Study of the relationship between bacteriophage and selective host cells according to different conditions of UV-C irradiation

Desalination 246 (2009) 397–408

Study of the relationship between bacteriophage and selective host cells according to different conditions of UV-C irradiation M. Ben Saida,*, A. Hassena, N. Saidia, H.W. Ackermannb a

Centre des Recherches et des Technologies des Eaux (C.E.R.T.E), Laboratoire de Traitement et Recyclage des Eaux E-mail: [email protected] b Departement de Biologie Médicale à la Faculté de Médecine, Laval University, Quebec, Canada Received 14 September 2007; revised 04 March 2008; accepted 11 March 2008

Abstract We investigate the potential for reactivation to restore infectivity to UV-damaged bacteriophage 2MPØ isolated from wastewater. The virus belongs to the Myoviridae family of the tailed phages. This Phage was chosen for detailed analysis, and was inspected by electron microscopy, host range determination. In addition, we explore the dynamics of bacteriophage host interactions in different conditions of UV-C irradiation. The inactivation kinetic or dose-response relationship was described by the method of plaque-forming ability. The UV-inactivation kinetic was represented in the exponential curve according to the model of Chick–Watson and Series-event kinetic model. In a 99.99% inactivation of 2MPØ bacteriophage which corresponded to 720 mW s/cm2 of UV-dose, high correlation was observed between the loss of infectivity and the increase of UV-C dose. The restore of infectivity of UVexposed bacteriophage was also determined under conditions which either activated or repressed the light-dependent photolyase enzyme in host cells, in order to examine the damage-dependent response of this bacterial repair system. The results of reactivation indicated that light-dependent repair (photoreactivation) was much more efficient than dark repair in restoring infectivity to bacteriophage damaged by UV-C radiation. Keywords: Disinfection; Ultraviolet light; Pseudomonas aeruginosa; Bacteriophage; Reactivation

1. Introduction Ultraviolet (UV) light disinfection is being increasingly used in the treatment of both wastewater and potable drinking water since such treat*Corresponding author.

ment does not produce disinfectant by-products [5]. The inactivation of microorganisms as a result of UV-C radiation is almost entirely attributable to photobiochemical reactions that are included within the microorganisms. The effectiveness of UV light in biological inactivation are due to the direct DNA damage by inducing the

Presented at the: MEDA WATER International Conference on Sustainable Water Management, Tunis, March 21–24 2007. 0011-9164/09/$– See front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.desal.2008.03.063

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formation of photoproducts, of which the cyclobutane pyrimidine dimer (CPD), and (6–4) photoproducts [1]. The accumulation of DNA photoproducts can be lethal to cell if the lesion blocks DNA synthesis and RNA transcription or can be mutagenic if the lesion is by passed by DNA polymerase. This photodimerization process has also been observed with uracil in RNA of viruses [9]. However, UV disinfection is noted to have some problems, one of which is reactivation. In response to UV damage, bacteria have developed different repair pathways, including photoenzymatic repair (PER), nucleotide excision repair (NER) also called dark repair, and recombinational repair also called post replication repair. PER involves direct monomerization process which is termed photoreactivation of CPDs [6–13]. The monomerization process performed by photolyases enzymes which bind to the dimers, capture the energy from photons via associated chromophores, and then use electron transfer to split the dimers and restore the DNA to its original form. The process of photoreactivation was originally described as the restoration of infectivity to UV-irradiated bacteriophages upon exposure to visible light in the presence of host bacteria [20]. For most DNA-viruses, the absence of associated photolyase genes precludes their ability to utilize visible light for repairing UV light-induced genomic damage [16]. Therefore, any DNA repair dependent on photoreactivation is conditional on successful infection of a host which can provide the necessary enzyme. Although some viruses such as bacteriophage T4 and possibly the Chlorella virus PBCV-1 encode enzymes which are involved in the excision repair of pyrimidine dimers resulting from UV light irradiation, these proteins would be active only when the virus is internalized in a bacterium and an alga, respectively [3–8]. The discovery that viruses may be the most abundant organisms in natural waters, surpassing the number of bacteria by an order of magnitude,

