords: Pseudomonas aeruginosa;Staphylococcus aureus; bacteriophage; dual-species; biofilms; antibiotic; synergy; simultaneous; sequential 1. Introduction Polymicrobial interactions are widespread in many biofilm-associated infections [ 1 ], accounting for a significant higher mortality and considerable high costs to the health-care systems [ 2 , 3 ]. Biofilms are communities of microbial cells adhered to biotic or abiotic surfaces and encased in a self-produced extracellular polymeric matrix that confer protection to the community against adverse environmental conditions and antimicrobials including the presence of antibiotics [ 4 ]. In an established polymicrobial biofilm, the co-existence of different species is a clear advantage for the overall biofilm population. These biofilms very often exhibit improved capabilities and functions compared to single-species ones [ 5 , 6 ], such as enhanced degradation of organic compounds [ 7 ], increased virulence [ 8 ], and increased tolerance against antimicrobials [9]. Pseudomonas aeruginosa and Staphylococcus aureus are versatile bacterial pathogens and common etiological agents of polymicrobial associated infections. These two opportunistic pathogens exhibit Antibiotics 2019,8, 103; doi:10.3390/antibiotics8030103 www.mdpi.com/journal/antibiotics Antibiotics 2019,8, 103 2 of 19 intrinsic and acquired antibiotic resistance [ 10 ]. When co-existing in a biofilm this tolerance largely increases, namely due to the decreased metabolic activity, increased bacterial doubling time, and increased level of mutations and upregulation of efflux pumps [11]. Bacteriophages (or phages), viruses that infect bacteria, are natural antibacterial agents that specifically infect and lyse bacteria. Phages are the most abundant biological entities on our planet and can be used as biocontrol agents targeting bacterial cells either in suspension or in biofilms [ 12 , 13 ]. Due to the phage bacterial host specificity and bacteriolytic activity against antibiotic-resistant strains, phage therapy has been suggested as a valuable approach to control numerous pathogenic bacteria. Phages can penetrate the inner layers of the biofilms and infect dormant cells [ 14 ], which is a clear advantage of phages compared to antibiotics in killing biofilms. Therefore, it has been proposed that phages may be a useful combination with antibiotic treatment [15–17]. Several studies demonstrated the efficiency of phage and antibiotic combinations in planktonic cultures of P. aeruginosa [ 17 , 18 ] and biofilms [ 19 ]. Besides, phages and antibiotics use different mechanisms of action, which make them effective against phage/antibiotic resistance pathogens [ 17 ]. Consequently, antibiotic and phage resistance have a low chance of evolving at the same time and, besides, bacteria resistant to one agent will be taken by the other agent. However, to our knowledge, this type of studies was never assessed on dual-species biofilms. In this study, we report the isolation and characterization of a new Pakpunavirus phage, named vB_PaM_EPA1, and the use of several phage-antibiotic combinations against mono and dual-species biofilms of P. aeruginosa and S. aureus. 2. Results 2.1. Isolation and Characterization of a New P. aeruginosa-Infecting Phage Six clinical strains were used for phage enrichment (Table S1), using raw sewage from Sifa Hospital (Izmir, Turkey) as phage sources. A phage vB_PaM_EPA1 (EPA1) was isolated, and its plaque morphology was characterized by clear and small plaques (0.8 mm in diameter) surrounded by halo rings on the host strain Sifa_Pa_1.5 (Table S1). The morphology of EPA1 particles was observed by Transmission Electron Microscopy (TEM). EPA1 has an icosahedral head with 69 nm in diameter and a contractile tail of 145 × 24 nm. According to Ackermann’s classification [ 20 ], EPA1 belongs to the Caudovirales order and Myoviridae family (Figure 1). Antibiotics 2019, 8, x 2 of 19 intrinsic and acquired antibiotic resistance [10]. When co-existing in a biofilm this tolerance largely increases, namely due to the decreased metabolic activity, increased bacterial doubling time, and increased level of mutations and upregulation of efflux pumps [11]. Bacteriophages (or phages), viruses that infect bacteria, are natural antibacterial agents that specifically infect and lyse bacteria. Phages are the most abundant biological entities on our planet and can be used as biocontrol agents targeting bacterial cells either in suspension or in biofilms [12,13]. Due to the phage bacterial host specificity and bacteriolytic activity against antibioticresistant strains, phage therapy has been suggested as a valuable approach to control numerous pathogenic bacteria. Phages can penetrate the inner layers of the biofilms and infect dormant cells [14], which is a clear advantage of phages compared to antibiotics in killing biofilms. Therefore, it has been proposed that phages may be a useful combination with antibiotic treatment [15–17]. Several studies demonstrated