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Full Research Article

Isolation, Identification and Characterization of Bacteriophages as a Potential Antibacterial Agent Against Multidrug-Resistant Pathogenic Escherichia coli Isolated from Swine in North Eastern Region of India

T
Tarakchi M. Sangma1
P
Parimal Rouchoudhury1
P
Prasant Kumar Subudhi1
F
Fatema Akter1
I
Indranil Samanta2
S
Samiran Bandyopadhayay3
T
Tapan Kumar Dutta1,*
1Department of Veterinary Microbiology, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl-796 015, Mizoram, India.
2Department of Veterinary Microbiology, West Bengal University of Animal and Fishery Sciences, Belgachia, Kolkata-700 037, West Bengal, India.
3ICAR-Eastern Regional Station, Indian Veterinary Research institute, Belgachia, Kolkata-700 037, West Bengal, India.

ABSTRACT

Background: Antimicrobial resistance (AMR) is a global concern for human and animal health. As the development and commercialization of a new class of antibiotics are difficult, time-consuming and extremely costly, exploration of alternatives like bacteriophages is considered one of the most suitable sources. There is a paucity of databases on the isolation of bacteriophages and exploration of their antibacterial properties against pathogenic MDR E. coli. The current study was undertaken to isolate and identify the bacteriophages from sewages to evaluate their lytic activity against MDR pathogenic Escherichia coli isolated from domestic animals in Mizoram.

Methods: Total 50 samples were collected to process for isolation, identification and characterization of bacteriophages. Simultaneously, a total of 192 rectal swabs from pigs were collected, followed by isolation and identification of Escherichia coli. All the isolates were subjected to detection of selected virulence genes by PCR and determination of their antimicrobial sensitivity profile using commercial disks. All the bacteriophage isolates were assessed for their lytic effect against the MDR pathogenic E. coli isolates.

Result: A total of six bacteriophages were isolated. Transmission electron microscopy revealed that bacteriophages belong to the Myoviridae family. Total 163 E. coli was isolated, out of which 59 isolates were pathogenic, which could be classified under STEC, EPEC, EHEC and ETEC pathotypes. By disk diffusion assay, majority of the E. coli showed resistance against commonly used antibacterial agents, viz., ampicillin, ciprofloxacin, cefpodoxime, ceftriaxone, nalidixic acid, tetracycline, doxycycline, gentamicin and streptomycin. Except one, all the bacteriophages exhibited lytic activity against pathogenic and MDR E. coli field isolates. Bacteriophages isolated and identified from sewages of Mizoram exhibited antimicrobial activities against field isolates of multidrug-resistant pathogenic E. coli isolated from pigs. Bacteriophages can be considered as a potential alternative to antibiotics to control the menace of AMR.

INTRODUCTION

Escherichia coli are regarded as major pathogens that affect both humans and animals and are linked to a number of illnesses. The ever-increasing AMR against conventional antimicrobial agents is another significant concern they pose. Furthermore, multidrug-resistant (MDR) pathogenic bacteria that form biofilms are regarded as a serious public health risk. Most of the antimicrobial medications that veterinarians and doctors have on hand are becoming ineffective (Dutta, 2020). The new generation of antimicrobials is too expensive and limited for everyday use by the average person in middle- and low-income nations (Ghosh et al., 2019). Bacteriophages and their derivatives are among the most effective and potent methods for fighting multi-drug resistant (MDR) bacteria (Czaplewski et al., 2016). Soil, sewage and animal secretions are the most common sources of phages, allowing them to be isolated for therapeutic purposes. Phage therapy may be one of the most extensively used therapies for infections brought on by antibiotic-resistant bacteria in the current period of limited antibiotic supply (Górski, 2018).
       
There is minimal information available on isolation, identification and antimicrobial activity of bacteriophages from India and no data are available from Mizoram. Accordingly, the present study was carried out to isolate, identify and molecularly characterize bacteriophages from sewage, evaluating their antimicrobial properties against multidrug-resistant (MDR) Escherichia coli isolated from pigs in Mizoram, India.