has inspired a resurgence of interest in viruses in the aquatic environment. Surprisingly little was known of the interaction of viruses and their hosts in nature [14]. Bacterial viruses have been hypothesized to play key roles in controlling bacterial host population density and diversity, and transfer of genes between hosts. Since the earliest studies of bacteriophage physiology, researchers have routinely reported data on the stability of purified viral isolates. Thus, most studies on the survival and fate of viruses in natural waters has focused directly on enteroviruses or on bacteriophages used as indicators of enteroviral pollution [2–4]. The detrimental effect of UV-C radiation on bacteriophage persistence and replication make a capacity for viral DNA damage repair ecologically relevant. In order to partially explain the capacity to repair or possibility escape UV-C radiation damage in an experimental environment or the tolerance of solar UV radiation in an aquatic environment, it has been hypothesized that viruses utilize several DNA repair mechanisms to allow for effective reproduction and by executing an alternative replicative strategy such as lysogeny [11]. In this study, we report; the isolation and partial characterization of Pseudomonas aeruginosa bacteriophage, designated 2MPØ, that have capacity to reactivate DNA damage; the evaluation of UV-C radiation against bacteriophage in an attempt to define the kinetics of virus inactivation in water. In addition, we propose the study of UV-C radiation effect on bacteriophage-host cell interactions and the potential for light-dependent repair (photoreactivation) and light-independent (dark repair) mechanisms to restore infectivity to the tested bacteriophage. 2. Materials and methods 2.1. Bacterial strains and bacteriophages The bacterial strain used in this study is P. aeruginosa (2MP) that is lysogenic for 2MPØ.

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This bacterial strain was isolated from wastewater, and has shown a high degree of phototolerance to the increasing dose of UV-C radiation. This bacterial strain was isolated from wastewater, and has shown a high degree of photo tolerance to the increasing dose of UV-C radiation. The 2MPØ bacteriophage was partially identified as a member of Myoviridae. Bacteriophage 2MPØ was detected by a plaque assay with lawns of the original bacterial host. The plaques were then screened for the ability to lyse both originally and others added indicator hosts. Bacterial strains used for bacteriophage propagation and titration were P. aeruginosa ATCC 15442 and P. aeruginosa PAO1, E. coli ATCC 1036, S. typhi ATCC 14029, and K. pneumoniae ATCC 13833 from the American Type Culture Collection.

2.2. Growth media and chemicals Luria–Bertani broth (LB) or LB agar (LBA) are used as growth bacteria media. Saline (0.85% [wt/vol] NaCl) was used for cell and bacteriophage suspensions during irradiation. The soft agar used for overlays contained 1% (wt/vol) trypton, 0.5 % (wt/vol) NaCl, and 0.65% (wt/vol) agar.

2.3. Bacteriophage purification and titration procedures Appropriately diluted samples containing bacteriophages were mixed with 0.1 mL of overnight bacterial host culture (2MP) and plated on 2.5 mL of top agar. Bacteriophage preparations were quantified by plating appropriately diluted samples on lawns of bacterial cells. Well-isolated plaque was picked up and immersed into 2 mL of nutrient broth (LB) and used to produce new bacteriophage lysates and to test its ability in reinfecting the original host cell (Fig. 2a). This procedure was repeated through three cycles or until lysates produced only a single-plaque morphology.

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2.4. Host range determination The host range of bacteriophage was determined against different species of bacterial strains obtained from the American Type Culture Collection (ATCC) and they were: P. aeruginosa ATCC 15442, P. aeruginosa PAO1, E. coli ATCC 1036, S. typhi ATCC 14029, and K. pneumoniae ATCC 13833. The plaque-forming ability of 2MPØ bacteriophage was tested by the spot-onthe-lawn technique. Ten μl of the purified phage stocks (107 PFU mL−1) were placed on the plates inoculated with a host cell. Bacterial suspensions were infected with phage at a multiplicity of infection (MOI). The lytic activity was observed after overnight incubation. 2.5. The efficiency of plating The efficiency of plating (EOP) was quantified by calculating the ratio of the bacteriophage plaque titers obtained with the heterologous host or homologous host, i.e., the different species or the same species of the original host, respectively, to those obtained with the original host. The ability of this bacteriophage to infect a range of bacterial species was assessed with a spot titration assay. 2.6. Electron microscopy of bacteriophage lysates The morphology of the bacteriophage 2MPØ was examined by electron microscopy. The bacteriophage was negatively stained with 2% (wt/vol) phosphotungstic acid (pH 7.2) and observed with a Philips model EM 300 transmission electron microscope, operating at 60 kV, with a magnification of 297,000 fold. 2.7. Batch laboratory irradiation device The laboratory UV-devise was built with the cooperation of the company Guy Daric S.A (Aubervilliers, France). This prototype contained

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M.B. Said et al. / Desalination 246 (2009) 397–408 Ballast

Extracteur

Lamp Lamp UV Réflector Petri dishe

Petri dishes

Fig. 1. Experimental setup for UV-C irradiation.

a sliding rack, with an irradiation board which could receive at the same time six Petri dishes 90 mm diameter. A germicidal low-pressure mercury vapour discharge lamp (length = 900 mm, diameter = 13 mm, power of UV emission at 253.7 nm = 55 W) with reflector could be adjusted in height above the irradiation board (Fig. 1). The lamp was supplied via electric ballast and the ozone produced in the irradiation room was removed by an extractor [5]. Incident intensity UV rays at 253.7 nm was measured using a selective detector for UV (253.7 nm) joined to a radiometer (Vilbert-Lourmat, Norme la Vallée, France). Dose was computed as the product of radiation intensity and time, and was calculated according the following equation: D=I·t