the efficiency of phage and antibiotic combinations in planktonic cultures of P. aeruginosa [17,18] and biofilms [19]. Besides, phages and antibiotics use different mechanisms of action, which make them effective against phage/antibiotic resistance pathogens [17]. Consequently, antibiotic and phage resistance have a low chance of evolving at the same time and, besides, bacteria resistant to one agent will be taken by the other agent. However, to our knowledge, this type of studies was never assessed on dual-species biofilms. In this study, we report the isolation and characterization of a new Pakpunavirus phage, named vB_PaM_EPA1, and the use of several phage-antibiotic combinations against mono and dual-species biofilms of P. aeruginosa and S. aureus. 2. Results 2.1. Isolation and Characterization of a New P. aeruginosa-Infecting Phage Six clinical strains were used for phage enrichment (Table S1), using raw sewage from Sifa Hospital (Izmir, Turkey) as phage sources. A phage vB_PaM_EPA1 (EPA1) was isolated, and its plaque morphology was characterized by clear and small plaques (0.8 mm in diameter) surrounded by halo rings on the host strain Sifa_Pa_1.5 (Table S1). The morphology of EPA1 particles was observed by Transmission Electron Microscopy (TEM). EPA1 has an icosahedral head with 69 nm in diameter and a contractile tail of 145 x 24 nm. According to Ackermann’s classification [20], EPA1 belongs to the Caudovirales order and Myoviridae family (Figure 1). Figure 1. TEM image of P. aeruginosa specific phage EPA1 obtained by negative staining with 2% (w/v) uranyl acetate. Scale bar represents 50 nm. 2.2. Host range, Efficiency of Plating and One-Step Growth Curve In total, seventeen drug-resistant clinical isolates (Table S1) and three reference P. aeruginosa strains were used to determine the host range and the efficiency of plating (EOP) of EPA1 (Table S1). Figure 1. TEM image of P. aeruginosa specific phage EPA1 obtained by negative staining with 2% (w/v) uranyl acetate. Scale bar represents 50 nm. Antibiotics 2019,8, 103 3 of 19 2.2. Host range, Efficiency of Plating and One-Step Growth Curve In total, seventeen drug-resistant clinical isolates (Table S1) and three reference P. aeruginosa strains were used to determine the host range and the efficiency of plating (EOP) of EPA1 (Table S1). EPA1 has a broad spectrum of activity (within the panel of strains used) and was able to propagate on 70% (14 out of 20) of the P. aeruginosa strains with moderate to high EOP. No lysis from without events were observed. Also, no correlation between phage susceptibility and antibiotic resistance was detected (Table S1). Due to the fact that EPA1 propagates better in P. aeruginosa PAO1 strain, we have used this strain to produce the phage for further experiments. Nevertheless, we are aware of the fact that PAO1 encloses filamentous phages that could influence phage production, however no filamentous phages were detected by plating methods or TEM [ 21 ]. One-step growth curve (OSGC) experiments were performed to examine the infection parameters of EPA1. The latent period of EPA1 was around 10 min, and the burst size was approximately of 34 progeny phages per infected cell (Figure S1). 2.3. Genome Analysis of EPA1 EPA1 has a linear double-stranded DNA genome containing 91,394 bp with an average 49.2% GC content. This phage encodes 175 putative CDSs, of which 35 have a putative function, and 140 are considered hypothetical/novel (Table S2). Most predicted gene products exhibit homology to phage known proteins belonging to the Pakpunavirus (ICTV 2015.029a-dB ratification) genus, mostly Pseudomonas phages JG004 (NC_019450.1), PAK_P4 (NC_022986) and vB_PaeM_C2-10_Ab1 (NC_019918). Moreover, seventeen tRNA genes coding for Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Lys, Leu, Met, Phe, Pro, Ser, Thr, Trp and Tyr were found. Regarding regulatory elements, 16 promoters were identified as well as 14 rho-independent terminators. The general characteristics of the phage genome are summarized in Table 1. Whole-genome comparisons through BLASTN show that EPA1 has a high overall nucleotide identity (>90%) with other P. aeruginosa phages, such as JG004 (NC_019450.1), vB_PaeM_SCUT-S2 (MK340761.1) and SRT6 (MH370478.1). EPA1 shares >145 proteins with these phages. Table 1. General features of EPA1 genome. Feature vB_PaM_EPA1 Genome size 91,394 bp G+C content 49,2% Number of predicted CDSs 175 Number of proteins with assigned functions 35 2.4. Characterisation of Mono and Dual-Species Biofilm Models In vitro mono and dual-species biofilms were formed in 24-well polystyrene plates for 48 h, and the number of viable bacteria cells were determined by colony forming unit (CFU) counting. It is well documented that P. aeruginosa inhibits S. aureus proliferation in dual-species biofilms. The reason for the lower density of S. aureus population has been attributed to the toxic effect of P. aeruginosa exoproducts [ 22 ], including LasA protease, 4-hydroxy-2-heptylquinoline-N-oxide (HQNO) [ 23 ], the Pel and Psl products [ 24 ], and phenazines such as pyocyanin [ 25 ]. Therefore, in order to successfully produce dual-species biofilms, biofilm formation has been initiated with an S. aureus cell culture, and 24 h later P. aeruginosa cells were added on the S. aureus biofilm, then incubated for another 24 h. A similar strategy of biofilm formation was also used by DeLeon et al. [ 26 ] where biofilms were initiated with S. aureus, and 48 h later P. aeruginosa cells were added [ 26 ]. Our results showed that the number of viable cells of S. aureus was 3.77 × 10 7 CFU/mL and 1.2 × 10 9 CFU/mL for P. aeruginosa in the mono-species biofilms. Regarding the dual-species biofilms, the concentrations were 1.28 ×107CFU/mL for S. aureus and 2 ×108CFU/mL for P. aeruginosa. Antibiotics 2019,8, 103 4 of 19 2.5. Biofilm Treatments The selected antibiotics (Table 2) and EPA1 were tested individually or in combinations within intact mono, and dual-species 48 h biofilms and treated for 24 h in total. Phage and antibiotics were simultaneously or sequentially added in combined treatments. Twenty-four hours post-treatment, CFUs were enumerated in order to assess the antibiofilm efficacy and to characterize the possible interactions between antimicrobials. Table 2. List of the antibiotics, MIC values of P. aeruginosa and S. aureus planktonic cells and their mechanism of action. Name of Antibiotics P. aeruginosa MIC Values S. aureus MIC Values Mechanism of Action Gentamicin 4 µg/mL 16 µg/mL Protein Synthesis Inhibitors 30S ribosomal subunit Kanamycin 10 µg/mL * Protein Synthesis Inhibitors Tetracycline 8 µg/mL * Protein Synthesis Inhibitors Chloramphenicol 32 µg/mL * Protein Synthesis Inhibitors 50S ribosomal subunit Erythromycin 128 µg/mL * Protein Synthesis Inhibitors Ciprofloxacin <1µg/mL <1µg/mL DNA Synthesis Inhibitor Meropenem 2 µg/mL 2 µg/mL Cell wall Synthesis Inhibitor * These antibiotics were not tested on S. aureus biofilm models. Further, the effects of phage, gentamicin at MIC and phage-gentamicin at MIC combinations (simultaneous and sequential) in mono and dual-species biofilms were also analyzed by confocal laser microscopy (CLSM). For that assessment, fluorescence probes were designed to specifically target differentially both bacterial species. Generally, microscopy analysis corroborated cell counting results. 2.5.1. Antibiotics and Phages Alone cause a Moderate Killing Effect on Biofilms Three antibiotics were selected (gentamicin, ciprofloxacin and meropenem), and their anti-biofilm ability was tested against P. aeruginosa and S. aureus mono-species biofilms. These antibiotics were selected depending on their mechanism of action (Table 2): protein synthesis inhibitor (gentamicin), DNA synthesis inhibitor (ciprofloxacin) and cell wall synthesis inhibitor (meropenem). The killing effect of the antibiotics against P. aeruginosa biofilms, used in different concentrations, ranged from 0.8 to 5 orders-of-magnitude (Figure 2). Regarding S. aureus, no significant reduction in the number of viable cells was observed when antibiotics were applied at their MIC (Figure S2). However, when gentamicin was applied with 8xMIC, the number of viable cells was reduced approximately 1.4 orders-of-magnitude (Figure S2). Additionally, EPA1 was individually tested (at multiplicity of infection, MOI, of 1) on P. aeruginosa biofilms for 6 h and 24 h. The observed reductions were 3.4 and 0.5 orders-of-magnitude, respectively (Figure 2, Figure 3b,c). The best reduction was observed at 6 h post-treatment; after that, P. aeruginosa cells started to regrow (Figure 2). CLSM images corroborated CFUs results. P. aeruginosa biofilms after being challenged for 6 h with EPA1 reduced their thickness from 22.4 µ m to 7.2 µ m, but after 24 h of phage contact an increase in biofilm thickness to 11.7 µm was observed (Figure 3a–c). Antibiotics 2019,8, 103 5 of 19 Antibiotics 2019, 8, x 4 of 19 CFUs were enumerated in order to assess the antibiofilm efficacy and to characterize the possible interactions between antimicrobials. Table 2. List of the antibiotics, MIC values of P. aeruginosa and S. aureus planktonic cells and their mechanism of action. Name of Antibiotics P. aeruginosa MIC Values S. aureus MI C Values Mechanism of Action Gentamicin 4 µg/mL 16 µg/mL Protein Synthesis Inhibitors 30S ribosomal subunit Kanamycin 10 µg/mL * Protein Synthesis Inhibitors Tetracycline 8 µg/mL * Protein Synthesis Inhibitors Chloramphenicol 32 µg/mL * Protein Synthesis Inhibitors 50S ribosomal subunit Erythromycin 128 µg/mL * Protein Synthesis Inhibitors Ciprofloxacin <1 µg/mL <1 µg/mL DNA Synthesis Inhibitor Meropenem 2 µg/mL 2 µg/mL Cell wall Synthesis Inhibitor * These antibiotics were not tested on S. aureus biofilm models. Further, the effects of phage, gentamicin at MIC and phage-gentamicin at MIC combinations (simultaneous and sequential) in mono and dual-species biofilms were also analyzed by confocal laser microscopy (CLSM). For that assessment, fluorescence probes were designed to specifically target differentially both bacterial species. Generally, microscopy analysis corroborated cell counting results. 