Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 56 issue 7 (july 2022) : 873-879

Isolation and Characterization of Lytic Bacteriophage against Methicillin Resistant Staphylococcus aureus from Pyoderma in Dog

Archana1, P. Kaushik1,*, Anjay1, A. Kumar2, S. Kumari3, P. Kumar3, P. Shekhar4
1Department of Veterinary Public Health and Epidemiology, Bihar Veterinary College, Bihar Animal Sciences University, Patna-800 014, Bihar, India.
2Department of Veterinary Biochemistry, Bihar Veterinary College, Bihar Animal Sciences University, Patna-800 014, Bihar, India.
3Department of Veterinary Microbiology, Bihar Veterinary College, Bihar Animal Sciences University, Patna-800 014, Bihar, India.
4Department of Veterinary Medicine, Bihar Veterinary College, Bihar Animal Sciences University, Patna-800 014, Bihar, India.
Cite article:- Archana, Kaushik P., Anjay, Kumar A., Kumari S., Kumar P., Shekhar P. (2022). Isolation and Characterization of Lytic Bacteriophage against Methicillin Resistant Staphylococcus aureus from Pyoderma in Dog . Indian Journal of Animal Research. 56(7): 873-879. doi: 10.18805/IJAR.B-4869.

ABSTRACT

Background: Pyoderma in dog poses complexity owing to involvement of multi drug resistant bacterial infection particularly Staphylococcus species. This may result into failure of antibiotic therapy in pyoderma. Therapeutic use of lytic bacteriophage (phage) may be an alternative to deal antimicrobial resistance in veterinary medicine. There is no report available on isolation and characterization of bacteriophage from clinical cases of pyoderma in animals, hence the study was performed with aim to isolate and characterize lytic phage against methicillin resistant S. aureus (MRSA). 

Methods: The phage was isolated from pus sample of dog suffering from severe pyoderma. The phage was isolated using MRSA as a bacterial host and was named as Staphylococcus phage BVC1 (SPBVC1). The morphology of the phage was determined using TME and was characterised by determining the host range and lytic potential of the phage at range of temperature and pH.

Result: The morphology of phage revealed an icosahedral head of diameter 81.31 nm. with sheath and a central tube and a tail of 92.08 nm. It showed strong lytic activity against Methicillin resistant S. aureus and was stable under a range of temperature varying from 4oC to 45oC and pH from 4 to 11. The phage has shown lytic activity against the MRSA however no lytic activity against the MSSA was shown by the phage. The high specificity of the phage for MRSA indicated its potential use as an alternative therapeutic approach against multidrug resistant staphylococcal infections.

INTRODUCTION

Staphylococcus aureus is a gram-positive cocci present as commensal skin microflora, in the nostrils and respiratory tract (Lee et al., 2009). It is an opportunistic pathogen commonly associated with humans and animals, capable of causing serious diseases and death including sepsis, pneumonia, meningitis in humans and mastitis, keratitis, osteomyelitis, pyoderma in animals (Mavrogianni et al., 2007; Pintado et al., 2019 and González et al., 2020). The occurrence of Staphylococcus pseudintermedius as an opportunistic pathogen on the skin is very common in dogs and the studies show 46 to 92% prevalence of S. pseudintermedius in dogs (Kawakami et al., 2010; Rubin and Chirino, 2011; Bannoehr et al., 2012 and Morris et al., 2017; Lynch and Helbig, 2021), however involvement of S. aureus in canine pyoderma has also been reported (Reddy et al., 2016 ; González et al., 2020). The increased incidence of methicillin resistant S. aureus (MRSA) has become a major cause of concern as S. aureus is one of the most common causes of nosocomial infections (Chambers et al., 2009; Dulon et al., 2011). Methicillin is a b-lactamase stable antibiotic which was introduced in 1959 to combat penicillin, drug used for the treatment of Gram positive bacterium, resistance. However, methicillin-resistant strains of S. aureus were isolated soon after its introduction (Jevons 1961; Knox 1961). The development of antibiotic resistance in bacteria to different groups of antibiotics has resulted into the search of alternative approach for treatment of such cases (O’Flynn et al., 2004; Kutter et al., 2015) and bacteriophage has been used as one of the alternative therapy against bacterial strain resistant to antibiotics (Watanabe et al., 2007; Lin et al., 2017). Bacteriophages (phages) are diverse and ubiquitous non living biological entities consisting of DNA or RNA enclosed in a protein capsid which are capable of infecting and replicating within bacterial cells, although they can not reproduce independently. They are widely distributed in nature and have been reported from sea and freshwater throughout the globe including hypersaline environments, the soil, deserts, polar regions and on as well as within other organisms (Díaz-Muñoz and Koskella, 2014). Phages typically bind to specific receptors on the bacterial cell surface, inject their genetic material into the host cell and then either integrate this material into the bacterial genome (“temperate” phages) and reproduce vertically, or hijack the bacterial replication machinery to produce phage progeny and lyse the bacterial cell (“lytic” phages) (Clokie et al., 2011). These lytic phages are used for conventional phage therapy to kill bacterial pathogens. Host specificity varies among phages, some of which are strain-specific, whereas others have a host range across the bacterial strains and even genera (Lin et al., 2017). The use of bacteriophage in the treatment of staphylococcal infections were first described by Bruynoghe and Maisin (1921) by injecting the phage preparation around surgically opened lesions with regression of infection within 24-48 hours. Keeping these facts and observation in mind the present study was conducted to isolate and characterize lytic bacteriophage against methicillin resistant S. aureus from pus of a dog with chronic skin disease.