(1)

where: D, UV dose (mW s cm−2); I, intensity of UV-light (mW cm−2); t, exposure time (s). UV-C dose was regulated by controlling the exposure time. 2.8. Bacteriophage inactivation with UV-C radiation Bacteriophage inactivation with UV-C radiation was performed according the method of Kadavy et al. [10]. The bacteriophage lysates were diluted 1000-fold in sterile saline (0.85%

[wt/vol] NaCl), and 0.5 mL samples were irradiated with a germicidal UV-C lamp (length = 900 mm, diameter = 13 mm, power of UV emission at 253.7 nm = 55 W) producing a flux of 4 mW/cm2. Phage PFU was quantified immediately after irradiation. 2.9. UV-C irradiated host cells (2MP) The host a cell was grow to early exponential phase, harvested by centrifugation, and suspended in an equal volume of saline solution. For UV-C exposure, 1 mL of suspended cells was added to each of 90-mm-diameter Petri dishes, and the suspended cells were irradiated with germicidal lamp UV-C radiation (length = 900 mm, diameter = 13 mm, power of UV emission at 253.7 nm = 55 W, UV-C flux = 4 mW/cm2). 2.10. Reactivation bacteriophage procedures in darkness or visible light condition Bacteriophage was irradiated with UV-C light to reduce the number of plaque-forming unit (PFU) to approximately 0.1% of the number present in the unirradiated lysate. A 100-µL sample of UV-C irradiated or unirradiated phage was added to 100 µL of unirradiated or irradiated host cell, the bacteriophage was allowed to attach 15 min, and the infected cells

M.B. Said et al. / Desalination 246 (2009) 397–408 Table 1 The relationship between bacteriophage and host cells in different states of UV-C irradiation Phage-host cells relationships C E1 E2 E3

Bacteriophage

Host cells

– + + –

– – + +

(+) UV-C Irradiated bacteriophage and/or host cells; (–) UV-C Unirradiated bacteriophage and/or host cells. (C) unirradiated bacteriophage infecting unirradiated host cells; (E1) irradiated bacteriophage infecting unirradiated host cells; (E2) irradiated bacteriophage infecting irradiated host cells; (E3) Unirradiated bacteriophage infecting irradiated host cells.

were plated by soft-agar overlay procedure onto LBA. The plates were incubated at 37°C, and the plaque formation ability of the phage was quantified by plaque essay. Due to the low survival of cell at a high UV doses, appreciatively 106 PFU of unirradiated cells were added at the time of plating (Table 1). This allowed formation of a confluent lawn and more effective detection of phage plaques. Titers of 2MPØ UV-irradiated lysates were performed in duplicate, with one set of plates incubated in the darkness and the matching set of plates incubated in the light, where DNA damage photoreactivation was possible. The comparative levels of UV-damaged bacteriophage reactivation in the host cells were calculated by determining the reactivation factor as recommended by Kadavy et al. [10], with some modification (Eq. (2)). This factor was the ratio of the number of PFU of 2MPØ obtained after infecting the host cells (2MP) with different recombinational states of UV-C radiation conditions (Table 1) to the number of PFU obtained with bacteriophage lysate and host cells without UV-C irradiation (Control test, E1). Reactivation factor (RF) = Plaques obtained after infection in different UV-C radiation condition Plaques obtained before UV-C radiation

(2)

401

3. Results Our overall experimental design was aimed to examine the plating efficiencies (PFU per millilitre) of UV-C-irradiated bacteriophages on P. aeruginosa hosts that had been maintained in different UV-C-radiation states immediately prior to infection and to investigate the capacity of irradiated bacteriophage to restore infectivity after rest time in visible light or/and in darkness condition. The observations reported here suggest that the evaluation of phage-host relationships will provide important knowledge about the dynamic microbial population in the natural water environment [15]. 3.1. Bacteriophage virion morphology and classification Electron micrographic examination revealed that the 2MPØ virion was structurally complex (Fig. 2). The morphology of 2MPØ most closely resembles the Myoviridae family of large and structurally complex bacterial viruses that possess contractile tails with fibres [19]. Myoviri ae, followed by Siphoviridae, were the most frequently isolated morphological types in raw sewage, treated sewage and river water collected a few metres downstream from a sewage outfall [22]. 3.2. Examination of the host range of P. aeruginosa bacteriophage 2MPØ The examination of isolated 2MPØ bacteriophage revealed that this virus could produce

Fig. 2. (a) Results of the phage purification experiment showing Petri plates with phage plaques and (b) Transmission Electron Microscopy (TEM) of bacteriophage 2MPØ.

