Efficacy of bacteriophage in removal of Pseudomonas aeruginosa from infectious surfaces

Nosocomial infections can be transmitted by contaminated hospital surfaces with resistant pathogens. conventional sanitations are not efficiently contributing to removing resistant pathogens. Bacteriophages suggest as decontaminating agents, safe, their selective ability to kill specific bacteria. This work aimed to assess the efficiency of a phage in removing pseudomonas aeruginosa from different hard surfaces. The decontamination ability of phages w was tested in vitro against Pseudomonas aeruginosa strain. Cystoviridae Phages with titer (2 × 10 PFU/mL) can efficiently reduce viable bacterial cells on contaminated surfaces. The treated surfaces with alcohol 70% and phage showed an evident drop of bacterial cell number from 1 h to 24 h. These results suggest that bacteriophages are biocontrol agents removing nosocomial infection pathogens transmitted by contaminated surfaces in the hospital environment.


Introduction
The presence of microbial agents on the surfaces and medical devices in different units of the hospital is an important detrimental factor for health. Those pathogens play a major role in causing nosocomial infections which they lead to longer duration and dissatisfaction of patients in hospitals. About 3.2 million patients suffer from nosocomial infections each year, with 37,000 dying directly from nosocomial infections [1][2].
The hospital surfaces persistently are contaminated by several drug-resistant pathogens including Staphylococcus spp, Enterobacteriaceae, and Pseudomonas spp [3][4][5]. P. aeruginosa is resistant to disinfectants, antiseptics, and preservatives even can grow in some disinfectants and transmits infection so causes nosocomial infections [5]. P. aeruginosa can grow in the environment as a biofilm and survives for as long as one year [5]. The mortality and morbidity rate associated with P. aeruginosa is higher than other pathogens [

2.2.
Isolation of bacteriophage The bacteriophage was isolated from P. aeruginosa supernatants. All of the above bacteria were incubated in 100 mL Luria-Bertani (Quelab, USA) for 24 hours at 37 ℃ in a shaker incubator. The three samples were centrifuged at 10,000 × g for 10 minutes. The supernatant was filtrated through a 0.22 µm syringe filter at sterile conditions [15-19].

2.3.
Double-Layer Plaque Assay (DLA Assay) 900 μl sterilized sodium chloride-magnesium sulfate (SM) buffer (100 mmol/L NaCl, 8 mmol/L MgSO4, 2% gelatin, and 50 mmol/L Tris-HCl [pH 7.5]) was added to 10 sterile tubes. Then 100 μl of phage was added to tube no. 1 and was vortexed. After vortexing 100 μl from tube no. 1 was taken out and added to tube no. 2. This procedure was repeated until tube no. 8. Tube no. 9 and tube no. 10 were selected as the positive and negative control respectively. From each of the diluted phage cocktail, 200 µl was transferred to 200 µl each of the above bacteria ATCC No (1.5× 10 8 CFU/ mL). The mixtures were added to the top agar and the top agars were added to the bottom agar then the plates were incubated overnight at 37 °C. The experiment was repeated three times [15-19].

2.4.
Transmission Electron Microscopy (TEM) In order to prepare the phage for TEM, the phage was centrifuged at 20,000×g for 60 min. The phage was deposited on carbon-coated copper grids and was stained by 2% uranyl acetate (pH 4-4.5). The phage cocktail was observed on a Zeiss EM 900 TEM at 130 kV [15-19].

2.5.
Determination the host range of phage The spot test was performed to determine the lytic activity of the phage. The overnight cultured P. aeruginosa was inoculated in top agar and were poured into the bottom agar. A certain volume of the phage cocktail supernatant (10 µl) was poured over the solidified agar. The plates were incubated at 37 °C overnight. The formation of the inhibition zone was checked [15-19].

2.6.
Phage Stability in vitro The stability of the phage was investigated under various environmental conditions such as temperatures and pH. Phage was incubated at 4, 22, 37, 50 °C for 60 minutes to determine thermal stability. The stability of the phage was determined at pH 3, 5, 7, 9, and 11 for 60 minutes. The lytic activity of the phage was detected by DLA assay. Also phage stability was measured after 1, 7, 14, 21, and 30 days at room temperature with DLA assay [15-19].

