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Therapeutic efficacy of lytic bacteriophage PSAE-1 against Salmonella Abortusequi in Guinea pig model.

Salmonella enterica subspecies enterica serovar Abortusequi, an host-adapted Salmonella serovar recognized as a cause of late term abortion in mares, still birth, birth of weak and debilitated new born and a range of other clinical conditions leading to huge economic losses to the equine breeders and stud owners (1,2). Salmonella Abortusequi not only associated with genital tract infections but also leading to major breeding inconsistencies and turning up fertile mare to barren (3). A number of antibiotics are available in the market against the infections caused by S. Abortusequi and other Salmonella sp. But, in recent time due to indiscriminate use of antibiotics, multi drug resistant S. Abortusequi isolates of Indian origin have been discovered and these isolates have also shown resistance against the antibiotics that were not even used earlier (4). Thus, the global emergence of MDR against the bacterial pathogens and their mild response to various antibiotics has motivated scientists to exploit alternative anti-infection strategies to combat the problem. Under these challenging circumstances, therapeutic uses of bacteriophages may turn out to be a valuable approach. Bacteriophages are the viruses infecting bacteria and have great potential to be used as therapeutic agents against the bacterial diseases. Use of phages against MDR bacteria, opens up a new possibility of treating bacterial diseases (5). Phage therapy has been proved successful and has emerged as a promising agent in the treatment of bacterial infections including Salmonella (6,7). In view of the problem of drug resistance and success reports of phage therapy, the present study was planned to evaluate safety and therapeutic efficacy of the bacteriophage against S. Abortusequi in guinea pig model.

MATERIALS AND METHODS

Bacterial strains

Salmonella Abortusequi strains E-156 (SAE-156), E-155, E-151, E-142 and E-189 were procured from Division of Biological standardization, IVRI, Izatnagar. Master seed lots of all strains were prepared after their revival, purification and confirmation by biochemical and serological tests. Viable counts of SAE-156 were carried out by standard serial dilution method in terms of Colony Forming Unit (CFU/ml). The organism was passaged 3 times in guinea pig to increase it virulence and re-isolation of the organism was done from heart blood, Salmonella Typhimurium, Salmonella Entertidis, Salmonella Pullorum E-19, Salmonella Gallinarum, Escherichia. coli used for host range determination of PSAE-1 were procured from National Salmonella Centre, Bacteriology and Mycology Division, IVRI, Izatnagar and Pasturellla multocida from B&M Division IVRI, Izatnagar whereas Staphylococcus aureus, and Streptococcus sp from Type Culture Lab of Division of Biological Standardization, IVRI, Izatnagar.

Bacteriophage

Previously isolated Salmonella Abortusequi phage (PSAE-1) (3) used for the present study were procured from Division of Biological Standardization.

Animals

Apparently healthy female adult guinea pigs (Dunkin-Hartley) were procured from LAR section, IVRI, Izatnagar. The animals were kept under conventional housing condition and provided feed and water ad libitum.

Revival, purification and preparation of phage stock

The stock suspension of the phage PSAE-1 was filtered by passing through 0.22 [micro]m PVDF filter (Millex-HV) and tested for presence of lytic activity against the selected SAE-156 by spot test as per the method by Rahaman et al. (8) and O'flaherty et al. (9) with some modifications. Briefly, 100 [micro]l of the 6-8 hrs pure growth of SAE-156 isolate in NZCYM broth (NewZeland Casamino Yeast Medium: Sodium chloride, Casein acid hydrolysate, Yeast extract, Magnesium sulphate-Difco U SA) was spread over the surface of separate NZCYM agar (2% agar) plate. After 5 minutes, 4 [micro]l of filtered phage suspension was aseptically placed over a surface of the agar and incubated at 31[degrees]C for 16-18 hrs. The plates were observed for zones of clarification (lytic zone). Phage PSAE-1 was further confirmed and tested for ability to produce plaque by soft agar over lay method (10). Plaques were further purified by liquid culture method (11). The purified phage was harvested by using about 2 ml of Salt Magnesium buffer (SM buffer: 50 mM Tris-Cl [pH: 1.5], 0.1 M NaCl, 8 mM MgSO. 1[H.sub.2]O, 0.01% Gelatin). The harvested suspension was clarified, filtered and used for future experiments.