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2MPØ

Indicator Host Group I P. aeruginosa (2MP) P. aeruginosa PAO1 P. aeruginosa ATCC 15442 Group II E. coli S. typhi K. pneumoniae

a

Titersa

EOP

2 × 1011

1.00

1 × 1010 5 × 10−2 5 × 106

3 × 10−5

3 × 108 1 × 107 5 × 103

2 × 10−3 5 × 10−5 3 × 10−8

Data shown are the means of triplicate independent experiments.

plaques on both the homologous hosts (P. aeruginosa ATCC15442 and P. aeruginosa PAO1) and heterologous hosts (E. coli ATCC 1036, S. typhi ATCC 14029, and K. pneumoniae ATCC 13833). We found that the 2MPØ bacteriophage was active against most of the bacterial strains tested (Table 2, titres). This result shows the multiplicity of infection (MOI) of 2MPØ with a difference in the level of infectivity in comparison with the potential of bacteriophage’s infectivity with the original host cell of P. aeruginosa (2MP).

3.4. Viral UV-C inactivation analysis or UV-dose-response The intrinsic kinetic of 2MPØ bacteriophage inactivation as a result of exposure to UV radiation was a function of UV dose. Several mathematical relations have been developed to describe micro-organisms responses to UV irradiation. In our case, Chick-Watson’s law can be used to describe the kinetics of viral inactivation by UVC radiation (Fig. 3). Viral UV-C inactivation was 6

1.2 y = 0.4529e

1

5

R2 = 0.9243 0.8

4

0.6

3

0.4

2

0.2

1 0

0 0

40 60 80 120 240 480 720 960 1200 Dose UV-C (mW.s/cm2)

3.3. The efficiency of plating (EOP) The tested bacteriophage (2MPØ) fell into two categories with respect to EOPs on their alternate hosts (Table 2). This bacteriophage originally isolated from P. aeruginosa showed a classic response to heterologous host systems (Group II) in that their EOPs on the alternate hosts were 10−3- to 10−8-fold lower than those on the originally host used to produce the lysates (Table 2). The EOPs on the homologous hosts (Group I) were 10−2- to 10−5-fold lower than those on the originally host cell (Table 2). These observations show that 2MPØ bacteriophage possess a capacity to infect a multiple bac-

–1.1379x

U-Log10

Bacteriophage

terial hosts in different levels of infectivity [22]. Indeed, as obligate parasites, viruses depend on the host cellular machinery to propagate. Several studies have shown that the species and the bacterial host growth stage may significantly influence the viral growth cycle [10–18]. Thus, the level of bacteriophage production changes in function of hosts systems.

Nt/N0

Table 2 Influence of bacterial host on plating efficiency or infectivity of the 2MPØ bacteriophage.

Nt/N0

U-log

Exponentiel (Nt/N0)

Fig. 3. UV inactivation kinetic of 2MPØ bacteriophage infecting P. aeruginosa (2MP) inactivation following exposure to UV-C radiation according to the first-order of Chick–Watson model where: y: reduction = Nt/N0 with Nt = the number of infectious viral particles at time t (time of UV exposure), N0 = the number of infectious viral particles at time zero (no UV radiation applied); A = event corresponding to bacteriophage retaining infectivity following UV-C irradiation; k = inactivation rate or slope of inactivation curve; x = InT with I,: UV-C intensity (mW/cm2); T, exposition time (s); n = threshold seriesevent model equal to 1. Where error bar are not shown, differences between duplicates were not detected.

M.B. Said et al. / Desalination 246 (2009) 397–408 Table 3 Ability of series–event model to 2MPØ bacteriophage dose-response behavior at varying values of n, where: n, threshold series–event model; k, inactivation rate; R2, coefficient of determination Equation of series event model

n

Nt/N0 = e−kIt Nt/N0 = e−kIt (1 + kIt) Nt/N0 = e−kIt (1 + kIt + (kIt)2/2) Nt/N0 = e−kIt (1 + kIt + (kIt)2/2 + (kIt)3/6)

1 2 3 4

K

R2

1.1379 0.984 0.8744

0.9243 0.9415 0.9463

0.794

0.9454

defined by the following equation: Nt/N0 = A exp(−k In t), where Nt = the number of infectious viral particles at time t (time of UV exposure), N0 = the number of infectious viral particles at time zero (no UV radiation applied); A, event corresponding to bacteriophage retaining infectivity following UV-C irradiation; k, inactivation rate or slope of inactivation curve; I, intensity of UV light energy (milliwatts per square centimetre); t = exposure time (s) and n, threshold level of series-event model (n = 1 in the first order of Chick–Watson model). The constants k and A were determined by linear regression.