2.7.
Decontamination test The efficiency of lytic activity of phage against P. aeruginosa was assessed on different kinds of hard surfaces by in vitro decontamination assays. P. aeruginosa was grown in tryptic soy broth (TSB, Merck Millipore), then 10 μl of the bacteria suspension (OD600nm =1) was spread on the different types of surfaces as plastic and ceramic (ceramic tiles sterilized by oven previous). The bacteria suspension was seeded and allowed to dry at room temperature. 50 μl of the phage lysate in SM buffer (2 × 10 12 PFU/mL), 50 μl of Alcohol 70% separately were spread on the surfaces and allowed to dry. Two groups as; bacteria suspension (OD600nm =1) , surfaces without bacteria suspension were used as a positive and negative controls respectively. After 15 min, 1, 3, 6, and 24 hours, surfaces were directly sampled by contact plates (Merck Millipore). Each plate, containing samples taken at the different time was incubated for 24 hours at 37°C and bacterial load was evaluated by enumerating plate CFU. Each sample was performed in triplicate [20, 13,14].

3.1.
Detection of Antimicrobial Resistance The Kirby-Bauer test showed that P. aeruginosa is sensitive to nitrofurantoin, cefotaxime, and cefixime but resistant to ampicillin, tetracycline, meropenem, gentamycin and ciprofloxacin.

3.2.
Characterization of phage Figure 1 shows the lytic activity of phage determined with the formation inhibition zone in spot test. The phage titer was calculated by DLA assay (2 ×10 12 PFU/mL). The morphology of the phage was shown by TEM, Cystoviridae with spherical shape (80-100 nm) with a lipid membrane around the capsomere (Figure 2). The viability of phage was considered in various conditions. The phage showed the highest titer at 37 °C, but no active phage was found at 50 °C. The highest and lowest titer of phage was at pH 11 and 3 respectively (Figure 3). The DLA assay results of incubation of phage after 1, 7, 14, 21, and 30 days at room temperature evidenced that phages efficiently can reduce viable bacterial cells.

Discussion
In this study, Cystoviridae phage against P. aeruginosa with a titer (2 × 10 12 PFU/ mL) was isolated from a bacterial supernatant. Phage lysate showed lytic activity against P. aeruginosa as documented with formation of the inhibition zone in spot test. The efficacy of lytic activity of phage at various hard surfaces (ceramic and plastic) against P. aeruginosa confirmed after 1hour with a decrease the number of bacteria (CFU). The highest lytic activity of phage determined at 37 °C and pH 11. We proved phage stability after 1, 7, 14, 21 and 30 days of incubation at room temperature, by PFU titration on the specific bacterial.
In present study phage lysate did not show destructive effects on the hard surfaces. Previous studies were showed phages have not side effect eukarutic cells [17][18][19][20][21]. Many chemical sanitizers have antimicrobials activity but are corrosive and toxic and unacceptable for treating tissue, food, and surfaces [23-24].
Our results showed with increasing the contact time between phage and contaminated surfaces decreased the number of bacteria (CFU). The highest and lowest bacterial concentrations observed in 15 minutes and 24 hours, respectively. The number of released progeny phage from the lysed host bacterium logarithmically increases so increases the lytic activity of phage. Bacteriophages are self-replications so they are economically affordable [17][18][19][20][21]. Gamma irradiation of beef, approved by the U.S. FDA in 1997, produces about a 1,000-fold reduction in the number of viable aerobic bacterial contaminants. However, it is expensive and requires batch processing. In addition, it is not specific [25]. Lytic bacteriophages are alternative approaches to aid the prevention of diseases caused by natural or intentional dissemination of pathogenic bacteria on various surfaces. Several authors [28][29] reported, treatment with bacteriophages significantly reduced the levels of major food-borne pathogens in various foods, with reduction levels ranging from 1.8 to 4.6 logs compared to those of untreated or placebo-treated controls [30][31]. LMP-102 is the first phage based preparation to be approved for a food safety application by a Western regulatory agency [14].

Conclusions
Phages potentially with stable lytic activity lead to effective and safe elimination of pathogens from the contaminated hard surfaces. Bacteriophages as biocontrol agents open new perspectives for the development of innovative products aimed at the prevention of nosocomial infections transmitted by contaminated surfaces in the hospital environment.