Host range of PSAE-1 and enumeration of plaque forming unit (PFU)

Host-range of purified phage PSAE-1 was determined by spot test as described earlier (9) against different homologus strains of S. Abortusequi (E-156 (SAE-156), E-155, E-151, E-142 and E-189) and heterologus strains of Salmonella viz; S. Typhimurium, S. Entertidis, S. Pullorum E-19, and S. Gallinarum. PSAE-1 was also tested for across genus lytic activity against E. coli, S. aureus, and Streptococcus sp. The phage count of the P SAE-1 was determined by combination of standard serial fold dilutions method and agar overlay technique in terms of plaque forming unit (PFU).

Determination of multiplicity of infection (MOI) of phage

The optimum phage to bacteria ratio required to achieve 100% lysis of the indicator strain within shortest period of time termed as MOI of the phage. MOI was conducted as per the method of Mishra et at. (12) with slight modifications. The lowest phage-bacteria ratio which showed complete lysis of the organism or highest lytic activity in shortest period of time was considered as MOI of PSAE-1.

Generation of therapeutic phage preparation

Phage preparation intended for therapeutic use was produced employing standardized optimum conditions. Briefly, 0.1 ml of suspension of 14-16 h old SAE-156 seeded in 100 ml NZCYM broth. After 24 h, a volume of phage PSAE-1 was directly added in culture broth, so that phage: bacteria ratio became equivalent to MOI and incubated at 37[degrees]C with intermittent shaking until the turbidity of suspension was reduced to about 90%. Preparation was centrifuged at 8000-10000g for 15 min and filtered through 0.22 im PVDF filter. This filtrate was used as therapeutic preparation in animal experimentation.

Safety of preparation in guinea pig

Safety tests of PSAE-1 preparation intended for in vivo trials were conducted in guinea pigs as per method of Abhishek et at. (13) with modifications. Six adult healthy guinea pigs two in each injected with a 1 ml of Phage containing [10.sup.8] PFU/ml through intra muscular (I/M), subcutaneous (S/C), and intra peritoneal (I/P) route respectively. Any untoward reaction or mortality was noted up to 20 days. Endotoxin content of preparation was determined by LAL assay using commercially available Kit (Lonza) as per the instructions of the manufacturer.

Therapeutic efficacy (TE) trial of phage

The therapeutic trail of the phage PSAE1 was determined as per method of Prajapati et at. (1) with modifications. Four group ([P.sub.UT]-Phage untreated, [P.sub.T6]-Phage treatment at 6 hrs, [P.sub.T12]-Phage treatment at 12 hrs and [P.sub.T24]-Phage treatment at 24 hrs) of healthy adult female guinea pig each containing six animals were infected with [10.sub.7] of SAE-156 in 0.1 ml of Phosphate buffer saline (PBS: NaCl. 8.5 g; KHP[O.sub.4] 1.0 g; KHP[O.sub.4] 2.0 g; distilled water: 1000 ml; pH 7.2). by I/P route. Each animal of [P.sub.T6], [P.sub.T12] and [P.sub.T24] group received [10.sup.5] PFU/of phage in 0.1 ml of PBS at different time intervals. [P.sub.UT] group inoculated with 0.1 ml of PBS I/P (Table1). All animals were sacrificed humanly under C[O.sub.2] and chloroform method 6 days post-infection and spleens were aseptically collected, weighed and homogenized individually in 9 times to weight of spleen in sterilized PBS. Four serial tenfold dilutions (1/10, 1/100, 1/1000, and 1/10000) of the homogenate were made. 0.1 ml of each dilution was spread plated on Hektoen Enteric agar (HE agar medium: Difco, USA) plates, incubated at 37[degrees]C for 12 hrs and average total spleen counts were determined. Numbers of Salmonella per spleen was first recorded as X and expressed as Y after the following transformation Y = log (X/log X) (14). The data of present study were analyzed using 'SPSS 20'software package using one way analysis of variance and level of significance among the treatments.