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In addition of Chick–Watson model, the inactivation kinetic or dose-response relation of 2MPØ bacteriophage is described by series-event kinetics. An ‘‘event’’ is assumed to represent a discrete unit of damage [21]. Bacteriophage continue to collect damage as long as it is exposed to UV radiation. It has been hypothesized that a threshold event level n exists; phage that accumulate damage at event level n or higher are assumed to be inactivated, while phage with damage at event level n–1 or lower are assumed to retain infectivity. According to series–event model (Table 3Fig. 4), application of UV-C dose equal to 720 mW.s/cm2 is required to reduce viral infectivity to 4 U-Log10; thus, the reduction of 99.99% of the viral infectivity, in the absence of reactivating conditions. The ability of the series-event model to fit measured data was quantified in terms of the higher value of the coefficient of determination R2 [21]. Based on this criterion, the series-event model with n = 3 was judged to provide the best fit to the data (R2 = 0.9463) with a coefficient of inactivation k equal to 0.874 (Table 3).

R2 = 0.9463

Nt/N0

1

5

0.8

4

0.6

3

0.4

2

0.2

1

U-Log10

y = 0.8136e

6

–0.8744x

0

0 0

40 60 80 120 240 480 720 960 1200

Reactivation Factor (RF)

1 1.2

C

E1

E2

E3

0,1 0,01 0,001 0,0001 RF (DARK CONDITION)

RF (PHOTOREACTIVATION)

Dose UV-C (mW.s/cm2)

n1

n2

n3

U-log

Exponentiel (n3)

Fig. 4. UV inactivation kinetic of 2MPØ bacteriophage infecting P. aeruginosa (2MP) inactivation following exposure to UV-C radiation according to the model of Series event where: y, reduction = Nt/N0 and x = IniT with, I, UV-C intensity (mW/cm2); T, exposition time (s); n, threshold series–event model; i,series event level.

Fig. 5. The reactivation factor of bacteriophage 2MPØ infecting P. aeruginosa (2MP), under darkness or visible light incubation. With the reactivation factor of: (C) Unirradiated bacteriophage infecting unirradiated host cells; (E1) irradiated bacteriophage infecting unirradiated host cells; (E2) irradiated bacteriophage infecting irradiated host cells; (E3) unirradiated bacteriophage infecting irradiated host cells.

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3.5. Reactivation of bacteriophage in visible light or darkness condition and restore of viral infectivity

Repair of viral DNA damage was quantified by reactivation experiments (Table 1 and Fig. 5). For the UV- inactivated bacteriophage, the reactivation factor obtained with these host cells exposed in visible light was 50-fold greater than that obtained in darkness condition. The photoreactivation level was not comparable to the control test but the result shown that infectivity of 2MPØ phage was photorestorable after absorption on their original host cells of P. aeruginosa (2MP). Under the influence of visible light, selected fraction of host bacteria; infected by inactive phage, become capable of producing active phage. The host cell therefore plays an essential role in the photoreactivation of phage and the restoration of viral infectivity by owing certain of its metabolic capabilities. As the light-activated enzyme photolyase will repair DNA damage caused by UV-C radiation [8], this enzyme was most likely responsible for restoring infectivity of tested bacteriophage in this experiment.

The UV-C radiation have a deleterious effects on both bacteriophage and bacteria replication. The decrease of virus infectivity was directly correlated with the biological state of the host bacteria influenced by UV radiation and the accumulation of the photoproduct either in bacteriophage or in the host cells. The reactivation of 2MPØ phage can be explained by the great significant resistance of the host cell to the conditions of stress materialized by UV-C radiation and its capacity to undertake the reactivation of the corresponding phage. These results strongly suggest that within the irradiated “infected cell”, the reactivation of the virus is closely related to its host cells [11]. If we keep in mind that, extracellular phage being not restorable, restoration of the phage is secured by some metabolism of the host cells. UV-C-irradiated host cells supported an approximately fourfold the induction of reactivation of UV-damaged 2MPØ in visible light condition compared with that obtained in darkness. These results demonstrate that the infectivity of virus which could not be repaired by original host cells in the darkness could be repaired by the same bacteria under photoreactivating condition. This data clearly demonstrate that light-dependent repair can restore infectivity to a significant viral particles damaged by UV-C radiation.

3.5.2. Reactivation and, enhanced plating efficiencies for UV-damaged bacteriophage infecting UV-irradiated host cells (E2)

3.5.3. Reactivation and, enhanced plating efficiencies for unirradiated bacteriophage infecting UV-irradiated host cells (E3)

The result of this experiment shown that, the reactivation coefficient in the visible light and in darkness of 2MPØ phage was less significant than for the first test (E1) when the bacteria was unirradiated by UV-C radiation (Fig. 5). The decrease of the reactivation level was explained by that not only the bacteriophage was inactivated, but the bacteria as well are damaged but they are still capable of a low phage production.

The concentration of unirradiated 2MPØ bacteriophage infecting the original host of P. aeruginosa post-irradiated by UV-C light, increased by 20 times in samples exposed to photoreactivating light relative to those in darkness (Fig. 5). The results showed the positive interaction between the bacteriophage and UV-damaged P. aeruginosa. The bacteria strain was found to operate under UV stress and can allow the viral life cycle.