MATERIALS AND METHODS

Isolation of host S. aureus from pus
 
The S. aureus was isolated from pus sample by selective plating on mannitol salt agar (MSA). A loopful of pus sample from dog skin surface was inoculated in tryptone soya broth (TSB) containing 10% sodium chloride salt (TSB-S) for enrichment at 37oC for 24 h. The positive sample was streaked on mannitol salt agar, incubated at 37oC for 24 h and observed for mannitol fermentation. Presumptive S. aureus colonies were identified on the basis of colony morphology and mannitol fermentation and were confirmed by biochemical test including the catalase activity tests and tube coagulase tests with human plasma to confirm S. aureus. To further verify the isolate, genomic DNA was extracted with phenol-chloroform and PCR was performed targeting 16S-rDNA using specific Forward-GTAGGTGGCAAGCGTT ATCC and Reverse- CGCACATCAGCGTCAG primer for S. aureus (Karmakar et al., 2016). The catalase-positive, coagulase-positive, mannitol-fermentation and PCR-positive S. aureus isolates were also screened for methicillin resistance both phenotypically and genotypically as per Braoios et al., (2009).
 
Bacteriophage isolation
 
The pus sample from dog skin was inoculated into 25 ml TSB with 10% NaCl. For enrichment of phage 0.5 ml of overnight grown culture of S. aureus isolate was added in TSB before incubation of sample at 37oC for 24 h. After enrichment, 1 ml of sample was transferred into 1.5 ml pre-sterilized microfuge tube and centrifuged at 5000 rpm for 10 min. The supernatant was filtered with 0.22 µm syringe filter and phage filtrate was preserved at 4oC. The presence of phage was confirmed by spot assay against the host bacteria. In brief, 100 µl of overnight grown culture of S. aureus was mixed with 3 ml of LB top layer agar (0.7% agar) and poured onto pre-dried nutrient agar (3%) plates and left to dry. Further 10 µl of phage filtrate was spotted over plates and allowed to be absorbed onto the plate. Once dried, plates were incubated at 37oC for 12-24 h and examined for presence of complete lysis/plaque produced.  Sample was considered positive for lytic phage on observation of complete lysis/clear plaque/confluent or opalescent lysis, while appearance of turbid zone in the spot was considered negative.
 
Purification of bacteriophage
 
The phage which produced clear plaques/lysis was purified by serial dilution and plating on soft agar overlays as per the method described by Adams (1959). The complete lysis zone/plaques formed was extracted using a sterile pipette-tip, re-suspended in 1 ml salt magnesium buffer (NaCl, 5.8 g; MgSO47H2O, 2 g; 1 M Tris Cl pH 7.5, 50 ml; 2% gelatin, 5 ml; add ddH2O to 1,000 ml) and incubated overnight at 4oC for eluting the phage in buffer. The phage suspension filtered through 0.22 µm syringe filter, was 10 fold serially diluted. 100 µl of diluted phage and 100 µl host bacterium were mixed with 3.0 ml molten soft agar (0.7% agar) and poured quickly on top of solidified nutrient agar plate (3% agar). The plates were gently rotated and incubated overnight at 37oC. The single plaque was passaged three times to obtain a pure phage.
 
