Introduction

Untreated wastewaters that are discharged into the Mediterranean Sea represent a serious threat to human health, both by swimming in polluted water and by the consumption of contaminated seafood (Prieto et al. 2001; Danovaro et al. 2009a, b). Mandated closures of beaches for swimming and closing shellfish harvesting have occurred after wastewater discharge in response to this public health threat, especially along the coasts of North Africa (Kamizoulis and Saliba, 2004).

Wastewater treatment processes include three stages aimed to improve the environmental quality of effluents; these stages are primary, secondary and tertiary treatment. Primary treatment involves mechanical scrapers that separate greases and oils from the wastewater streams. Secondary wastewater treatment engages microorganisms to digest particulate organic matter in the wastewater stream. Tertiary wastewater treatment is a final stage to remove pathogens such as coliforms, nutrients and other pollutants to meet treatment performance criteria as determined by regulations for effluent water quality. Tertiary wastewater treatment is essential for producing clean and high-quality effluent that can be discharged into receiving waters. Effluent standards are addressed in the Sustainable Development Goals. Nevertheless, in some cases, environmental regulations and public health concerns require further performance standards in tertiary treatment. Appropriate choices of all stages of wastewater treatment affect the overall wastewater stream cost and efficacy. Community-degrading microorganisms present in the wastewater stream can be affected by the presence of antibiotics naturally present in wastewater. Although wastewater systems are not specifically designed to remove pharmaceuticals, pharmaceuticals as pollutants are increasingly recognized by the negative environmental impacts on receiving waters. Advanced tertiary treatment methods like activated carbon, ozonation and advanced oxidation have shown efficiency in pharmaceutical removal (Rivera-Utrilla et al. 2013).

Biological treatments in most wastewater plants address the removal of some types of pharmaceuticals; however, antibiotics persist in treated wastewater, inducing bacteria resistance. Growing environmental regulations and public health concerns implemented to achieve the Sustainable Development Goals1 have called for tertiary treatment of wastewater before discharge into receiving waters to remove both pathogens and nutrients that contribute to eutrophication. In the past, chlorination was the most common method to disinfect wastewater. However, it has been shown that chlorine disinfection creates by-products that negatively impact both the environmental and human health (Al-Abri et al. 2019; Tsamba et al. 2020). Tunisia wastewater treatment plants (WWTPs) managers wish to initiate more sustainable and effective practices for advanced wastewater treatment that may include ultra-violet (UV) disinfection to reduce antibiotic-resistant bacteria (Mansour 2018). Some harmful microorganisms can escape UV treatment. For example, P. aeruginosa is a ubiquitous opportunistic pathogen of both plants and humans (Ebrahimi et al. 2017). This pathogen causes respiratory, urinary tract and bloodstream infections in humans (Brizou 1972; Slekovec et al. 2012). P. aeruginosa strains are antibiotic resistance for many common antibiotics, disinfectants and biocides (Pukatzki et al. 2002; Schwartz et al. 2015). P. aeruginosa may produce persistent biofilms (Saidi et al. 2011) as well as show resistance to common antibiotics (Abedon 2020).

Phage therapy is well established in clinical settings to remove pathogenic bacteria; however, the use of phages to remove pathogenic bacteria in a WWTP setting could be a viable alternative to both UV disinfection and chlorination. The culture of phage was accomplished in simple culture medium including only host cells. The easy, fast and economical methods available for phage multiplication are feasible both in industrialized and developing countries. Phages could kill bacteria or/and to impact the dissemination of genes encoding antibiotic resistance. Phage therapy can be used as a preventive measurement against community infection from exposure to wastewater (Cisek et al. 2017). Methods to reduce community infections that may not be treatable with antibiotics are critical moving forward. The cost of WWTP phage therapy is likely significantly lower than the public health costs from any novel community infections from wastewater effluent exposure (Bradbury 2004). Phages are an efficient alternative to eliminate pathogenic bacteria in specific environmental settings (Ackermann and DuBow, 1987; Summers, 2005). Phages present an interesting tool for scientific research given the ease and speed of laboratory phage cultivation (Ackermann 2001; Merril et al. 2003; Labrie et al. 2009). Two categories of bacteriophage are recognized: temperate and virulent. During lytic infection, virulent phages inject their nucleic acid into the host cell following attachment to the cell membrane, and then, they exploit the cellular machinery of the host to synthesize new phage capsule material to complete the lytic cycle with new host cells infections (Withey et al. 2005). In contrast, during lysogenic infection, temperate phage nucleic acid recombines with the host cell genome forming a dormant prophage; this prophage does not induce the lytic cycle in the bacteria cell. In wastewater treatment, only lytic phages were considered for improving water quality since the goal was to kill the bacteria host cell.