3.5.1. Reactivation and, enhanced plating efficiencies for UV-damaged bacteriophage infecting unirradiated host cells (E1)

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4. Discussion 4.1. Bacteriophage virion morphology and classification Electron microscopy examination revealed that the 2MPØ virion had a complex structure and most closely resembled bacteriophages of Myoviridae family. Myoviridae are a diverse group of phages that are characterized by their contractile tails. Myoviridae are able to infect both Gram-positive and Gram-negative bacteria [22]. Virions not enveloped, tailed, head. Head separated from tail by a neck, tail complex. Reactivation of UV-irradiated bacteriophage 2MPØ of P. aeruginosa was quantified in cells that were in distinct irradiation conditions at the moment of infection (UV-irradiated or unirradiated host cells). The reactivation is a phenomenon whereby inducible host cell DNA repair systems respond to UV-C irradiation and eliminate damage from the chromosomes of both the irradiated host and infecting UV-C-inactivated bacteriophages, ultimately allowing replication of the now-repaired virus and the death of the host cell [18–14]. 4.2. The inactivation kinetic of P. aeruginosa: UV dose-response A UV dose-response relationship for inactivation of 2MPØ bacteriophage is shown in Fig. 3. Inactivation behavior was described by series– event kinetics. Table 3 and Fig. 4 illustrate the ability of the series–event model to fit observed dose-response behavior. The UV-C inactivation or the loss of infectivity of 2MPØ bacteriophage was expressed as a function of the initial PFU count (Nt/N0). The results of the dose/inactivation curve of 2MPØ bacteriophage showed that the first instants of exposure to UV-C radiation represent an essential factor in ensuring a high inactivation rate. Indeed, the rate of bacteriophage inactivation was very important in this interval of time, especially at dose lower than 720 mW s/cm2 when we can note

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the reduction of 4 U-Log10 of 2MPØ infectivity. When we increase the value of UV dose beyond 720 mW s/cm2, and the inactivation rate was slowed down and tends to become constant. The lethal dose determined in this study was relatively higher and it’s directly related to the original host cell of P. aeruginosa that shown a high tolerance to UV-C radiation. 4.3. Reactivation of bacteriophage in visible light or darkness condition and restore of viral infectivity Given that the infectivity of virus is extremely sensitive to UV radiation, and that damage to viral infectivity is proportional to the UV radiation received (UV dose) [20], what is the potential of bacteriophage for reversing DNA damage radiation, restoring of infectivity and replication phases? 4.3.1. Reactivation and, enhanced plating efficiencies for UV-damaged bacteriophage infecting unirradiated host cells (E1) During the exposure of 2MPØ bacteriophage to UV-C radiation, the DNA-phage was damaged. Consequently, the phage was unable to be reactivated except in the presence of the host cell. In this situation, the virus will penetrate inside the host cell. While referring to the bibliographical data which affirms that one of the mechanisms of persistence of the viruses in the water environment in spite of the significant quantity of solar irradiation is the lysogenic replication [10–17] you can predict the fate of the nucleic acid bacteriophage damaged by UV-C radiation injected inside the host cell. Probably, this last will be inserted in the bacterial genome which must necessarily be recA + [12], this insertion will induce the S.O.S system of DNA-repair host cell. By this and other mechanisms of DNA repair systems such as photoreactivation loaded by the enzyme photolyase, the phage can finish its viral cycle

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and repair its genetic material. The reactivation capacity expressed by bacteriophage 2MPØ suggests that DNA-repair host cells systems were play an important role in virus replication and the maintenance of viral infectivity [7]. This reactivation cannot concern all phages. Indeed, the most resistant viruses were which finish their cycle of reactivation and release free infectious virions. When P. aeruginosa host cell (2MP) was infected with UV-irradiated bacteriophage and incubated under conditions in which photoreactivation was possible, a nearly 50-fold increase in plaque-forming ability was observed in comparison to samples of the same infected cells incubated in darkness. These experiments revealed that cyclobutane pyrimidine dimers were created by UV-C irradiation of bacteriophage 2MPØ and that this class of DNA damage was subject to repair by host cells photoreactivation mechanism. Photoreactivation is currently defined as the removal of cyclobutane pyrimidine dimers by photolyase activity [16-20]. This indicates that photoreactivation was much more efficient than dark repair in restoring infectivity to viruses damaged by UV radiation. It is possible that there was too little damage to the phage DNA to induce the host dark-repair system. Photoreactivation uses photons and an enzyme to directly reverse UVDNA damage and is energetically less costly than dark repair, which requires the formation of new nucleotides and the combined action of several enzymes [20]. 4. 3. 2. Reactivation and, enhanced plating efficiencies for UV-damaged bacteriophage infecting UV-irradiated host cells (E2) After infecting a UV-C-irradiated host, the irradiated bacteriophage 2MPØ was able to undertake inducible dark repair and photoreactivation to recover infectivity. In this situation, the virus will penetrate inside the cell since the exposure to UV-C does not have an influence on the