MATERIALS AND METHODS

Sewage samples (n=50) were collected in sterile containers from various places in Mizoram following the method described by Jothikumar et al., (2000) and carried to the laboratory maintaining a cold chain for processing. All the samples were inoculated in 5 mL nutrient broth (10×) with 1 mL of overnight grown E. coli (ATCC 43888), 400 µL of 0.1M CaCl2 and 0.1M MgCl2. The entire mixture was incubated aerobically at 37oC overnight in a shaking incubator at 150-200 rpm. Following incubation, the enriched broth was centrifuged at 10000 rpm for 15 minutes followed by collection of supernatant and sequential filtration utilizing 0.45 µm and 0.2 µm membrane filters. The filtrate was stored at 4oC for further use. A double agar overlay method (Adams, 1959) was performed to isolate the bacteriophage using hard agar at the bottom as the base and a mixture of phage particles and host cells (Escherichia coli ATCC 43888) in soft agar at the top. The phage isolates were characterized by turbidity reduction test in Escherichia coli (Shende et al., 2017) and spot assay (Adams, 1959) (Fig 1). Estimation of phage titer was done by enumerating the visible plaques in a double agar overlay plate (Fig 2) and PFU was used to measure the viable bacteriophage concentration using the following equation (Pelzek et al., 2013).

PFU/ml = No. of visible plaques ×  1000 µl of phage stock plate × (dilution factor)

Fig 1: Spot assay on Luria-bertani agar for demonstration of bacteriophages isolated from different sewage samples collected from various places of Aizawl.



Fig 2: Plaque assay for demonstration of bacteriophages isolated from different sewage samples collected from various places of Aizawl.

 
All the phage isolates were concentrated with 1M NaCl and PEG 8000 (10% w/v) and was kept overnight at 4oC to obtain the bacteriophage pellet, which was further dissolved in phage buffer (Anand et al., 2017). Phage DNA was extracted (Jothikumar et al., 2000), checked for purity and quantity by Nano-drop spectrophotometer and stored at -80oC. Restriction fragment length polymorphism (RFLP) was performed (Goodridge et al., 2003) for identification of bacteriophage isolates. Phage DNA was digested with EcoRI, BamHI, XhoI and HindIII enzymes following the guidelines of the manufacturer. Electrophoresis in a 1% agarose gel was used to separate the digested DNA fragments. The fragments were visualized in a UV transilluminator and documented using a gel documentation system. Fragment sizes were determined from co-migrating EcoRI and HindIII digests of lambda DNA (Fig 3). Morphological documentation bacteriophage isolates by Transmission Electron Microscopy (Fig 4) (Model No.: FEI TECHNAI G2 F20 X-Twin) to observe and document the phage morphology (Goodridge et al., 2003).

Fig 3: Agarose gel electrophoresis of restriction endonuclease digestion of bacteriophages isolated from sewages of various places of Aizawl.



Fig 4: (A) TEM observation of the phage virion revealed that the phage had an icosahedral capsid and a contractile tail, which comes under Myoviridae family. (B) Determined Myovirus with an icosahedral head and a contractile tails.


               
Total 192 porcine rectal swabs were collected from different places Mizoram and used for isolation and identification of E. coli. All the isolates were characterized phenotypically and genotypically (Mandakini et al., 2021; Phillips et al., 1995; Paton and Paton, 1998) and subjected to AST by disk diffusion assay on Mueller-Hinton agar (HiMedia, Mumbai) as per the guidelines of CLSI (2020) using 23 commercially available antimicrobial disks. Antibacterial property of bacteriophage isolates was observed by pour plate method in LB (Fig 5) (Kutter and Sulakvelidze, 2005). The titer of the phage was determined by plaque assay (Pelzek et al., 2013). 

Fig 5: Demonstration of antibacterial property bacteriophages against the Escherichia coli isolated from animals.

RESULTS AND DISCUSSION

In the present study, 6 bacteriophages were isolated. Bacteriophages could not be recovered from soil and pig effluents and are isolated more frequently during the summer season before monsoon (Comeau et al., 2005), which might be due to the high concentration of bacterial substrate in the sewages or other effluents. During rainy seasons, the bacterial load decreases in the soil of effluents, which may reduce the propagation of bacteriophages. In our present study, all the bacteriophages were isolated during the summer months, which are in corroboration with the previous reports (Shende et al., 2017). All the bacteriophages were recovered using one bacterial host (E. coli, ATCC 43888). In previous reports, Bacillus cereus and Bacillus subtilis were found to be the best hosts for the growth of bacteriophages (Shende et al., 2017). Many reports indicated that either B. subtilis and/or E. coli can be used as host for bacteriophage isolation (Gillis and Mahillon, 2014). In comparison to E. coli, B. subtilis supports the growth of multiple morphotypes of phages (Shende et al., 2017). The B. subtilis phage appears to be the best option for isolation work because of its potent activity at both high and low pH and temperature. The size of the bacteria may be connected to lower E. coli phage recovery (Shukla and Hirpurkar, 2011).

