Isolation and Characterization of Lytic Phages TSE1-3 Against Enterobacter Cloacae

Open Life Sci. 2016; 11: 287–292

Research Article Open Access

Komal Ameer Khawaja, Zaigham Abbas, Shafiq ur Rehman* Isolation and characterization of lytic phages TSE1-3 against Enterobacter cloacae

DOI 10.1515/biol-2016-0038 aureus and Pseudomonas aeruginosa [1]. The E. aerogenes Received November 5, 2015; accepted August 25, 2016 and E. cloacae are among the two most common Enterobacter Abstract: The emergence of antibiotic resistant bacterial species, responsible for causing human infections (90–99% pathogens is becoming a major challenge for patient of Enterobacter infections) [2]. The Enterobacter cloacae care. The utilization of alternative therapies for infectious mainly causes urinary and respiratory tract infections. The diseases other than antibiotics is an urgent need of today majority of Enterobacter spp. are resistant to antibiotics of medical practice. The utilization of lytic bacteriophages common use and present a global clinical problem [1]. The and their gene products as therapeutic agents against Enterobacter cloacae is associated with different types of antibiotic resistant bacteria is one of the convincing outbreaks in hospital settings [3-5]. alternative approaches. Here we present the isolation The bacteriophages were first discovered by Félix and characterization of three lytic bacteriophages TSE1-3 d’Herelle in 1917 [6]. The therapeutic potential of against Enterobacter cloacae from sewage effluent. The bacteriophages in controlling the bacterial infections in isolates maintained antibacterial activity for 10 hours humans were first employed in 1919 [6]. Consequently, of incubation followed by the development of phage the anti-plague activity of bacteriophages was reported by resistance. Their stability at different temperatures and d’Herelle in 1925 [7]. The concept of phage therapy ceased pH, established their possible application in phage in western medicine due to the emergence of antibiotics, therapy. The highest activity of the phages was observed while it is still researched and applied in Russia and at 37°C and pH 7.0, while they gave lytic activity up to neighboring countries [8]. The West remained reluctant 60°C. The latent period of all the TSE phages was 20 in using the bacteriophages as therapeutic agents due to minutes, while the burst size was 360 for TSE1, 270 early unreliable trials; however now phage therapy has for TSE2 and 311 for TSE3. The phages were harboring also gained attention in the developed world. double-stranded DNA larger than 12kb in size. Further Increasing emergence of antibiotic resistance poses research into the phages genome and proteins, animal a challenge among the scientific community to identify experiments, delivery parameters and clinical trials may potential modalities for controlling the antibiotic lead to their utilization in phage therapy. resistant Enterobacter spp. The bacteriophages against the Enterobacter spp. can be utilized as an alternative Keywords: Enterobacter cloacae, Phage therapy, therapy for controlling the antibiotic resistant isolates. Bacteriophage, TSE1 The bacteriophages selectively infect their bacterial host down to individual strains. The bacteriophages are thought of as a potentially valuable tool in controlling 1 Introduction bacterial infections, which in certain cases of antibiotic resistance, might be the only available effective modality. The Gram-negative bacteria belonging to Enterobacter spp. Additionally a single dose of phage can destroy a specific are the third leading cause of hospital-acquired infections bacterium, while multiple doses of antibiotics are needed of blood stream and respiratory tract after Staphylococcus to treat bacterial infections. Currently no harmful effects of phages to eukaryotic cells have been reported. The United States Food and Drug Administration have approved the *Corresponding authors: Shafiq ur Rehman, Department of use of Listeria phage in foods, which has re-established Microbiology and Molecular Genetics, University of the Punjab, the use of phages as antimicrobial agents [9]. The current Lahore, Pakistan, E-mail: [email protected] effort was done to isolate and characterize the lytic phages Komal Ameer Khawaja, Zaigham Abbas, Department of Microbiology against Enterobacter cloacae to be used in phage therapy. and Molecular Genetics, University of the Punjab, Lahore, Pakistan

2 Materials and Methods bacteriophages was incubated at varying temperatures (28, 35, 37, 40, 45, 50, 55, 60 and 65°C) for 1 hour in L-broth (pH 7.0) followed by calculating phage titer by the double 2.1 Host bacterial strains layer agar method [11].