2.5.1. Antibiotics and Phages Alone cause a Moderate Killing Effect on Biofilms Three antibiotics were selected (gentamicin, ciprofloxacin and meropenem), and their antibiofilm ability was tested against P. aeruginosa and S. aureus mono-species biofilms. These antibiotics were selected depending on their mechanism of action (Table 2): protein synthesis inhibitor (gentamicin), DNA synthesis inhibitor (ciprofloxacin) and cell wall synthesis inhibitor (meropenem). The killing effect of the antibiotics against P. aeruginosa biofilms, used in different concentrations, ranged from 0.8 to 5 orders-of-magnitude (Figure 2). Regarding S. aureus, no significant reduction in the number of viable cells was observed when antibiotics were applied at their MIC (Figure S2). However, when gentamicin was applied with 8xMIC, the number of viable cells was reduced approximately 1.4 orders-of-magnitude (Figure S2). Additionally, EPA1 was individually tested (at multiplicity of infection, MOI, of 1) on P. aeruginosa biofilms for 6 h and 24 h. The observed reductions were 3.4 and 0.5 orders-of-magnitude, respectively (Figure 2, Figure 3b,c). The best reduction was observed at 6 h post-treatment; after that, P. aeruginosa cells started to regrow (Figure 2). CLSM images corroborated CFUs results. P. aeruginosa biofilms after being challenged for 6 h with EPA1 reduced their thickness from 22.4 μm to 7.2 μm, but after 24 h of phage contact an increase in biofilm thickness to 11.7 μm was observed (Figure 3a–c). Figure 2. Treatment of P. aeruginosa PAO1 48 h biofilms with different antimicrobial agents individually or in combinations; phage EPA1 and ( a ) gentamicin; ( b ) ciprofloxacin; and ( c ) meropenem for 24 h. A prefix PHAGE indicates EPA1 in MOI 1, 6 H and 24 Hindicates treatment time period for 6 and 24 h, MIC indicates the dose of antibiotics with 1-time MIC value of P. aeruginosa 8 × MIC indicates the dose of antibiotics with 8-times MIC value of P. aeruginosa, PHAGE +antibiotic indicates simultaneous treatment, and PHAGE 6 H+antibiotics indicates phage was added first then antibiotic was added with 6 h delay. * Under detection limit (<10 2 ). ( ˆ ) Statistical differences between the control and treated biofilms were determined by two-way repeated-measures analysis of variance (ANOVA) with a Tukey’s multiple comparison test. Antibiotics 2019, 8, x 5 of 19 Figure 2. Treatment of P. aeruginosa PAO1 48 h biofilms with different antimicrobial agents individually or in combinations; phage EPA1 and (a) gentamicin; (b) ciprofloxacin; and (c) meropenem for 24 h. A prefix PHAGE indicates EPA1 in MOI 1, 6 H and 24 Hindicates treatment time period for 6 and 24 h, MIC indicates the dose of antibiotics with 1-time MIC value of P. aeruginosa 8 × MIC indicates the dose of antibiotics with 8-times MIC value of P. aeruginosa, PHAGE + antibiotic indicates simultaneous treatment, and PHAGE 6 H+ antibiotics indicates phage was added first then antibiotic was added with 6 h delay. * Under detection limit (<102). (^) Statistical differences between the control and treated biofilms were determined by two-way repeated-measures analysis of variance (ANOVA) with a Tukey’s multiple comparison test. Figure 3. 3D reconstructions of confocal stacks of images of mono-species P. aeruginosa biofilms. (a) Control, (b) 6 h phage treatment, (c) 24 h phage treatment, (d) 24 h Gentamicin treatment, (e) 24 h simultaneous treatment, (f) 24 h sequential treatment. All biofilms were stained with EPA1_TFP (with mCherry) recombinant protein. Scale bar represents 50 μm. 2.5.2. Combined Treatments with Simultaneous Application of Phage and Antibiotics have Synergistic Effects for Low Concentrations of Antibiotics The efficacy of the combinations of phage and antibiotics was also tested on P. aeruginosa monospecies biofilms. These maturated intact biofilms were treated in one of two ways; simultaneously (phage and antibiotic were added at the same time) and sequentially (phage was added first, then antibiotic was added with a delay of 6 h). The combination of phage EPA1 with gentamicin (Figure 3e), ciprofloxacin, or meropenem when applied simultaneously with a lower dose (MIC) resulted in population reductions of 4.7, 4.1 and 2.6 orders-of-magnitudes (Figure 2), respectively. These results show a clear synergistic effect between antimicrobial agents in most cases (Table 3). When the antibiotic concentrations were increased, we 317.13 µ m 22.4 µm CONTROL (a) 317.13 µ m 7.2 µm 6H PHAGE (b) 317.13 µ m 11.7 µm 24H PHAGE (c) 317.13 µ m 8.3 µm 24H GENTAMICIN (d) 317.13 µ m 9.6 µm SIMULTANEOUS (e) 317.13 µm 19.2 µm SEQUENTIAL (f) 50 µm50 µm 50 µm50 µm 50 µm 50 µm Figure 3. 