Based on the virulence gene profile, 59/163 pathogenic E. coli isolates that were isolated and identified from pigs. Previously from this laboratory, 2291 E. coli from 790 fecal samples from pigs in India’s Northeast region was reported (Mandakini et al., 2015). Similarly, Lalzampuia et al., (2013) also identified 102 E. coli from 53 Mizoram pig fecal samples. Forty two E. coli from 19 chickens that died from severe diarrhoea were also reported by Dutta et al., (2011). 

The type of samples, the media employed, the researchers’ laboratory procedures, the host’s treatment status and other factors can affect the rate of E. coli isolation from fecal samples. There is considerable variation in the number of E. coli colonies obtained from a same sample. In the present study, four distinct pathotypes of E. coli including STEC (n=32), EPEC (n=6), EHEC (n=8) and ETEC (n=13) were recorded. Earlier, from the same laboratory, various pathotypes of E. coli were also reported (Das et al., 2021; Das et al., 2024; Kylla et al., 2017). The pathogenic E. coli isolated from livestock and poultry are always associated with public health significance due to their proximity to humans. As the human dwellings are very close to the pig housings and share a common source of water, transmission of such organisms between pigs to humans is possible (Puii et al., 2019).
       
Majority of E. coli isolates were multi-drug resistant (MDR) and exhibited resistance to at least three classes of antibiotic. Among the 59 pathogenic E. coli isolates, imipenem was recorded as the most sensitive (88.44%). Maximum resistance was recorded against amoxicillin (91.53%) followed by ciprofloxacin (86.44%) and cefpodoxime (81.36%). Previously, Kylla et al., (2017) also reported a similar type of observation in E. coli isolates from pigs in 4 NE states of India. Carballo et al., (2013) observed that the most gram-negative bacteria antimicrobial resistances were to tetracycline (66.7%), ampicillin (52.4%), cephalothin (46.0%), chloramphenicol (44.4%), nalidixic acid (39.7%) and trimethoprim-sulphamethoxazole (33.3%).
       
The bacteriophage lytic activity was tested against 59 pathogenic E. coli isolates, of which 28.81% were positive. The agar overlay and spot test methods revealed that E. coli phage had clear spots or plaques against 17 E. coli isolates. One of the critical factors influencing phage antimicrobial activity is host range, which is likely to be influenced by phage tail morphology. The phage isolates were effective against multiple E. coli pathotypes, whereas majority of the phage isolates showed the lytic spectrum against STEC pathotypes. The efficacy of bacteriophage isolates against different E. coli pathotypes was variable. The P-5 isolate could not exhibit any bacteriolytic activity against any of the E. coli pathotypes, which may be due to the non-availability of any specific surface attachment molecules on the E. coli isolates. Manohar et al., (2019) characterized Escherichia phage myPSH2311 and reported broad host range activity belonging to 6 different pathotypes including EAEC, EHEC, EIEC, EPEC, ETEC and UPEC besides few unknown pathotypes, which are in corroboration of our report, where the bacteriophages exhibited lytic activity for multiple pathotypes of E. coli. In the present study, isolated phages showed lytic activity against the five isolates of EHEC, which is also in corroboration of the report of Viazis et al., (2011), who reported 7/8 phages as highly effective against 16 EHEC strains.
               
Although the high specificity of the bacteriophages is their greatest strength, it also poses a challenge to their widespread use. Accurate targeting is ensured by the great specificity. Therefore, one crucial step in achieving the application is suitably expanding and altering the phage’s host range. Genetic engineering has been shown to alter the host spectrum of phages, but only for a small number of phages that elucidate the mechanism of receptor-binding protein binding on the host (Yoichi et al., 2005). Bacteriophage replication is essentially host genus-specific however; members of the family Enterobacteriaceae are so closely related that polyvalent phages are common, particularly in the E. coli-Shigella-Klebsiella group. There are only a few phages that have broad host specificity across bacterial genera. Accordingly, the observation in the present study might be considered as one of the highly specific broad-spectrum bacteriophages recovered from sewage. Further studies will be required to characterize the phage isolates and to determine their host range before recommending them as potential antibacterial agents against major MDR bacterial pathogens.