This research explores methods to improve wastewater quality via bacteriophage use to reduce antibiotic-resistant pathogens. This study aims (i) to isolate and to identify lytic bacteriophages from wastewater; (ii) to use the phage as a biocontrol agent for P. aeruginosa strain ATCC27853 when added in PWW and SWW; and (iii) to document the impact of phage on the P. aeruginosa strain ATCC 27853 antibiotic resistance for selected antibiotics (e.g., gentamicin, streptomycin, rifamycin, spiramycin and tetracycline).

Material and methods

Bacteriophage host range

The strain P. aeruginosa ATCC 27853 was used in this research. PA25 was isolated from domestic SWW from Charguia pilot, Tunisia. The physicochemical characteristics of the PWW and SWW effluents are presented in Table 1.

Table 1 Physicochemical characteristics of primary wastewater (PWW) and secondary wastewater (SWW) used in these experiments
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Phages isolation followed the protocols outlines in Petrovski et al. (2011). A 50 mL sample of wastewater was added to 50 mL Tryptic Soy Broth (TSB, Difco, medium). One mL of an overnight culture of P. aeruginosa strain ATCC 27853 added to the assay mixture was incubated at 37 °C for 2 h for PA25 multiplication. Following incubation, 2 mL of chloroform was added to the assay mixture to lyse bacteria cells. Vacuum evaporation of solvent was followed by centrifugation at 1800 × g for 15 min to collect cell debris, and then, the supernatant was filtered through 0.22 µm pore-size filters to extract PA25. PA25 suspension was stored at – 20 °C. A volume of 100 µl of the suspension was added to 5 mL of TSB medium previously inoculated with 100 µl of overnight P. aeruginosa ATCC strain 27853 culture and then poured over agar plates and incubated 12 h at 37 °C for plaque formation (Kropinski et al. 2009). A single plaque was picked from the plate and eluted into1 mL of fresh culture of P. aeruginosa ATCC strain 27853. Optical density at 600 nm (OD600) was determined for samples incubated with and without PA25. The mixture was then layered on the agar for plaque formation. Titers of PA25 were determined by plaque-forming units PFU/mL, using the agar overlay method on TSA (Armon and Kott 1993). Serial dilutions were performed, followed by the drop count method (Adams, 1959).

Transmission electron microscopy (TEM)

Electron microscopy (TEM) is an important tool for high-resolution structure determination in applications ranging from condensed matter to biology. Particularly, TEM has provided the fastest diagnostic technique in virology and in phage identification. The major contribution of electron microscopy is the demonstration of the capsid of tailed phages shape and dimension that is very specific to establish differences in phage taxonomy. Methods used are those described by Ackermann (2001). In summary, a volume of 1.5 mL of PA25 suspension was centrifuged at 17,000 × g (4 °C) for one hour (High Speed Brushless Centrifuge MPW-350R). After discarding the supernatant, 1.5 mL of 0.1 M ammonium acetate was added to 100 µl of the pellet, and the mixture was urged again at 17,000 × g (4 °C) for one hour. The pellet was prepared for the microscopic observation with coloration by uranyl acetate 2%, on a carbon grid, mixed with a drop of the PA25 particle. Excess liquid was removed with filter paper, then left to dry in room temperature, and examined at 80 kV by using the JEOL JEM-1230 transmission electron microscope.

One-step growth curve

One-step growth curve was constructed to analyze the life cycle of PA25. PA25 was added to 1 mL of log-phase P. aeruginosa ATCC strain 27853 at a multiplicity of infections of 0.01 and then incubated for 2 h at 37 °C. The unabsorbed PA25 particles were removed by centrifugation at 10 000 × g for 5 min. After suspending in 50 mL of TSA medium, the mixture was incubated at 37 °C with continuous shaking. Samples were collected at intervals of 30 min, and viral abundance was immediately quantified with a double-agar plaque assay.