3D reconstructions of confocal stacks of images of mono-species P. aeruginosa biofilms. ( a ) Control, ( b ) 6 h phage treatment, ( c ) 24 h phage treatment, ( d ) 24 h Gentamicin treatment, ( e ) 24 h simultaneous treatment, ( f ) 24 h sequential treatment. All biofilms were stained with EPA1_TFP (with mCherry) recombinant protein. Scale bar represents 50 µm. Antibiotics 2019,8, 103 6 of 19 2.5.2. Combined Treatments with Simultaneous Application of Phage and Antibiotics have Synergistic Effects for Low Concentrations of Antibiotics The efficacy of the combinations of phage and antibiotics was also tested on P. aeruginosa mono-species biofilms. These maturated intact biofilms were treated in one of two ways; simultaneously (phage and antibiotic were added at the same time) and sequentially (phage was added first, then antibiotic was added with a delay of 6 h). The combination of phage EPA1 with gentamicin (Figure 3e), ciprofloxacin, or meropenem when applied simultaneously with a lower dose (MIC) resulted in population reductions of 4.7, 4.1 and 2.6 orders-of-magnitudes (Figure 2), respectively. These results show a clear synergistic effect between antimicrobial agents in most cases (Table 3). When the antibiotic concentrations were increased, we were expecting an increase in the killing efficacy of simultaneous combined treatments, however, interestingly 8 × MIC did not increased the overall biofilm killing (in certain cases, we observed an antagonistic effect) (Table 3). Table 3. General overview of the efficacy of combined treatments in 48 h P. aeruginosa mono-species biofilm. Synergistic—the biofilm reduction using phage-antibiotic combinations is greater than the sum of their individual treatments. Additive—the biofilm reduction using phage-antibiotic combination is similar to the sum of their individual treatments. Antagonistic—the biofilm reduction using phage-antibiotic combinations is lower than the sum of their individual treatments. Treatments Gentamicin Ciprofloxacin Meropenem Simultaneously MIC Synergistic Synergistic Synergistic Simultaneously 8 MIC Additive Antagonistic Antagonistic Sequentially MIC Synergistic Synergistic Synergistic Sequentially 8 MIC Synergistic Synergistic Antagonistic 2.5.3. Antibiotics that Target Protein and DNA Synthesis Mechanisms Interfere with Phage Replication In order to understand why increasing the antibiotic concentration did not lead to an increased killing activity, we tested the effect of the antibiotics on phage replication. Phage titer was enumerated after 24 h of simultaneous treatment and compared with the control. Unsurprisingly, the titer of phages, when combined with gentamicin and ciprofloxacin was significantly lower than the titer of phages in control samples (Figure S3). Conversely and as expected, the phage replication was not affected by the presence of meropenem. This antibiotic is affecting bacteria cell wall synthesis and thus does not interfere in phage replication. 2.5.4. Combined Treatments with Sequential Application of Phage and Antibiotics have a better Killing Efficacy than when Applied Simultaneously The fact that protein and DNA synthesis inhibitors interfere with phage replication, led us to assess a sequential treatment in which the phage was applied first and six hours later the antibiotic. This six hour period was chosen based on previous biofilm/phage interaction studies that refer that after six hours of phage interaction, a regrowth of phage-resistant phenotypes is observed [ 27 ]. Our CFU and CLSM results have also corroborated this phenomenon (Figures 2a and 3b,c). The same phage-drug combinations tested before were applied in sequential treatments. The results showed an almost eradication of the biofilm with gentamicin (MIC, 8 × MIC) (Figure 3f) and ciprofloxacin (8xMIC). Besides, other combinations with ciprofloxacin or meropenem (with MIC) also showed an increased killing effect, 4.7 and 2.8 orders-of-magnitudes, respectively. In accordance, increasing the antibiotic concentration of meropenem in sequential treatment (to 8 × MIC), resulted in an antagonistic effect (3.7 orders-of-magnitudes), contrarily to what was observed for the other antibiotics (Figure 2). CLSM results also confirmed that almost all biofilm was eradicated except a cluster (Figure 3e). To understand the impact of antimicrobial application order in sequential interaction, the same combinations were applied in the reverse order. Gentamicin was applied first, and then Antibiotics 2019,8, 103 7 of 19 phage was applied six hours after. The collected data showed that killing efficacies of combinations were reduced when gentamicin MIC and 8 × MIC were applied first, with reductions of 2.5 and 3.6 orders-of-magnitude, respectively (Figure S4). The data suggest that biofilm exposure to phage prior to antibiotics is more effective than simultaneous treatment in eliminating biofilm-associated cells. Considering the overall results, when gentamicin was administered at MIC sequentially after six hours of phage addition, it almost eradicated biofilms (Figure 3f). 