Host range determination
 
The host range of the bacteriophage was determined against a number of methicillin resistant (62) and methicillin sensitive (42) S. aureus isolates as per the methods described by Jamalludeen et al., (2007). Lawn of a single bacterial isolate was inoculated on a BHI agar plate and 10 μl of phage suspension (109 pfu/ml) was dropped in the centre of bacterial lawn after the plate dried up. Following incubation at 37oC for 24 h the plates were examined for lysis. A clear zone in the bacterial lawn was recorded as complete lysis.
 
Transmission electron microscopy (TEM)
 
Purified bacteriophage was visualized by TEM (Jeol JEM-1011, Japan) at the division of plant pathology, Indian Agricultural Research Institute, New Delhi, India. In order to visualize the phage by TEM, the bacteriophage suspension was concentrated as per the method described by Davis et al., 1986 with some modifications. In brief, 10 ml of high titre bacteriophage filtrate was mixed gently with 10 ml of TM buffer and incubated for 15 minutes at room temperature. After incubation, 2 ml of 5 M NaCl and 2.2 g of solid PEG-8000 was added to the mixture and dissolved completely in a centrifuge tube. The tube was kept at 4oC for 2 h followed by centrifugation at 12000 g at 4oC for 45 min. The supernatant was poured off and pellets were dissolved in 300 µl of TM buffer which were further treated with equal volume of chloroform and centrifuged at 12000 rpm for 5 min after proper mixing. The supernatant was stored into different aliquot at 4oC and processed for transmission electron microscopy.
 
Determination of thermal and pH stability of phage
 
The thermal tolerance of bacteriophage was evaluated at five different temperatures 4, 25, 37, 45 and 65oC in SM (Salt Magnesium) buffer. 100 µl of the bacteriophages suspension (6.2×108 PFU/ml) was added to a microfuge tube containing 900 µl SM buffer and placed in water bath at 25, 37, 45 and 65oC and at 4oC in refrigerator. The tubes were incubated at the required test temperature for 60 min and then placed on ice for 10 min before titration by double-layer agar plate method. A ten-fold serial dilution was prepared and 100ml phage from each dilution was added with 100 µl host bacterium and were mixed with 3.0 ml molten soft agar (0.7% agar, w/v) and poured quickly on top of solidified nutrient agar plate (3% agar, w/v). The plates were gently rotated and left to dry at room temperature for 20 min. The plates were incubated overnight at 37oC. The number of plaques formed was recorded and plaque forming units (pfu) per ml in the bacteriophage suspension was calculated. To evaluate the stability of phage at different pH conditions, it was incubated in SM buffer with different pH value. The pH of buffer was adjusted with 4 N HCl (Hi-media) or 2 N NaOH (Hi-media) to a pH range of 1-12 and the phage was treated with each SM buffer solution. After incubation at 37oC for 12 h, phage titration was determined by double-layer agar plate method as described above. All experiments were conducted in duplicate.

RESULTS AND DISCUSSION

Pyoderma in dogs is very common clinical presentation globally. The treatment efficacy of canine pyoderma using antimicrobial agents has become limited and challenging with the emergence of multidrug resistant staphylococci, including resistance to the semi-synthetic penicillinase-resistant penicillins such as methicillin (Jones et al., 2007; Kawakami et al., 2010 and Morris et al., 2017). Also the rate of emergence of resistant pathogens is not proportionate to the rate and economics involved in the discovery of new antibiotics. Therefore, under these circumstances, to overcome the problem of evolving antibiotic resistance in staphylococci, bacteriophage may act as a potent alternative (Kwiatek et al., 2012; Han et al., 2013). Different studies have indicated the systemic and topical use of antimicrobial agents, with varying efficacy in the therapeutic management of pyoderma in dogs (Tomlin et al., 1999; Morris et al., 2006; Loeffler et al., 2011; Bryan et al., 2012 and Beco et al., 2013), however very limited reports are available about characterization of S. aureus specific phages that has been isolated from canine pyoderma. There is no report available on characterisation of S. aureus specific phage isolated from canine pyoderma or dermatitis cases.
       