Bacterial challenge test

Two types of wastewater were used from Charguia Pilot Tunis: Upstream PWW samples and Downstream SWW samples. PA25 bacteriolytic activity was determined in (a) distilled water (DW), (b) sterilized wastewater (StWW) and (c) unsterilized wastewater (UstWW) following the method of Wang et al. (2016). P. aeruginosa ATCC 27853 was incubated into 500 mL TSB medium and grown overnight at 37 °C. Bacteria were harvest from 250 mL saturated TSB culture by centrifugation at 18 000 × g during 30 mn. The pellet was resuspended in three 5-L containers with DW, UstPWW and StPWW, to a density expressed in Colony-forming Unit (CFU) per ml of 6.0 × 103, 1.4 × 105 and 3.0 × 108, respectively. The same experiment was repeated using sterilized secondary wastewater StSWW and unsterilized secondary wastewater UstSWW with initial load of P. aerugnosa of 1.58 × 104 and 2.0 × 105 CFU/mL, respectively. 10 mL of PA25 suspension was added to each 5-L container, corresponding to 105 PFU/mL. All experiments were performed at room temperature around 25 °C during 48 h. Bacteria enumeration with or without PA25 treatment from both PWW and SWW were assayed via serial dilution total plate count (TPC) method using TSA medium (Cabral (2010). All experiments were performed in triplicates. Statistical significance was determined using student-t-test with a P-value threshold of < 0.05.

Effect of PA25 in antibiotic resistance of P. aeruginosa ATCC strain 27853

P. aeruginosa ATCC strain 27853 antibiotic resistance was assayed using two treatments: (1) experimental containers considering 90 mL of sterile wastewater to which was added 10 mL of pellet overnight P. aeruginosa ATCC strain 27853 culture in TSB medium, along with 1 mL of PA25 suspension corresponding to 105 PFU/mL and (2) Control containers with no PA25. Both treatments were incubated for 48 h at 37 °C.

Antibiotics susceptibility of P. aeruginosa ATCC strain 27853 subsequent PA25 contact

Antimicrobial susceptibility was assayed via the agar diffusion method with a disk diffusion test (Acar 2000) using TSA medium and inoculums of 108 CFU/mL (Control treatment) and 104 CFU/mL (Experimental treatment). The following disks for susceptibility testing were used: (a) gentamicin (GM), 15 µg/disk, (b) streptomycin (S10 10 µg/disk); (c) tetracycline (TE), 30 µg/disk; (d) rifampicin (RIF), 30 µg/disk and (e) spiramycin (SP) 100 µg/disk (Oxoid). P. aeruginosa ATCC strain 27853 was plated in Mueller Hinton agar (Biokar, Diagnostic), and the antibiotic disks were placed over the bacteria layer (Nassar et al. 2019). Plates were incubated at 37 °C overnight. After this period, the inhibition zone diameter was measured, and strains classified according to the sensitivity to each antibiotic.

Statistical analysis

All experiments were performed in triplicate. The Student–Newman–Keuls analysis (SPSS 20) software was used to determine the significant differences of removal efficiencies of phage treatment to check the influence of load bacteria variation with a P-value threshold of < 0.05.

Results and discussion

Results

Characterization of bacteriophages PA25 by plaque morphology, and transmission electron Microscopy TEM

Plaque assay of PA25 on P. aeruginosa showed the presence of medium plaques (0.7–0.8 cm) that determined the titer expressed by plaque-forming units PFU/ mL, estimated to be of 105 PFU/mL. (b) Electron micrographs of TEM IBIS, JEOL JEM 1230 of PA25 showed clearly that PA25 appeared as a doubled stranded DNA phage, with Isometric head 61 and 91 nm and Thin, long, noncontractile, and flexible tail with a base plate 110 and 170 nm, classified to belong to the Siphoviridae family, PA25 successfully isolated from SWW was able to undergo the lytic life cycle in P. aeruginosa ATCC27853. The results of OD600 of bacteria culture evolution showed that a 3-h contacting time was necessary to reduce OD600 from 1.6 to 0.6. The titer was determined, by PFU/ mL, estimated to be of 105 PFU/mL (Fig. 1a). PA25 was first characterized by plaque morphology showing medium sized plaques and gave clear-plaques expressing lytic cycles. Based on JEM-1230 transmission electron micrographs (JEOL, Tokyo, Japan), isolated PA25 appeared as a doubled stranded DNA phage, with a head and long tail, classified to belong to the Siphoviridae family (Ackermann 2007; 2009; 2012; Aprea 2015. 2018), similar to lytic Pseudomonas phages isolated previously from central Mexico, reported by Sepúlveda-Robles et al. (2012) describing a novel phage in the Siphoviridae family (Fig. 1b).