2.5.5. The Phage Killing Efficacy with the Sequential Treatment of Phage and Gentamicin cannot be Extrapolated to other Protein Synthesis Inhibitors An impressive biofilm biomass reduction was observed with a protein synthesis inhibitor (gentamicin) at MIC. To understand if this effect can be extrapolated to other antibiotics of the same class, we also tested kanamycin, tetracycline, erythromycin and chloramphenicol (Table 3), in simultaneous and sequential combinations. Contrarily to what we were expecting, the effect observed for gentamicin was not reproduced with the other tested antibiotics (Figure 4). In fact, those antibiotics alone had a low to moderate effect against biofilms, lower than 3 orders-of-magnitude in the overall biomass reduction. Gentamicin alone caused ten times more biomass damage, which could be one of the reasons for the better performance of sequential treatments with gentamicin compared to the other antibiotics. Antibiotics 2019, 8, x 7 of 19 An impressive biofilm biomass reduction was observed with a protein synthesis inhibitor (gentamicin) at MIC. To understand if this effect can be extrapolated to other antibiotics of the same class, we also tested kanamycin, tetracycline, erythromycin and chloramphenicol (Table 3), in simultaneous and sequential combinations. Contrarily to what we were expecting, the effect observed for gentamicin was not reproduced with the other tested antibiotics (Figure 4). In fact, those antibiotics alone had a low to moderate effect against biofilms, lower than 3 orders-of-magnitude in the overall biomass reduction. Gentamicin alone caused ten times more biomass damage, which could be one of the reasons for the better performance of sequential treatments with gentamicin compared to the other antibiotics. Figure 4. Treatment of P. aeruginosa PAO1 48 h biofilms with protein synthesis inhibitor antimicrobial agents individually or in combinations for 24 h. (a) Kanamycin is a 30S protein synthesis inhibitor; (b) Tetracycline is a 30S protein synthesis inhibitor; (c) Erythromycin is a 50S protein synthesis inhibitor; (d) Chloramphenicol is a 50Ss protein synthesis inhibitor. A prefix PHAGE indicates EPA1 in MOI 1, 6 H and 24 H indicates treatment time period for 6 and 24 h, MIC indicates the dose of antibiotics with 1-time MIC value of P. aeruginosa, PHAGE + antibiotic indicates simultaneous treatment and PHAGE 6 H + antibiotics indicates that phage was added first, then antibiotic was added with 6 h delay. (^) Statistical differences between the control and treated biofilms were determined by two-way repeated-measures analysis of variance (ANOVA) with a Tukey’s multiple comparison test. 2.5.6. The Efficacy of Sequential Antibiofilm Treatments is Dependent on the Antibiotic Concentration We also investigated the effect of the different gentamicin concentrations on the simultaneous and sequential treatment efficacy (Figure 5). A direct correlation was observed between the concentration of gentamicin and the biofilm killing efficacy. An almost complete biofilm eradication (below the detection limits) was observed only when antibiotic concentrations were equal or above the MIC (Table S1). Figure 4. Treatment of P. aeruginosa PAO1 48 h biofilms with protein synthesis inhibitor antimicrobial agents individually or in combinations for 24 h. ( a ) Kanamycin is a 30S protein synthesis inhibitor; ( b ) Tetracycline is a 30S protein synthesis inhibitor; ( c ) Erythromycin is a 50S protein synthesis inhibitor; ( d ) Chloramphenicol is a 50Ss protein synthesis inhibitor. A prefix PHAGE indicates EPA1 in MOI 1, 6 H and 24 H indicates treatment time period for 6 and 24 h, MIC indicates the dose of antibiotics with 1-time MIC value of P. aeruginosa, PHAGE +antibiotic indicates simultaneous treatment and PHAGE 6 H +antibiotics indicates that phage was added first, then antibiotic was added with 6 h delay. ( ˆ ) Statistical differences between the control and treated biofilms were determined by two-way repeated-measures analysis of variance (ANOVA) with a Tukey’s multiple comparison test. 2.5.6. The Efficacy of Sequential Antibiofilm Treatments is Dependent on the Antibiotic Concentration We also investigated the effect of the different gentamicin concentrations on the simultaneous and sequential treatment efficacy (Figure 5). A direct correlation was observed between the concentration of gentamicin and the biofilm killing efficacy. An almost complete biofilm eradication (below the detection limits) was observed only when antibiotic concentrations were equal or above the MIC (Table S1). Antibiotics 2019,8, 103 8 of 19 Antibiotics 2019, 8, x 8 of 19 Figure 5. Treatment of P. aeruginosa PAO1 48 h biofilms with gentamicin at different concentrations. A prefix PHAGE indicates EPA1 in MOI 1, 1/2 MIC indicates the dose of antibiotics with 1/2× MIC value, MIC indicates the dose of antibiotics with 1× MIC value of P. aeruginosa, 2 MIC indicates the dose of antibiotics with 2× MIC value of P. aeruginosa, 8 MIC indicates the dose of antibiotics with 8× MIC value of P. aeruginosa, PHAGE + antibiotic indicates simultaneous treatment and PHAGE 6 H + antibiotics