RESULTS AND DISCUSSION

All S. Abortusequi strains used in the present study, on revival exhibited typical morphological, biochemical and serological characteristics (4, 12: e, n, x antisera) of S. Abortusequi (Fig1a and 1b). The average viable count of 12-14 h incubated NZCYM broth cultures of selected indicator strains SAE-156 was determined as ~[10.sup.8] CFU/ml.

Lytic range of phage is important criteria in the selection of phage intended to be used for therapeutic application. Phage PSAE-1 was found lytic to all S. Abortusequi strains tested but not to other heterologus strains of Salmonella viz S. Typhimurium, S. Entertidis, S. Pullorum E-79, S. Gallinarum and other bacterial pathogens E. coli, P. multocida, S. aureus, and Streptococcus sp. The first mandatory step for developing a universal phage-based therapeutic system is the availability and ability of phages that can lyse the target pathogen/pathogens. Phage PSAE-1 was reoprted to be first lytic phage against Salmonella Abortusequi in India (3) was revived, purified and produced in bulk. Phage PSAE-1 showed clear lytic zone against SAE-156 and other strains by spot test (Fig 2) and produced clear plaques (Fig 3). The phage count/titre (PFU/ml) of the phage PSAE-1 was found to be [10.sup.8] PFU/ml.

The effectiveness of the phages in reducing bacterial colonization/infections is depending on phage concentration and it may vary at different incubation temperatures and duration of incubation (15). Therefore, to standardize the optimum phage to bacteria ratio an experiment on MOI was conducted. The MOI of phage required to achieve 100% lysis of the indicator strain SAE156 within 180-200 minutes was 1:[10.sup.4]. In a similar study by Ramchandra et at. (16) who reported optimum lytic efficacy of Pasturetta phage against P muttocida was 1:100. The findings of Mishra et at. (12) supports our work who reported that minimum of 0.01 MOI is required for optimum lysis of Staphytococcus aureus associated with goat mastitis. Hungaro et at. (7) conducted MOI trails on different temperatures reported significant difference in the reduction of growth of Satmonetta Entertidis depending upon MOI. The study on MOI of phages indicates that it may vary depending upon the phage and the target bacteria.

About 15 ml of the therapeutic preparation was generated employing standardized optimum conditions and re-tested for presence of lytic effect by spot test. The endotoxin content of preparation was found less than 2 EU/ml.

In clinical settings, phages appear to be safe as there have been no reports of complications associated with their use but it becomes imperative to ascertain the safety of phage before subjecting them to in vivo trials. Safety test of the Phage PSAE-1 was conducted in guinea pigs and it was found safe through various routes. The present study showed the concordance with the previous studies conducted on mice and human to test the safety of E. coli T-4 phages (17,18). Likewise, Chilamban et al. (11) reported that Staphylococcus aureus phage do not showed any untoward reaction in mice through intra-mammary route. The findings of Carlton et al. (19) are also in accordance with our observations who tested safety of Listeriaphage P100, in rats with high doses and no measurable effects. Abhishek et al. (13) reported that crude & purified phage preparations, as well as lytic enzyme of phage SA1 against Staphylococcus aureus (IVRI-82) did not showed any adverse effects on mice through subcutaneous route. None of the reports mentioned significant phage related undesirable side effects. But, it would be prudent to ensure further the safety of therapeutic phages before using them as therapeutic agents.

