Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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Indian Journal of Animal Research, volume 56 issue 10 (october 2022) : 1279-1282

Bacteriophages: A Potential New Therapeutic Alternate to Antibiotics to Treat Chronic Septic Wounds in Large Animals

Sanjay Shukla1,*, Anju Nayak1, R.K. Sharma1, P.C. Shukla1, R.V. Singh1
1Department of Veterinary Microbiology, College of Veterinary Science and A.H., Nanaji Deshmukh Veterinary Science University, Jabalpur-482 001, Madhya Pradesh, India.
Cite article:- Shukla Sanjay, Nayak Anju, Sharma R.K., Shukla P.C., Singh R.V. (2022). Bacteriophages: A Potential New Therapeutic Alternate to Antibiotics to Treat Chronic Septic Wounds in Large Animals . Indian Journal of Animal Research. 56(10): 1279-1282. doi: 10.18805/IJAR.B-4317.
Background: Overuse of antibiotics is a major problem in the treatment of chronic infections. The antibiotic treatment is frequently non-curative due to emergence of antibiotic resistance, thus alternative treatment is necessary. The natural ability of pathogens to develop resistance is not only a threat to animal health but also leads to accumulation of antibiotic resistance determinants or resistant bacteria livestock products. Phage therapy has been developed as an alternative therapeutic approach against antibiotic resistance microorganisms.

Methods: In present study 150 samples of sewage were collected from livestock farm, NDVSU, Jabalpur (M.P.). Bacteriophages were isolated and characterized from the collected sewage samples. “Cocktail” of the recovered phage lysate isolates (ØVS1, ØVS5, ØVS9 and ØVS27) was prepared for assessment of therapeutic utility of S. aureus phages in chronic septic infections. Therapeutic trial was performed in large animals (cattle and buffalo) at livestock farm, Adhartal, NDVSU, Jabalpur (MP.). Pathogens isolated from chronic septic wounds were showing antibiotic resistance but the bacteria were sensitive to phage lysate. Phage ‘cocktail’ was applied topically over the chronic septic wounds as single dose. Follow up of the cases was done at regular intervals (0, 5 and 10 days) which included clinical examination of wound by appearance, status of discharge and formation of granulation tissue.