indicates phage was added first then antibiotic was added with 6 h delay. * Under detection limit (<10 2 ). (^) Statistical differences between the control and treated biofilms were determined by two-way repeated-measures analysis of variance (ANOVA) with a Tukey’s multiple comparison test. 2.5.7. Sequential Application of Phages and Gentamicin have a great Antibiofilm Effect in DualSpecies Biofilms The killing capacity of gentamicin (MIC) and EPA1 was tested individually and in combination (simultaneous and sequential treatments) in a dual-species biofilm model comprising P. aeruginosa and S. aureus (Figure 6). Intact biofilms were grown for 48 h and treated for 24 h in total. In the control, it was possible to observe a predominance of P. aeruginosa (1.4 × 10 9 CFU/mL), in comparison with S. aureus (2.3 × 10 5 CFU/mL) (Figure 7). Although S. aureus was the first colonizer, CLSM images indicate that both species were randomly distributed throughout the biofilm 3D structures (Figure 6). Figure 5. Treatment of P. aeruginosa PAO1 48 h biofilms with gentamicin at different concentrations. A prefix PHAGE indicates EPA1 in MOI 1, 1/2 MIC indicates the dose of antibiotics with 1/2 × MIC value, MIC indicates the dose of antibiotics with 1 × MIC value of P. aeruginosa, 2 MIC indicates the dose of antibiotics with 2 × MIC value of P. aeruginosa, 8 MIC indicates the dose of antibiotics with 8 × MIC value of P. aeruginosa, PHAGE +antibiotic indicates simultaneous treatment and PHAGE 6 H + antibiotics indicates phage was added first then antibiotic was added with 6 h delay. * Under detection limit (<10 2 ). ( ˆ ) Statistical differences between the control and treated biofilms were determined by two-way repeated-measures analysis of variance (ANOVA) with a Tukey’s multiple comparison test. 2.5.7. Sequential Application of Phages and Gentamicin have a great Antibiofilm Effect in Dual-Species Biofilms The killing capacity of gentamicin (MIC) and EPA1 was tested individually and in combination (simultaneous and sequential treatments) in a dual-species biofilm model comprising P. aeruginosa and S. aureus (Figure 6). Intact biofilms were grown for 48 h and treated for 24 h in total. In the control, it was possible to observe a predominance of P. aeruginosa (1.4 × 10 9 CFU/mL), in comparison with S. aureus (2.3 × 10 5 CFU/mL) (Figure 7). Although S. aureus was the first colonizer, CLSM images indicate that both species were randomly distributed throughout the biofilm 3D structures (Figure 6). The individual treatments with gentamicin with MIC and 8 × MIC resulted in a significant reduction of approximately 3.3 orders-of-magnitude and 4.6 orders-of-magnitude of P. aeruginosa cells, respectively. Phage treatment was less effective than gentamicin, resulting in a reduction of 0.7 orders-of-magnitude of P. aeruginosa cells (Figure 8b). None of the individual treatments showed a significant impact on the S. aureus population (Figure 7). Antibiotics 2019,8, 103 9 of 19 Antibiotics 2019, 8, x 9 of 19 Figure 6. 3D reconstructions of confocal stacks of images of dual-species of P. aeruginosa and S. aureus biofilms. (a) 48 h old intact biofilms were stained by using recombinant proteins, LM12_AMI-SH3 (with GFP) specific for S. aureus and (b)EPA1_TFP (with mCherry) specific for P. aeruginosa. (c) 48 h old intact biofilms were stained by using both recombinant proteins. Scale bar represents 50 μm. Figure 7. Treatment of 48 hours dual-species biofilm. (a) P. aeruginosa number of viable cells. (b) S. aureus number of viable cells. A prefix PHAGE indicates EPA1 in MOI 1, 6 H and 24 H indicates treatment time period for 6 and 24 h, MIC indicates the dose of antibiotics with 1× MIC value of P. aeruginosa, 8 MIC indicates the dose of antibiotics with 8× MIC value of P. aeruginosa, PHAGE + antibiotic indicates simultaneous treatment and PHAGE 6 H + antibiotics indicates phage was added first then antibiotic was added with 6 h delay. * Under detection limit (<10 2 ). (^) Statistical differences between the control and treated biofilms. (#) Statistical differences between the simultaneously and sequentially treated biofilms. Statistical differences were determined by two-way repeated-measures analysis of variance (ANOVA) with a Tukey’s multiple comparison test. The individual treatments with gentamicin with MIC and 8× MIC resulted in a significant reduction of approximately 3.3 orders-of-magnitude and 4.6 orders-of-magnitude of P. aeruginosa cells, respectively. Phage treatment was less effective than gentamicin, resulting in a reduction of 0.7 317.13 µ m 19.5 µm GREEN FILTER (a) 317.13 µ m RED FILTER (b) 317.13 µ m MIX (c) Figure 6. 3D reconstructions of confocal stacks of images of dual-species of P. aeruginosa and S. aureus biofilms. ( a ) 48 h old intact biofilms were stained by using recombinant proteins, LM12_AMI-SH3 (with GFP) specific for S. aureus and ( b )EPA1_TFP (with mCherry) specific for P. aeruginosa. ( c ) 48 h old intact biofilms were stained by using both recombinant proteins. Scale bar represents 50 µm. Antibiotics 2019, 8, x 9 of 19 Figure 6. 