In recent time bacteriophage therapies have been reemerging as potential candidate as an alternate to antibiotics (20,21). Phages have been used widely for treatment of several bacterial infections including Salmonella infections (22). Purified phage PSAE-1 was administered at different time interval viz. 6 hour, 12 hour and 24 hrs after challenge with a dose of [10.sup.5] PFU/ml through I/P route. Therapeutic index of phage treated group are significantly higher ([P.sub.T6]: 0.91, [P.sub.T12]: 1.36, [P.sub.T24]: 0.70) in comparison to untreated control (Table 3). Phage treatments at different time duration are significantly reducing the average total spleen counts of infected organism but the reduction is maximum when the therapeutic inoculation of phage was given at mid phase of infection i.e with in 12 hrs (Table 3). The average total spleenic counts of group [P.sub.T6] and [P.sub.T24] were significantly reduced as compare to untreated control ([P.sub.UT]) though this reduction was little bit more significant in [P.sub.T6] group (Table 2). All untreated-challenged controls showed presence of its challenge organism in spleen in a range >[10.sup.6]. Observations of our study is supported by the work of Smith and Huggins (23), who reported that anti-K1 bacteriophages were able to lyse K1+ E. coli when administered by intramuscular injection at the same time or eight hours after, infection with E. coli. Biswas et al. (24) reported that vancomycin-resistant Enterococcus faecium-infected mice were successfully treated with phage therapy just 45 minutes after infection and five hours after infection but only 50% recovery was seen up to 18 or 24 hours infection. Capparelli et al. (25) found that 100% of mice were died in control group challenged with a lethal dose of CFU ([10.sup.7]) of S. enterica serovar Paratyphi B whereas all mice in phage [empty set] 1 treated group were survived. The observations of therapeutic trial of Phage Lys SA4 in mice by Mishra et al. (26) partially supports our findings who reported that mean viable count of Staphylococcus aureus in the spleen of phage treated mice were considerably low when phage treatment was given after 24 hrs of challenge. Hong et al. (21) reported that mortality and re-isolation of Salmonella Gallinarum decreased significantly in challenged chickens treated with the bacteriophages. The findings of Prajapati et al. (14) on Brucella phage in reducing spleen count of phage treated group after 48 hrs of challenge with B. abortus544 partially supports our work. Recently, large no of studies on phages has been conducted against various bacterial agents have proved the therapeutic potentials of lytic phages (22). Our work supports past studies and indicates that further research with Salmonella Phages may widen the scope of useful applications of this biological agent in field conditions as therapeutics.

CONCLUSION

The Present study indicates that phage can be used as an effective mean to reduce the colonization of S. Abortusequi in the guinea pigs. Phage preparation was found safe in experimental animals through various routes of inoculation at the titre of [10.sup.8] p.f.u/ml. Therapeutic trails suggest that phage preparation can be used for first phase trials in target animals. Preliminary studies are encouraging and further study is required to establish more safety, specificity, appropriate dosage and long term effectiveness of phages in target animals.

ACKNOWLEDGMENTS

The authors are thankful to the Director, Indian Veterinary Research Institute, Izatnagar, U.P., for providing sufficient fund and laboratory facilities to conduct this research.

Ethical approval

The animal ethics committee of (IAEC) Indian Veterinary Research Institute, deemed university approved the study. All animals were kept under standard management conditions and sacrificed at the end of experimentation for ethical reasons by standard method recommended by IAEC/CPCSEA.

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(21.) Hong, S. S., Jeong, J., Lee, J., Kim, S., Min, W., Myung, H. Therapeutic Effects of Bacteriophages Against Salmonella gallinarum Infection in Chickens. J. Microbiol. Biotechnol. 2013; 23(10): 1478-1483

(22.) Bielke, L.R., Tellez, G., Hargis, B.M. Successes and Failures of Bacteriophage Treatment ofEnterobacteriaceae Infections in the Gastrointestinal Tract of Domestic Animals, Bacteriophages, 2012:159-178 Dr. Ipek Kurtboke (Ed.), ISBN: 978-953-51-0272-4, InTech, Available from: http://www.intechopen.com/books/bacteriophages/ successes-and-failures-of-bacteriophagetreatment-ofenterobacteriaceae-infections-in-the-gastrointe

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(26.) Mishra, A.K., Rawat, M., Abhishek., Kannan, Suresh. Charcterization and lytic activity of endolysin induced by bacteriophage SA4 against mastitogenic isolates of Staphylococcus aureus of bovine origin. Indian Vet. J., 2012; 89(6): 24-26

Abhishek [1], R.K. Agarwal [1], B. Kumar [2], A.K. Mishra [3], Anjay [4], A. Priyadarshini [1], S. Kumar [1], C. Prakash [1], J.K. Chaudhary [5] and M. Rawat [6]

[1] Division of Veterinary Bacteriology and Mycology, Indian Veterinary Research Institute Izatnagar, Bareilly (U.P.)--243122, India.