Fig. 1
figure 1

Characterization of bacteriophages PA25 by plaque morphology, and transmission electron microscopy (TEM): a Plaque assay of PA25 showing medium plaques (0.7–0.8 cm) on P. aeruginosa that determined the titer expressed by plaque-forming units PFU/ mL, estimated to be of 105 PFU/mL b TEM micrographs of IBIS, JEOL JEM 1230 of PA25 as a doubled stranded DNA phage, with isometric head 61 to 91 nm; a thin, long, noncontractile, and flexible tail with a base plate 110–170 nm, classified as the Siphoviridae family (Ackermann 2007; 2009; 2012; Aprea 2017;2018), Bar length = 200 nm

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One-step growth of PA25 lytic of P. aeruginosa ATCC strain 27853

A constructed one-step growth of PA25 showed exponential curves with a short latent period less than 20 min (Fig. 2). The latent PA25 period is defined as the period required between PA25 adsorption and the beginning of the cell disruption.

Fig. 2
figure 2

One-step growth of PA25 lytic phage on P. aeruginosa ATCC strain 27853. A constructed one-step growth of PA25 showed exponential curves with a short latent period less than 20 min (Fig. 2). The latent phage PA25 period is defined as the period required between phage PA25 adsorption and the beginning of the cell disruption

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Bacterial challenge test

PA25 was added to treatment of PWW, SWW and sterile distilled water (StDW); all treatments artificially contaminated by P. aeruginosa ATCC 27853. A control without PA25 addition was included. The results showed that bacterial load remain unchanged with PA25 addition to StDW; the discharge was weak, and the bacteria load was 7.00 × 103 and 3.00 × 103, for StDW and StDW + Pg, respectively (Fig. 3). This result may be due to the limited medium for bacteria growth that limited phage load. With the treatments of StSWW and StSWW + Pg, the PA25 does not induce cell lysis and bacteria load was 1.5 × 104 and 1.00 × 104, respectively. However, bacteria density in SWW effluent was reduced when PA25 was added to UStSWW + Pg, P. aeruginosa ATCC strain 27853 density was reduced from 2.00 × 105 to 6.00 × 103 CUF/mL. The effluent bacteria density was greatly reduced when PA25 was added to UStPWW; P. aeruginosa ATCC strain 27853 density to 3.00 × 108 to 2.00 × 103 CUF/mL, for untreated control (no phage) and treatment with PA25, respectively. In contrast, in StPWW the P. aeruginosa ATCC strain 27853 density deceased only slightly from to 1.40 × 105 to 5.00 × 104. The obtained results showed clearly that the sterilization of the sample and artificially inoculation of P. aeruginosa ATCC strain 27853 induced a less efficient reduction of bacteria cells by PA25. This result was consistent for both PWW and SWW. These results showed that PA25 may be added to both treated and untreated wastewater. The greatest reduction in P. aeruginosa ATCC strain 27853 density in effluents comes from adding PA25 to UStPWW compared to UStSWW. This difference in efficiency of PA25 when added to PWW compared to that of SWW may be related to the high organic content of PWW, higher at upstream of the pilot wastewater treatment stream. PA25 appears to thrive in the endogenous microbial community and chemistry of the wastewater samples; P. aeruginosa ATCC strain 27853 lyses easily occurs at the level of PWW. Other methods such chlorination or UV light are limited in their application to PWW, and require additional levels of pre-treatment to be effective. The wastewater disinfection was effective in this study, and PA25 application deserves attention to reduce effluent pollution.