Result: Six animals showed complete recovery (60%) out of 10 animals from wounds as predicted by progressive healing status of wounds till the formation of granulation tissue within 10 days after phage application. Thus, recovery was much higher than the conventional antibiotic therapy normally used for the treatment of chronic septic wound infections in animals.
World health organization published a list of global priority pathogens comprising twelve species of bacteria categorized into critical, high and medium priority based on their level of resistance (Tacconelli et al., 2018). The current rate of antibiotic resistance development exceeds with the level of antibiotic discovery and represents a global public health challenge. It highlights the serious problem regarding therapeutic options for multi-drug resistant (MDR) bacterial infections (Bassetti et al., 2017).
S. aureus accounts for a large proportion of the morbidity and mortality due to surgical wound infections (Foster, 2005). It is also an extremely important pathogen causing a variety of infections including soft tissue abscesses, wound infection, endocarditis, osteomyelitis and bacteraemia and economically the most significant disease like mastitis in dairy animals (Mann, 2008). Amongst the antimicrobial resistant bacteria, methicillin-resistant S. aureus (MRSA) is the most challenging to treat. The most widely prevalent livestock associated MRSA (LA MRSA), belonged to CC 398 lineage, was described in pigs and other food animals in different parts of the world. The LA MRSA was originated as methicillin sensitive S. aureus strains in human which acquired the methicillin and tetracycline resistance attributes from pigs and transferred back to human as MRSA (Samanta and Bandyopadhyay, 2019).
Antimicrobial resistance in bacteria pathogen is a concern not only in veterinary medicine, but also in general worldwide (Balsalobre et al., 2014). Recent studies also raise anxiety on the collateral damage of antibiotics to the microbiota of animals (Modi et al., 2014). The current resurgence of interest is persisting in light of the growing urgency of the antimicrobial resistance (AMR) crisis, which predicts that AMR will be the leading cause of death by 2050, causing 10 million global deaths per year (O’Neill, 2016).
It is difficult to develop effective new antibiotics due to requirement of sufficient time and funds. There is a demand for new strategies to treat bacterial infections, such as antimicrobial peptides, phage therapy and phage endolysins. Phage therapy is particularly promising as it involves a phage that specifically infects and kills bacteria (Reardon, 2015). The use of phages as antibacterial drugs frequently referred to as “phage therapy” has been discussed and deployed since these bacterial viruses were discovered in the early 1900s (Summers, 2012).
Phages lysates have been extensively used to prevent and treat many numbers of chronic septic bacterial infections with great recovery rate (Heo et al., 2009; Mishra et al., 2012 and Breyne et al., 2017). Present study was conducted to isolate, characterized and assess the antimicrobial activity of phage lysate in chronic septic wounds. Six cases were completely cured out of ten in topical application of phage lysate whereas only one case was cured out of six in topical application of vetbacin antibiotic ointment. Recovery was found to be sixty per cent with bacteriophage therapy and seventeen percent in chronic septic wounds infections. The results of phage therapy were very good as compare to topical antibiotic therapy. The outcome of the research is promising and encouraging and this will help to form strategies for successful treatment of chronic septic wounds infections in animals not responding to the conventional therapy due to development of antibiotic resistance.
The proposed research work was carried out during the year 2018-19 (12 months period) in the Department of Veterinary Microbiology and Livestock Farms, College of Veterinary Science and Animal Husbandry, NDVSU, Jabalpur, Madhya Pradesh, India. Freshly prepared different culture media like basal agar (tryptone broth with 1.5% agar), soft agar (tryptone broth with 0.7% agar), mannitol salt agar, muller-hinton agar, nutrient agar and nutrient broth were used during the study for isolation of microbial organism.
Propagation of Staphylococcus bacteriophages
Isolation of bacteriophages against S. aureus (ATCC25923) from sewage samples was done by soft agar overlay method as described by Synnot et al., (2009) with slight modification. Sewage samples for bacteriophage isolation were collected from livestock farms (cattle, buffalo, pig and goat), N.D.V.S.U., Jabalpur (M.P.).  
Titration of phage lysate
Recovered phage isolates after characterization were subjected for titration to obtain high titer lysate preparation so as to ensure maximum number of phages in lysate to trigger lytic activity for phage therapy. Titration was done by preparing tenfold serial dilution of the phage lysate. For titration, 10-1 to 10-12 dilutions were made in normal saline. Each dilution was subjected to formation of plaques.  Phage lysates 1×1010 (ØVS1, ØVS5 ØVS9 and ØVS 27) were used in for the preparation “cocktail” of bacteriophage (Fig 1).

Fig 1: Transmission electron microscopic image of S. aureus phage (200 nm).

Therapeutic trial of lytic phage in chronic wounds
The clinical trial of recovered bacteriophage was performed in large animals (cattle and buffalo) to evaluate the efficacy of bacteriophage in chronic wounds at Livestock Farm, Adhartal, NDVSU, Jabalpur, Madhya Pradesh, India. The clinical trial was carried out to study the therapeutic effectiveness of phage lysate on chronic septic wound of animals. The test animals were divided into two groups. Group I was subjected to topical application of phage lysate cocktail. Group II was topically treated with topical application of Vetbacin ointment (combination of neomycin and bacitracin antibiotics). The observations in both groups were recorded on day 0, 5 and 10.
The pus samples were collected from the chronic septic wounds and then transported to laboratory for microbiological investigation for the isolation and identification of bacterial pathogens associated with wounds. Isolated bacterial pathogens were subjected to antibiotic sensitivity test to know the antibiotic resistant pattern. These bacterial isolates were also subjected to bacteriophage sensitivity to know the susceptibility of pathogens. In trail wounds of animals were cleaned properly with sterile normal saline before the application phage lysate. One ml (1010 PFU) of phage cocktail was applied topically over the wound and observed daily up to 10 days. Recovery statuses of wounds were recorded at regular time interval (0, 5 and 10 days). Recovery was assessed by two ways like physical appearance of wound and microbial investigation. Physical assessment of wound was done by appearance of wounds, discharges of wounds and formation of granulation tissue over wounds after treatment. Microbiological investigations of wounds were done to see the presence of bacterial agent before and after treatment. 
Clinical samples were processed for isolation and identification of bacterial pathogens associated with septic wounds. Result of bacterial isolation of septic wounds indicated that most of the chronic septic wounds were infected with multiple bacterial pathogens in which Staphylococcus spp., Pseudomonas spp. Escherichia spp. and Bacillus spp. were predominantly associated. The findings of multiple pathogens associated with wounds were supported with the findings of other worker like Vinodkumar et al., (2008) and Heo et al., (2009). Most common organism of chronic wound was Staphylococcus spp. which did not showed the sensitivity towards antibiotic like penicillin methicillin ampicillin ciprofloxacin and cephalexin used in study. The findings of antibiotic drug resistance showed the similarity with the findings of Shrivastava et al., (2017) who isolated and observed the multiple drug resistance in S. aureus. However the isolated Staphylococcus had shown the sensitivity towards the recovered bacteriophages lysates. This finding is supported by Lehman et al., (2019) with similar findings that S. aureus pathogens were showing the sensitivity towards the bacteriophage lysate.
Result of physical and microbial status of wounds in phage therapy treated group indicated that the good appearance of six cases out of ten were showing the formation of granulation tissue due to complete  healing process to confirmed the full recovery of wounds within 10 days (Fig 2). There was not even a single wound showing the presence of Staphylococcus at 5th day in phage therapy treated group. While in the conventional topical antibiotic therapy (topical application of vetbacin cream, himex and topicure spray) only one case showed the formation of granulation tissue and rest all were continuously showing the discharge of pus from the wounds up to 10 days.