A characterized environmental isolate of Enterobacter cloacae (accession no: KF975427) is used as the 2.5 Analysis of bacteriophage growth bacteriophage host (donated by Dr. Yasir Rehman, parameters Assistant Professor in the Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, The one step growth curve of the bacteriophages was Pakistan) in this study. The host bacterial strain was performed in duplicates following an already reported streaked on L-agar plates and cultured in L-broth for use method [10]. Actively growing cells of host strain (O.D600 after 24 hours incubation at 37°C. of 0.4-0.6), were suspended in 500 μL of L-broth and mixed with bacteriophages, and incubated for one minute at 37°C. Centrifugation of the mixture was done at 2.2 Isolation and purification of 11000 × g for 30 seconds and the pellet was re-suspended bacteriophages in 100 mL fresh broth followed by incubation at 37°C with shaking. Viral titer was counted by double layer The bacteriophage was isolated from sewage outlet of agar technique from samples taken at 5 minute intervals Tech Housing Society, Lahore. Isolation and purification for an hour. of the bacteriophages was performed according to an already published protocol by our group [10]. Purified bacteriophage lysate was stored at 4°C when storing for 2.6 Assessment of bacteriophage antibacte- short time or further processing, while stored at -20°C rial activity for longer time. The viral titer in each phage lysate is expressed as plaque forming units per millilitre (PFU/mL). The potential of the bacteriophages against the respective host bacterial strain was checked according to already published work [10]. Briefly, a 24 hour bacterial culture 2.3 Determination of host specificity (1 × 108 CFU) and respective bacteriophage (1 × 108 PFU) were added in one flask, while the other flask containing The host infectivity of each isolated phage against the same concentration of bacteria but no phages was Aeromonas caviae (accession no: KF975428), taken as control. The optical density was taken at intervals Pseudomonas stutzeri (accession no: KF975434), and of 2 hours for a 24 hour period after incubation at 37°C Klebsiella pneumoniae (accession no: KF975429) (gift from with shaking (120 rpm). Dr Yasir Rehman, Assistant Professor at the Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore Pakistan) was determined through the 2.7 Stability of bacteriophages during double layer agar technique. The host specificity against storage two hospital isolates of Enterobacter cloacae (strain number EC11 and EC12) was also tested. The purified phages (1 × 1010 CFU/ml) were incubated for one month at different storage temperatures (-20, 4, 37°C) followed by assay for phage infectivity. 2.4 Examining bacteriophage stability at wide range of pH and temperature 2.8 Isolation of bacteriophage genomic DNA The viability of Enterobacter cloacae phages at varying pH and temperatures was investigated. Briefly, known The DNA of the bacteriophages was extracted as previously concentrations of purified bacteriophages were incubated mentioned [10] followed by electrophoretic analysis in in L-broth with different pH values (5-10), incubated 0.7% TBE agarose gel. The isolated bacteriophage genome for 1 hour at 37°C. Similarly, to study the temperature was treated with endonuclease I to assess either it is single stability of bacteriophages, a known concentration of or double stranded DNA. Isolation and characterization of lytic phages TSE1-3 against Enterobacter cloacae 289