Fig. 3
figure 3

taken from the primary domestic and from the secondary wastewater according to the addition or not of the phage PA25. (Pg): phage PA25, StDW: Sterilized distilled water + P. aeruginosa. StDW + Pg: Sterilized distilled water + P. aeruginosa + Phage PA25 StPWW = Sterilized Primary wastewater + P. aeruginosa StPWW + Pg: Sterilized Primary wastewater + P. aeruginosa + Phage PA25. UstPWW: Unsterilized primary wastewater + P. aeruginosa UstPWW + Pg: Unsterilized primary wastewater + P. aeruginosa + Phage PA25. StSWW = Sterilized Secondary wastewater + P. aeruginosa StSWW + Pg = Sterilized Secondary wastewater + P. aeruginosa + Phage PA25. UstSWW:Unsterilized secondary wastewater + P. aeruginosa UstSWW + Pg Unsterilized secondary wastewater + P. aeruginosa + Phage PA25

Load of P. aeruginosa ATCC strain 27853 over 48 h, in sample

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PA25 infection significantly impacted P. aeruginosa ATCC27853 regression. The bacterial density reduction was most dramatic in unsterilized wastewater. It can be explained by the presence of endogenous P. aeruginosa present previously in wastewater associated with P. aeruginosa ATCC27853 strain artificially inoculated in wastewater. Several studies reported linear relationship between phages and bacteria load (Leverentz et al. 2004; Wong et al. 2020). Burmeister et al. (2020) highlighted the abundance and the significant roles played by phage thereby modifying bacterial population structure, composition and dynamic as well as density. Untreated wastewater has a high organic content, thus supports high microbial growth (Al-Rekabi et al. 2007). However, SWW that has aerobic conditions can more easily be treated with phages (Gerba and Pepper 2019; Tan et al. 2020). In fact, phage requires aerated and suspended conditions to effectively attach bacteria hosts; suspended solids and high organic content of wastewater can remove phage from suspension, thus decreasing its efficiency in bacteria removal (Watts et al. 2020). This suggests that phage act on the endogenous P. aeruginosa present normally in wastewater microbial communities. Indeed, bacteria and phages are in a perpetual competition in the environment and continually evolve by modification of cell receptors (bacteria) and binding proteins (phages) (Van Houte et al. 2016; Broniewski, et al. 2020). In fact, phage can influence the abundance, diversity and evolution of bacterial communities (León and Bastías 2015). Several phages have been reported to add virulence factors to their host and to increase bacterial virulence (Brussow et al. 2004). However, lytic phage can also exert a selective pressure allowing the proliferation of strains with reduced virulence (Yuan et al. 2019). In reality, phage use structures present on the bacterial surface as receptors, which can be virulence factors in different bacterial species. Therefore, some strains may modify in these receptors and will be resistant to phage infection and may also exhibit reduced virulence. Silva et al. (2014) showed that the efficiency of phage therapy is highly dependent on different environmental factors such as salinity and the availability of organic matter. In this study, PWW with high organic content showed better performance of the phage in decreasing P. aeruginosa ATCC strain 27853 cells suggesting the use of phage in PWW. Phage is highly specific for bacterial species and multiplies at the expense of the cell, eventually reducing the number of viable bacterial (Sulakvelidze and Morris 2001). Even though the major advantage of phage treatment is phage specificity, since the non-target bacterial populations should remain unaffected, phages should be able of lysing the majority of strains of a given bacterial species.

Antibiotics susceptibility of P. aeruginosa ATCC strain 27853 subsequent to PA25 contact

The results showed that P. aeruginosa ATCC 27853 untreated with PA25 was resistant to tetracycline (TE), streptomycin (S10) and spiramycin (SP), and showed limited sensitivity to gentamicin and rifampicin. However, when PA25 was added to the bacteria suspensions, bacterial resistance to streptomycin (S10) declined. The inhibition zone diameter increased with addition of PA25; indeed, diameter of sensitivity of gentamicin increased from 19 to 21 mm, and from 11.3 to 20 mm for rifampicin. Streptomycin inhibition zone diameter increased to 20 mm (Fig. 4). Antibiotics resistance results are shown in Table 2; PA25 appears to have effectively attacked P. aeruginosa ATCC strain 27853 and the stressed bacteria became more sensitive to antibiotics (considered separately gentamicin and rifampicin).