Fig 2: Recovery status of wound.

In present study in chronic cases of wounds the recovery percent in bacteriophage therapy was sixty while in conventional antibiotic therapy was seventeen percent.  Thus, recovery was much higher in phage therapy than the conventional antibiotics therapy chronic septic wound infection used during the present study.  In chronic septic wounds infections the efficacy of bacteriophage therapy was found to be sixty percent which was appreciable as compared to the conventional antibiotic therapy. Thus, the application of lytic phage in single dose proved to be innovative and effective therapy for treatment of septic chronic wounds. Our findings related with bacteriophage therapy showed agreement with the findings of other workers like Slopek et al., (1987); Alisky et al., (1998); Mathur et al., (2003); Wills et al., (2005); Mishra et al., (2012) and Breyne et al., (2017) they reported the success in bacteriophage therapy was 60-100% against suppurative infections and abscesses. Bacteriophage may be inducing IFN gamma and some other interferon’s which leads to good healing process. Thus, the phage cocktail can be prescribed in the treatment of chronic septic wounds.
It is opined that the suppurative chronic abscess generally get heavily infected with common pus forming bacteria. Such wound provides favorable microenvironment for propagation of sensitive phages. Treatment of pus forming pathogens and Staphylococcal infections with antibiotics is becoming very difficult in due to widespread presence of multiple drug resistence.  In wounds treatment, a phage ‘cocktail’ (mixture of phages) collectively provides a wider antibacterial range and proves to be more effective treatment of chronic septic infections in animals (Estrella et al., 2016) and Verstappen et al., 2016).
The present study highlights bacteriophage therapy as a possible solution to drug resistance. The prospects of the phage therapy are bright particularly in the antibiotic resistance crisis. These studies revealed good effect of phage application through antibacterial power. Phage therapy in the medical science particularly in the area of treatment of burn and wound has been accelerated. The therapeutic potential of phage in the veterinary field did not get adequate attention, though the treatment with phage is more economic and efficient. Present study highlights methodology of isolation and initiated search of bacteriophage which might prove useful as antibacterial agents. 
In chronic wounds therapeutic efficacy of phage lysate was better than conventional antibiotic therapy. Recovery of chronic septic wounds with phage therapy was 60 per cent.  Thus, recovery was much higher than the conventional antibiotics therapy normally used in animals with chronic wound infection treatments within 10 days.  Excessive use of antibiotics is a major problem in the treatment of chronic septic infections. The result of present study was very encouraging to use the bacteriophage as therapeutic agent against S. aureus infections. The natural ability of pathogens to develop resistance is not only a threat to animal health but also leads to accumulation of antibiotic residues in livestock products. Phage therapy has been developed as an alternative method of therapy against antibiotic resistance microorganisms.

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