3 Results occurred after 18 hours post infection, and attained the optical density similar to uninfected control at 24 hour post The bacteriophages TSE1, TSE2 and TSE3 against infection (Figure 7), which indicates the development of Enterobacter cloacae were isolated from the sewage phage resistant bacterial cells. exhaust of the Tech housing society Lahore and are shown in Figure 1 and Figure 2. The plaque morphology along with the PFU/mL were recorded for each stock of the virus. The plaque morphology of TSE1 and TSE3 phages appeared as pinpoint, while morphology of the TSE2 was small circular on double layer agar plates (Figure 2). The plaque size of TSE1 and TSE3 appeared less than 1mm, while it was 1mm for the plaques of TSE2. The virus titer of TSE1, TSE2 and TSE3 were determined as 2.0 × 108, 4.8 × 106 and 2.5 × 108 PFU/mL respectively. The host range of the isolated phages was checked Figure 1. Isolation of bacteriophages from the sewage exhaust of against different bacterial strains, including Aeromonas Tech housing Society Lahore, against Enterobacter cloacae. The caviae, Pseudomonas stutzeri, and Klebsiella pneumoniae figure shows different types of bacteriophages isolated on a single plate using the double layer agar technique. and hospital isolates of Enterobacter cloacae. All the TSE phages exhibited a narrow host range as they were unable to infect any tested bacteria other than Enterobacter cloacae. The purified phages (1 × 1010 CFU/ml) were incubated for one month at different temperatures followed by analysis of viral titer. All the TSE phages remained stable at both tested storage temperatures (-20°C and 4°C), while none remained stable at 37°C (Figure 3). The phage survival in the adverse environment is also a desired characteristic for their use in phage therapy. The PFU of all the phages was maximum at pH 7 (2.2 × 105 for TSE1, 4.6 × 104 for TSE2 and 2.3 × 104 for Figure 2. The plaque morphology of purified bacteriophages against TSE3), while reduction of PFU was observed at both pH 6 Enterobacter cloacae obtained through double layer agar technique. (2.8 × 103 for TSE1, 7.0 × 102 for TSE2, 1.4 × 103 for TSE3) (A) bacteriophage TSE1 with an average size of less than 1 mm. (B) bacteriophage TSE2 with the size of 1 mm. (C) bacteriophage TSE3 and at pH 8 (2.8 × 103 for TSE1, 3.6 × 103 for TSE2 and with the size less than 1 mm. 1.3 × 103 for TSE3). No plaques were observed at pH 5, 9 and 10 (Figure 4). The PFU was highest after treatment at 37°C while fewer plaques were observed at other temperatures (28°C to 60°C), however no lytic activity was observed after treatment at 65°C (Figure 5). The latent period, and burst size of all the TSE phages was calculated from a one-step growth curve. The latent period for all TSE bacteriophages was calculated as 20 minutes. However the burst size of TSE1, TSE2 and TSE3 was 360, 270 and 311 particles per cell respectively (Figure 6). The potential of the isolated TSE phages to reduce the growth of their host bacteria and development of phage resistant bacteria were analyzed by plotting the bacterial growth reduction curve. The OD600 of non-infected E. cloacae increased throughout the 24 hours incubation which indicates their normal growth pattern. The bacteriophages Figure 3. Storage conditions of different temperatures (-20°C, 4°C reduced the growth of target bacteria during 8-18 hours post and 37°C) checked for average PFU/mL of bacteriophages after infection. However the development of resistant bacteria incubation for a month. 290 K. Ameer Khawaja, et al.

Figure 4. The effect of pH on bacteriophage stability. The phage Figure 5. The effect of temperature on bacteriophage stability. The lysate treated at different pH values (5, 6, 7, 8, 9 and 10) for one phage lysate treated at different temperatures (28, 37, 45, 55 and hour followed by calculating phage titer. 60°C) for an hour followed by determining phage titer.

Figure 6. One step growth curve experiment performed for Figure 7. The bacteriophages TSE1-3 showed a pronounced estimation of the bacteriophage latent period, burst size and growth reduction in growth of their host Enterobacter cloacae at 8 hours to pattern. 18 hours post infection on bacterial growth reduction assay.

The genome of all the bacteriophages was isolated environmentally isolated Enterobacter cloacae (accession and observed under UV after running the 0.7% agarose no: KF975427) strain were isolated and characterized. The gel for one and half hours. The genome of all the phages TSE phages were isolated from sewage exhaust which has appeared double stranded DNA as it was not digested by been reported as a good source for bacteriophage isolation endonuclease I enzyme, indicative of double stranded [13]. The TSE phages under study showed a narrow host DNA. The genome of all the phages appeared more than range, a characteristic of majority of bacteriophages 12 kb in size (Figure 8). already reported [15]. The ability of phages to infect specific host(s) makes them suitable for phage therapy [16]. 4 Discussion The stability of studied TSE phages at varying temperatures and pH was also investigated, this data is Hospital acquired infections (HAIs) are global, but are less helpful in determining their suitability for use in phage studied in developing nations like Pakistan [12]. In the therapy through the oral route. The TSE phages showed present study, bacteriophages against the Enterobacter their viability at pH of 6-8, while at neutral pH (7.0) cloacae which usually show marked antibiotic resistance maximum phage titer was recorded (Figure 4). The ability were under investigation. The bacteriophages against an of phages to tolerate at varying pH levels indicates that Isolation and characterization of lytic phages TSE1-3 against Enterobacter cloacae 291