[2] Division of Biological Products, Indian Veterinary Research Institute Izatnagar, Bareilly (U.P.)--243122, India.

[3] Goat Health Division, Central Institute for Research on Goats (CIRG), Makhdoom, Farah-281122.

[4] Division of Veterinary Public Health, Indian Veterinary Research Institute Izatnagar, Bareilly (U.P.)--243122, India.

[5] Division of Livestock economics, statistics and Information Technology Indian Veterinary Research Institute, Izatnagar, Bareilly (U.P.)--243122, India.

[6] Division of Biological Standardization, Indian Veterinary Research Institute Izatnagar, Bareilly (U.P.)--243122, India.

(Received: 07 April 2015; accepted: 16 June 2015)

* To whom all correspondence should be addressed. Tel.: +91-9456600623; E-mail: abhivbm@gmail.com

Table 1. Schedule for challenge and treatment of phage.

Group                No of    Challenge dose
                    animals      (0.1 ml)

Untreated control               101 CFU/ml
  [P.sub.UT]
[P.sub.T6]             6        101 CFU/ml
[P.sub.T12]            6        101 CFU/ml
[P.sub.T24]            6        101 CFU/ml

Group                  Route of      Time of phage treatment
                    administration   after challenge (0.1ml)
                                     [10.sup.5] PFU/ml, I/P

Untreated control        I/P           No phage treatment
  [P.sub.UT]
[P.sub.T6]               I/P               After 6 hrs
[P.sub.T12]              I/P              After 12 hrs
[P.sub.T24]              I/P              After 24 hrs

Table 2. Total spleen counts of Satmonetta Abortusequi
of individual guinea pig

Group Animal no     Total spleen counts(X) of Satmonetta
                    Abortusequi at 1:1000 dilution

                      1        2        3       4        5        6

Untreated control   240000   285000    NC     290000   288000   221000
  ([P.sub.UT])
[P.sub.T6]          60000    10000    18000   34000    30000    29000
[P.sub.T12]          1000     4000    1000    13000    15000     9000
[P.sub.T24]         13000    61000    91000   23000    19000    31000

Table 3. Transformation value (Y) = log(X/logX) for different
treatment groups

Group                    Transformation value (Y) = log(X/log X)
Animal no                    of each guinea pig in groups

                    1          2          3          4          5

Untreated        4.649412   4.118062      NC      4.125015   4.122248
  control
  ([P.sub.UT])
[P.sub.T6]       4.098891   3.39194    3.626345   3.815239   3.826122

[P.sub.T12]      3.260191   3.045509   3.260191   3.351119   3.555321

[P.sub.T24]      3.908456   4.142481   4.288952   3.122069   3.641436

Group            Transformation          Mean           Therapeutic
Animal no            value           Y [+ or -] SE      response=
                  (Y) = log(X/                           (Mean Y
                 log X) of each                         control -
                 guinea pig in                          Y treated)
                     groups

                       6

Untreated           4.621183      4.68 [+ or -] 0.020      Nil
  control                                 (c)
  ([P.sub.UT])
[P.sub.T6]          3.81283       3.11 [+ or -] 0.091      0.91
                                         (ab)
[P.sub.T12]         3.499685      3.32 [+ or -] 0.015      1.36
                                          (a)
[P.sub.T24]         4.11639       3.98 [+ or -] 0.106      0.10
                                          (a)

Different superscript (a, b) differ significant (P>0.05)

Mean value bearing Different superscripts (a, b, c,) differ
significantly (P > 0.05)
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Author:Abhishek; Agarwal, R.K.; Kumar, B.; Mishra, A.K.; Anjay; Priyadarshini, A.; Kumar, S.; Prakash, C.;
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Sep 1, 2015
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