Although S. pseudintermedius has been reported primarily as the cause of canine pyoderma (Rubin and Chirino 2011; Bannoehr et al., 2012 and Morris et al., 2017), we have isolated S. aureus from pus sample of dog in the present study and was confirmed by biochemical test and PCR amplification (228 bp) of 16S rDNA (Fig 1). On further characterization of bacterial isolate, it was found to be methicillin resistant S. aureus (MRSA), showing resistance to oxacillin and detection of mecA gene of size 310 bp (Fig 2) in the isolate. This confirmed the involvement of MRSA in the case under current study which may have resulted in the treatment failure of antibiotic response. Earlier reports also indicate the involvement of multidrug resistant staphylococci including S. aureus and MRSA in canine and human patient suffering from pyoderma globally (Qekwana et al., 2017; Gagetti et al., 2019; González et al., 2020 and Kengne et al., 2020). The confirmed bacterial isolate was used as host for isolation of bacteriophage. The phage was isolated and named as SPBVC1 and was purified for further characterization. The purified phage produced clear medium sized plaques (2-2.5 mm in diameter) when propagated on MRSA host as shown in Fig 3.
 

Fig 1: PCR amplification of 16S rRNA gene with a product size 228 bp, M: 100 bp DNA ladder.


 

Fig 2: PCR amplification of mecA gene with a product size 310 bp, M: 100 bp DNA ladder.


 

Fig 3: Plaques produced by bacteriophage SPBVC1.


 
The morphology of the SPBVC1 phage under transmission electron microscopy (TEM) showed an icosahedral head of diameter 81.31 nm. and a tail of 92.08 nm. Based on the International Committee on Taxonomy of Viruses (Murphy et al., 1995), TEM characteristic of SPBVC1 phage was suggestive of the order Caudovirales (Fig 4). The ultrastructure of phage showing a contractile tail consisting of a sheath and a central tube suggested it to be a member of family Myoviridae (Deghorain et al., 2012).
 

Fig 4: Transmission electron micrograph of bacteriophage, SPBVC1. 120000X.


       
The host range of the phage SPBVC1 was determined using a range of methicillin sensitive and methicillin resistant staphylococci. The lytic pattern of phage showed lysis of 38.8 percent of MRSA isolates only (Table 1), however none of the staphylococcal isolates other than MRSA were lysed by the phage under the study indicating the specificity of phage for MRSA only which is an important characteristic of bacteriophage intended for therapeutic or biotechnological use (Deghorain and Van, 2012). Although the isolated phage SPBVC1 had a limited host range, it had shown lytic activity against MRSA strains which are multidrug resistant staphylococci. Yazdi et al., (2018) also reported the isolation of Phage vB-SsapS-104 with a narrow host range for S. saprophyticus clinical isolates only. The use of methicillin resistant S. aureus as host during isolation of phage or the high density of the methicillin resistant staphylococci population in the pus sample could be one of the possible reason (Richard et al., 2008) resulting in the high specificity of the phage SPBVC1 in the present study. These findings suggest that the SPBVC1 phage reported here may be a useful candidate for phage cocktails intended for therapeutic or biocontrol use in future.
 

Table 1: Lytic potential of bacteriophage SPBVC1 against MRSA strains isolated from different origin.


       
The stability study of phage for their resistance to heat and pH will be helpful in minimizing the phage loss and maintenance of phage viability under various conditions that has a role in various aspects of phage therapy (Li and Zhang, 2014). Therefore, biological stability of the phage SPBVC1 at different temperature and pH was evaluated in the present study which showed that the phage remained lytic at a range of temperature varying from 4oC to 45oC and became completely inactive at 65oC (Fig 5a). The phage was able to lyse in a range of acidic and alkaline pH value from 4 to 11 however the lysis potential declined significantly below pH 5 (Fig 5b). This indicated that the phage was stable in a pH range from 5 to 11. The results of bio stability of phage are in concordance with earlier observations by different workers (Cui et al., 2017; Ganaie et al., 2018 and Yazdi et al., 2018). The study concludes that the isolated phage (SPBVC1) may be used as a potential alternative against MRSA infections and can be exploited for therapeutic management of pyoderma caused by MRSA in pets however, a detail insight into the phage genome and interaction of phage and bacteria would expand their applicability in phage therapy.
 

Fig 5a: SPBVC1 Phage thermos stability assay.


 

Fig 5b: SPBVC1 Phage stability at varied pH.

CONCLUSION

The study concludes that the isolated phage (SPBVC1) may be used as a potential alternative against MRSA infections and can be exploited for therapeutic management of pyoderma caused by MRSA in pets however, a detail insight into the phage genome and interaction of phage and bacteria would expand their applicability in phage therapy.

CONFLICT OF INTEREST

None.

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