therapy. The bacteria become resistant to phages by downregulating the expression of the phages receptors (flagella and capsules), which make them less virulent [22]. The genome of the studied phages was larger than 12 kb, and it was DNA evident by digestion with the DNase. According to the size of the genome, we can deduce that the genome is double-stranded, as the size categorization placed them among medium-to large-sized genomes, which are mostly double-stranded [23]. The reported small size phage genomes are around 5.5 kb, while the larger size genomes may be greater than 250 kb [24]. Currently, there are great number of studies on bacteriophage isolation and characterization against Figure 8. The bacteriophage DNA extraction: The genome of TSE1, E. cloacae. The emergence of phage resistant strains poses TSE2 and TSE3 showin in lanes 1-3 respectively compared with 1kb a great challenge for phage therapy, while there is need to plus ladder in the labeled (lane 4) on 0.7% agarose gel . investigate its role in decreasing the bacterial virulence. The concept of using bacteriophages in contrast to they may be suitable for administration through the oral antibiotics for controlling bacterial infections looks route after completing the clinical trials [16, 17]. The best promising. The financial investment for phage therapy temperature for phage stability was determined as 37°C, is considerably less compared with identification of a which is also reported for other bacteriophages against novel antibiotic. Further work on bacteriophage genome Enterobacter cloacae [8,18]. The TSE phages survived at all characterization, dose and lytic activity against more the tested temperatures from 28°C to 60°C, however the clinical isolates might lead to utilization of theses phages phage titer decreased when treated at 37°C to 60°C (Figure in therapy. 4 and Figure 5). For the application of bacteriophages in phage therapy, their prolonged stability is most important. Conflict of Interest: Authors declare to have no conflict The stability of the isolated TSE bacteriophages was of interest for this work. tested at three different temperatures; the bacteriophages remained stable at 4°C and -20°C for a month, while none survived at 37°C. The best storage temperature for the TSE References phages was -20°C, as reported by previous authors [14]. [1] Bornet C., Davin-Regli A., Bosi C., Pages J. M., Bollet C., The growth pattern of the TSE phages was studied Imipenem resistance of enterobacter aerogenes mediated by by plotting their one step growth curve, all three phages outer membrane permeability, J. Clin. Microbiol., 2000, 38, showed similar behavior in the growth curve for the 1048-1052. same latent period (20 minutes) and moderate burst [2] Marcos M., Inurrieta A., Soriano A., Martinez J. A., Almela M., Marco F., et al., Effect of antimicrobial therapy on mortality in size (Figure 6). This moderate burst size favors their use 377 episodes of Enterobacter spp. bacteraemia, J. Antimicrob. in the phage therapy and coincide with other phages Chemother., 2008, 62, 397-403. against Enterobacter species [19]. The physiological stage [3] Andersen B. M., Sorlie D., Hotvedt R., Almdahl S. M., of the host cell will also have effects on the burst size of Olafsen K., George R., et al., Multiply beta-lactam resistant phages and this factor should explored in future research Enterobacter cloacae infections linked to the environmental [20]. The bacterial growth reduction assay indicated flora in a unit for cardiothoracic and vascular surgery, Scand. J. Infect. Dis., 1989, 21, 181-191. the phage’s potential to reduce the growth of the target [4] Ayus J. C., Sheikh-Hamad D., Silent infection in clotted bacteria during the incubation time. The application hemodialysis access grafts, J. Am. Soc. Nephrol., 1998, 9, of phages in phage therapy and their feasibility can be 1314-1317. predicted by the results of the bacterial growth reduction [5] Musil I., Jensen V., Schilling J., Ashdown B., Kent T., assay [21]. The TSE phages reduced the growth of the Enterobacter cloacae infection of an expanded polytetrafluoro- ethylene femoral-popliteal bypass graft: a case report, J. Med. target bacteria at eight hours post infection until 18 hours Case Rep., 2010, 4, 131 doi: 10.1186/1752-1947-4-131. had elapsed, after which resistant bacterial cells started to [6] Hermoso J. A., Garcia J. L., Garcia P., Taking aim on bacterial emerge (Figure 7). The development of resistant bacterial pathogens: from phage therapy to enzybiotics, Curr. Opin. strains after phage exposure is a great hindrance in phage Microbiol., 2007, 10, 461-472. 292 K. Ameer Khawaja, et al.

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