3D reconstructions of confocal stacks of images of dual-species of P. aeruginosa and S. aureus biofilms. (a) 48 h old intact biofilms were stained by using recombinant proteins, LM12_AMI-SH3 (with GFP) specific for S. aureus and (b)EPA1_TFP (with mCherry) specific for P. aeruginosa. (c) 48 h old intact biofilms were stained by using both recombinant proteins. Scale bar represents 50 μm. Figure 7. Treatment of 48 hours dual-species biofilm. (a) P. aeruginosa number of viable cells. (b) S. aureus number of viable cells. A prefix PHAGE indicates EPA1 in MOI 1, 6 H and 24 H indicates treatment time period for 6 and 24 h, MIC indicates the dose of antibiotics with 1× MIC value of P. aeruginosa, 8 MIC indicates the dose of antibiotics with 8× MIC value of P. aeruginosa, PHAGE + antibiotic indicates simultaneous treatment and PHAGE 6 H + antibiotics indicates phage was added first then antibiotic was added with 6 h delay. * Under detection limit (<10 2 ). (^) Statistical differences between the control and treated biofilms. (#) Statistical differences between the simultaneously and sequentially treated biofilms. Statistical differences were determined by two-way repeated-measures analysis of variance (ANOVA) with a Tukey’s multiple comparison test. The individual treatments with gentamicin with MIC and 8× MIC resulted in a significant reduction of approximately 3.3 orders-of-magnitude and 4.6 orders-of-magnitude of P. aeruginosa cells, respectively. Phage treatment was less effective than gentamicin, resulting in a reduction of 0.7 317.13 µ m 19.5 µm GREEN FILTER (a) 317.13 µ m RED FILTER (b) 317.13 µ m MIX (c) Figure 7. Treatment of 48 h dual-species biofilm. ( a) P. aeruginosa number of viable cells. ( b) S. aureus number of viable cells. A prefix PHAGE indicates EPA1 in MOI 1, 6 H and 24 H indicates treatment time period for 6 and 24 h, MIC indicates the dose of antibiotics with 1 × MIC value of P. aeruginosa, 8 MIC indicates the dose of antibiotics with 8 × MIC value of P. aeruginosa, PHAGE +antibiotic indicates simultaneous treatment and PHAGE 6 H +antibiotics indicates phage was added first then antibiotic was added with 6 h delay. * Under detection limit (<10 2 ). ( ˆ ) Statistical differences between the control and treated biofilms. (#) Statistical differences between the simultaneously and sequentially treated biofilms. Statistical differences were determined by two-way repeated-measures analysis of variance (ANOVA) with a Tukey’s multiple comparison test. Antibiotics 2019,8, 103 16 of 19 4.11. Statistical Analysis The results of assays were compared using two-way analysis of variance (ANOVA) by applying the Tukey’s multiple comparisons test using Prism 6 (GraphPad, La Jolla, CA, USA). Means and standard deviations (SD) were calculated with the software. Differences among conditions were considered statistically significant when p<0.001. 4.12. Nucleotide Sequence Accession Number The genome sequence of Pseudomonas phage vB_PaM_EPA1 was deposited in the GenBank database under the accession number MN013356. Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6382/8/3/103/s1, Figure S1. One-step growth curve of phage vB_PaM_EPA1 in P. aeruginosa Sifa_Pa_1.5 at 37 ◦ C. Shown are the PFU per infected cell; Figure S2. Treatment of S. aureus 48 h biofilms population with different antimicrobial agents (gentamicin, ciprofloxacin and meropenem) for 24 h; Figure S3. The effect of the antibiotics (gentamicin, ciprofloxacin and meropenem) on phage replication for 24 h post-treatment; Figure S4. Treatment of P. aeruginosa PAO1 48 h biofilms population with reverse sequential combinations. Table S1. Bacterial strains feature and susceptibility to phage EPA1; Table S2. The genome annotation of phage vB_PaM_EPA1. Author Contributions: J.A., L.M. and E.A. conceived and designed the experiments; E.A. and S.C. performed the experiments; E.A., L.M., S.S., H.O., S.K. and J.A. analyzed the data; E.A. wrote the paper. The overall editing was performed by L.M. and J.A. All authors read and approved the final manuscript. Funding: This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684) and the Project PTDC/BBB-BSS/6471/2014 (POCI-01-0145-FEDER-016678). This work was also supported by BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Ergun Akturk acknowledges FCT for grant PD/BD/135254/2017. Conflicts of Interest: The authors declare no conflict of interest. References 1. Røder, H.L.; Sørensen, S.J.; Burmølle, M. Studying Bacterial Multispecies Biofilms: Where to Start? Trends Microbiol. 2016,24, 503–513. [CrossRef] [PubMed] 2. Wolcott, R.D.; Rhoads, D.D.; Bennett, M.E.; Wolcott, B.M.; Gogokhia, L.; Costerton, J.W.; Dowd, S.E. Chronic wounds and the medical biofilm paradigm. J. Wound Care 2014,19, 45–53. [CrossRef] [PubMed] 3. 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Notes 2012 ,5, 244. [CrossRef] [PubMed] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).