Fig. 4
figure 4

Photographs of Petri dishes of P. aeruginosa ATCC27853 treated with 5 antibiotics a in the absence of phage PA25 and b in the presence of phage PA25, (G): gentamicin (S): streptomycin; (T):tetracycline (R): rifampicin (S1): spiramycin all discs (Oxoid). P. aeruginosa ATCC 27853 untreated with PA25 phage was resistant to tetracycline: (TE), streptomycin: (S10) and spiramycin: (SP), and showed limited sensitivity to gentamicin and rifampicin. However, when phage PA25 was added to the bacteria suspensions, bacterial resistance to streptomycin (S10) declined. The inhibition zone diameter increased with addition of phage PA25

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Table 2 Effect of phage PA25 on the sensibility of P. aeruginosa to antibiotics (R resistant to considered anribiotic) _ (S sensible to considered anribiotic), b value represent the average of triplicates ± standard deviation
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The effect of phage strains on changing bacterial virulence is still poorly understood (León and Bastías 2015). Recently, there has been an interest in research of the use of phage therapy against pathogenic bacteria (Moghadam et al. 2020). Current study indicates clearly that bacteria associated with phage have altered their sensitivity to streptomycin (S10) compared to resistance without phage treatment. However, Olivares et al. (2020) showed that P. aeruginosa resistant to β-lactams antibiotic may lose this resistance by another way by increasing doses of antibiotics. P. aeruginosa show low resistance to aminoglycoside; studies have found gentamicin (an aminoglycoside) resistance rates ranging from 12 to 22%. Gentamicin is the least active of the aminoglycosides, with lower rates of resistance being reported for tobramycin and amikacin (Lister et al. 2009). But, this kind of resistance is usually enzymatic, thus bacteria do not respond to dose increases. However, Gene PA14_40260-40,230 is part of an operon that encodes a novel efflux pump, and deletion of this operon in P. aeruginosa resulted in a decrease of the resistance of the bacterium to gentamicin and ciprofloxacin in biofilm (Zhang and Mah 2008). This may suggest that the selected phage may contribute to Gene PA14_40260-40,230 deletion. Further research is needed to confirm this hypothesis. Concerning streptomycin antibiotic, it has a unique fixation site which is the ribosomal protein S12. The addition of the phage affects the pathogen load (Levin and Bull 1996) and also limits bacteria resistance to antibiotics (Torres-Barceló et al. 2014). The effect of the phage on bacteria is likely a genetic modification that induces a loss of antibiotic resistance (Levin and Bull 1996). Rifampicine is one of the most potent and broad spectrum antibiotics used against bacterial pathogens and is a key component of anti-tuberculosis therapy. Rifampicine effectiveness stems from its inhibition of the bacterial RNA polymerase (RNAP) (Campbell et al. 2001). P. aeruginosa has been found to be resistant to tetracycline (Dean et al. 2003), streptomycin (Cervantes-Vega et al. 1986) and spiramycin and has a limited sensitivity to rifamycin (Yee et al. 1996), and gentamycin (Kadurugamuwa et al. 1993). In addition, the apparition of strain sensitivity to streptomycin could be due to bacterial response to phage infection (Levin and Bull 1996). Bacterial resistance against these antibiotics has been associated with their overuse in livestock veterinary practices; phages could be a new tool to mitigate antibiotic resistance in P. aeruginosa found in wastewater.

The phage therapy was shown as a potential solution for the antibiotic resistance crisis in clinical medium (Golkar et al. 2014; Berglund, 2015). In wastewater treatment systems, microbial communities may increase genetic diversity linked to antibiotic resistance, and wastewater effluents may consequently impact microorganisms entering the environment and ecological systems (Sorum, 2006), thus impacting pathogen transmission to humans and animals (Brüssow and Kutter, 2005; Cabello, 2006). Inadequate treatment of municipal wastewater may increase the conditions that promote microbial antibiotic resistance through new mutations (Courvalin, 2001). Synergy between judicious antibiotic use generally and phage treatment of wastewater specially could address the threat of bacterial pathogens released in wastewater effluent to improve environmental and human health (Sulakvelidze and Kutter, 2005; Comeau et al. 2008), and that PA25 may be added at the level of PWW and SWW indicating efficiency of PA25 as tool in primary or secondary plant treatment. This study supports the use of PA25 as a tool to fight bacterial infections and should serve as a baseline for additional research for broader wastewater treatment applications to prevent pathogen spreading (Alisky et al. 1998).

Conclusion

In this manuscript, the phage PA25 was isolated from secondary domestic wastewater to remove P. aeruginosa ATCC strain 27853. The effect of PA25 on reducing P. aeruginosa ATCC strain 27853 discharge was more effective when the phage was added in PWW. Also, the isolated PA25 was able to reduce antibiotic resistance in the target bacteria. The result provided should be useful for limiting pathogens spread in environment and should be used as new tool for both wastewater primary treatment and wastewater disinfection.