attachment and the penetration of the phage inside the host cell. Indeed, UV-C do not have an action on host cell surface receptors and thus on the adsorption to the host cells. The result of this experiment shows a reactivation of bacteriophages infectivity with a coefficient of reactivation less significant than registered for the first test (E1). The phages’ reactivation can be explained by the hyper-resistance of the host cell to the stress conditions materialized by the radiations UV-C and its potential to undertake the reactivation of the UV- damaged phage. After UV radiation of both bacteriophage and host bacteria, the induction of various mechanisms of DNA-repair host cell, increase directly the level of DNA repair in the host cell and indirectly increase the reactivation of bacteriophage that make use of the host cells machinery for self-restoration. According to Kadavy et al. [10], the lysogenic bacterial was more resistant to UV than the not lysogenic one. In fact, the lysogenic state makes the bacterial cell more competitive, and adapted to the stress conditions and thus can survive longer compared to a nonpermissive bacterial cell. 4.3.3. Reactivation and, enhanced plating efficiencies for unirradiated bacteriophage infecting UV-irradiated host cells (E3) The increasing of plaque-forming ability after infecting post- irradiated host cell can be explain in part by the induction of lytic cycle in the irradiated host cells. However, the induction of the lytic cycle concerns a part of lysogenic bacteria [17]. For consequence, this hypothesis was rejected to explain the increasing of ability forming plaques after infection irradiated host cell with unirradiated bactriophage. It is possible that an apparent enhancement in bacteriophage reactivation after infection the irradiated host cells could result from (i) more

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productive interactions between bacteriophages and bacterial cell receptors or (ii) more efficient transit of the periplasm by the phage DNA, due possibly to the existence of a more appropriate membrane potential for optimal entry of phage DNA in these hosts [14]. The enhanced of DNA damage repair capacity (photoreactivation and dark repair), actually expressed either in irradiated or unirradiated host cells at the time that infected by phage explain in part, the persistence and the abundance of all viruses in the hostile environment. In fact, the different pathways of DNA repair mechanisms of host cells and the bacteriophage parasitism’s, extend the infective lifetimes of bacteriophages in environments exposed to different factors of stress such as UV radiation. In spite of their small size and their inability of survival without their respective host cells, bacteriophage has shown resistance and flexibility under the hostile environment such as UV-C radiation.

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and the large spectrum of infectivity of bacteriophages isolated from aquatic environment (The Efficiency of Plating (EOP) or Multiplicity of Infection (M.O.I)), gives the idea to use bacteriophages as a bio-disinfectant. The bio-disinfection procedure can have many advantages if you can construct bacteriophages genetically modified which have a direct action on the bacterial resistant to disinfecting process such as P. aeruginosa by the inhibition of the regulation genes like the recA gene which has a central role in the induction, and the starting of the several DNA-repair systems and bacterial reactivation, and mediating mortality of their host. Acknowledgments We gratefully acknowledge the cooperation of Ackermann H.W for help with the Electron Microscopy of bacteriophage lysates. I also thank Hassen A. for valuable discussions and helpful criticism.

5. Conclusion Our results clearly demonstrate that photoreactivation mechanism’s host cells of P. aeruginosa is important and may be essential for maintaining high concentrations of infectious bacteriophage in different conditions of UV-C irradiation. Photoreactivation could be the mechanism allowing for the persistence of viruses in surface waters. The data presented here indicate that the loss of viral infectivity resulting from solar radiation may be largely offset by in first order host-mediated light-dependent repair and secondary by host-mediated light-independent repair of the damaged DNA. 6. Perspectives The use of the bacteriophages was developed as alternative anti-infection modalities has become one of the highest priorities of modern medicine and biotechnology. The phagotherapy

References [1] B.H. Al-Adhami. R.A.B. Nichols. Kusel. J.R. O’Grady and Smith H.V. Detection of UV-induced thymine dimers in individual Cryptosporidium parvum and Cryptosporidium hominis oocysts by immunofluorescence microscopy. Appl. Environ. Microbiol., 73 (2007) 947–955. [2] K.R. Calci. W. Burkhardt III. W.D. Watkins and S.R. Rippey. Occurrence of male-specific bacteriophage in feral and domestic animal wastes, human feces, and human-associated wastewaters, Appl. Environ. Microbiol., 64 (1998) 5027–5029. [3] N.J. Fuller. W.H. Wilson. I.R. Joint and N.H.Mann. Occurrence of a sequence in marine cyanophages similar to that of T4 g20 and its application to PCRbased detection and quantification techniques, Appl. Environ. Microbiol., 64 (1998) 2051–2060. [4] C.P. Gerba. D.M. Gramos and N. Nwachuku. Comparative inactivation of enteroviruses and adenovirus 2 by UV light, Appl. Environ. Microbiol., 68 (2002) 5167–5169. [5] A. Hassen, M. Mahrouk, H. Ouzari, M. Cherif, A. Boudabous and J. Damelincourt, UV disinfection of treated waste water in a large-scale pilot plant and

408

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

M.B. Said et al. / Desalination 246 (2009) 397–408 inactivation of selected bacteria in a laboratory UV devise, Bite, 1464 (2000) 1–10. J.L. Jacobs and G.W. Sundin. Effect of Solar UV-B radiation on a phyllosphere bacterial community, Appl. Environ. Microbiol., 67 (2001) 5488–5496. C.E. James, K.N. Stanley, H.E. Allison, H.J. Flint, C.S. Stewart, R.J. Sharp and J.R Saunders. Lytic and Lysogenic infection of diverse Escherichia coli and Shigella strains with a verocytotoxigenic bacteriophage, Appl. Environ. Microbiol., 67 (2001) 4335–4337. E.C. Jensen, H.S. Schrader, B. Rieland, T.L. Thompson, K.W. Lee, K.W. Nickerson and T.A. Kokjohn, Prevalence of broad-host-range lytic bacteriophages of Sphaerotilus natans, Escherichia coli, and Pseudomonas aeruginosa, Appl. Environ. Microbiol., 64 (1998) 575–580. F. Joux, H.J. Wade, P. Lebaron and D.l. Mitchell, Marine bacterial isolates display diverse responses to UV-B radiation, Appl. Environ. Microbiol., 65 (1999) 3820–3827. D.R. Kadavy, J.J. Shaffer, S.E. Lott, T.A. Wolf, C.E. Bolton, W.H. Gallimore and E.A. Kokjohn, Bacteriophage reactivation levels, Appl. Environ. Microbiol., 66 (2000) 5206–5212. O. Bergh, G. Borsheim, G. Bratbak, and M. Heldal, High abundance of viruses found in aquatic environments. Nature, 340 (1989) 467–468. J.J. Kim and G.W. Sundin, Construction and analysis of photolyase mutants of Pseudomonas aeruginosa and Pseudomonas syringae: contribution of photoreactivation, nucleotide excision repair, and mutagenic DNA repair to cell survival and mutability following exposure to UV-B radiation, Appl. Environ. Microbiol., 67 (2001) 1405–1411. K. Oguma, H. Katayama, H. Mitani, S. Morita, T. Hirata and S. Ohgaki, Determination of pyrimidine dimers in Escherichia coli and Cryptosporidium parvum during UV light inactivation, photoreactivation, and dark repair, Appl. Environ. Microbiol., 67 (2001) 4630–4637.

[14] J.J Shaffer, L.M. Jacobsen, J.O. Schrader, K.W. Lee, E.L. Martin and T.A. Kokjohn. Characterization of Pseudomonas aeruginosa bacteriophage UNL-1, a bacterial virus with a novel UV-A-inducible DNA damage reactivation phenotype, Appl. Environ. Microbiol., 65 (1999) 2606–2613. [15] F. Torrella and R.Y. Morita, Evidence by electron micrographs for a high incidence of bacteriophage particles in the waters of Yaquina Bay, Oregon: ecological and taxonomical interpretations, Appl. Environ. Microbiol., 37 (1979) 774–778. [16] K.E. Wommack and R.R. Colwell, Virioplankton: viruses in aquatic ecosystems, Microbiol. Mol. Biol. Rev., 64 (2000) 69–114. [17] K.E. Wommack. R.T. Hill, T.A. Muller and R.R. Colwell, Effects of sunlight on bacteriophage viability and structure, Appl. Environ. Microbiol., 62 (1996) 1336–1341. [18] M.A. Woody and D.O. Cliver, Effects of temperature and host cell growth phase on replication of Fspecific RNA coliphage Q, Appl. Environ. Microbiol., 61 (1995) 1520–1526. [19] H.W. Ackermann, Tailed bacteriophages: the order caudovirales, Adv. Virus Res., 51 (1998) 135–201. [20] G. Markus, W. Weinbauer Steven, A. Wilhelm Curtis, Suttle and D. Randy Garza, Photoreactivation compensates for UV damage and restores infectivity to natural marine virus communities, Appl. Environ. Microbiol., 63 (1997) 2200–2205. [21] K. Chiu. D.A. Lyn, P. Savoye, E.R. Blatchley III and Associate Member, ASCE. Integrated UV disinfection model based on particle tracking. Environ. Eng., 125 (1999) 7–16. [22] D. Prangishvili, H.P. Arnold, D. Gotz, U. Ziese, I. Holz, K. Jakob Kristjansson and A. Wolfram Zillig, A novel virus family, the Rudiviridae: structure, virus–host interactions and genome variability of the Sulfolobus viruses SIRV1 and SIRV2, Genet. Soc. Amer., 152 (1999) 1387–1396.