CONCLUSION

Therefore, it may be concluded that the bacteriophages isolated and identified from sewages of Mizoram exhibited antimicrobial activities against field isolates of multidrug-resistant pathogenic E. coli. Bacteriophages could be potential candidates in the future as alternatives to conventional antimicrobial agents to contain the menace of AMR in the environment.

ACKNOWLEDGEMENT

The authors are thankful to the DBT sponsored project on “DBT-NER Institutional Level Biotech Hubs at College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram (Phase-II)” (BT/NER/143/SP44310/2021) for financial support and the Dean, CVScandAH, Central Agricultural University, Aizawl, Mizoram - 796015, India for providing research facility during this study.
 
Author contributions
 
Tarakchi M. Sangma: laboratory activities, sample collection; Tapan Kumar Dutta: Conceptualization, manuscript preparation; Parimal Rouchoudhury C: Data analysis; Prasant Kumar Subudhi: Manuscript editing; Indranil Samanta: Manuscript editing; Samiran Bandyopadhayay: Manuscript editing.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of the affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish or preparation of the manuscript.

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    Full Research Article

    Isolation, Identification and Characterization of Bacteriophages as a Potential Antibacterial Agent Against Multidrug-Resistant Pathogenic Escherichia coli Isolated from Swine in North Eastern Region of India

    T
    Tarakchi M. Sangma1
    P
    Parimal Rouchoudhury1
    P
    Prasant Kumar Subudhi1
    F
    Fatema Akter1
    I
    Indranil Samanta2
    S
    Samiran Bandyopadhayay3
    T
    Tapan Kumar Dutta1,*
    1Department of Veterinary Microbiology, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl-796 015, Mizoram, India.
    2Department of Veterinary Microbiology, West Bengal University of Animal and Fishery Sciences, Belgachia, Kolkata-700 037, West Bengal, India.
    3ICAR-Eastern Regional Station, Indian Veterinary Research institute, Belgachia, Kolkata-700 037, West Bengal, India.

    ABSTRACT

    Background: Antimicrobial resistance (AMR) is a global concern for human and animal health. As the development and commercialization of a new class of antibiotics are difficult, time-consuming and extremely costly, exploration of alternatives like bacteriophages is considered one of the most suitable sources. There is a paucity of databases on the isolation of bacteriophages and exploration of their antibacterial properties against pathogenic MDR E. coli. The current study was undertaken to isolate and identify the bacteriophages from sewages to evaluate their lytic activity against MDR pathogenic Escherichia coli isolated from domestic animals in Mizoram.

    Methods: Total 50 samples were collected to process for isolation, identification and characterization of bacteriophages. Simultaneously, a total of 192 rectal swabs from pigs were collected, followed by isolation and identification of Escherichia coli. All the isolates were subjected to detection of selected virulence genes by PCR and determination of their antimicrobial sensitivity profile using commercial disks. All the bacteriophage isolates were assessed for their lytic effect against the MDR pathogenic E. coli isolates.

    Result: A total of six bacteriophages were isolated. Transmission electron microscopy revealed that bacteriophages belong to the Myoviridae family. Total 163 E. coli was isolated, out of which 59 isolates were pathogenic, which could be classified under STEC, EPEC, EHEC and ETEC pathotypes. By disk diffusion assay, majority of the E. coli showed resistance against commonly used antibacterial agents, viz., ampicillin, ciprofloxacin, cefpodoxime, ceftriaxone, nalidixic acid, tetracycline, doxycycline, gentamicin and streptomycin. Except one, all the bacteriophages exhibited lytic activity against pathogenic and MDR E. coli field isolates. Bacteriophages isolated and identified from sewages of Mizoram exhibited antimicrobial activities against field isolates of multidrug-resistant pathogenic E. coli isolated from pigs. Bacteriophages can be considered as a potential alternative to antibiotics to control the menace of AMR.

    INTRODUCTION

    Escherichia coli are regarded as major pathogens that affect both humans and animals and are linked to a number of illnesses. The ever-increasing AMR against conventional antimicrobial agents is another significant concern they pose. Furthermore, multidrug-resistant (MDR) pathogenic bacteria that form biofilms are regarded as a serious public health risk. Most of the antimicrobial medications that veterinarians and doctors have on hand are becoming ineffective (Dutta, 2020). The new generation of antimicrobials is too expensive and limited for everyday use by the average person in middle- and low-income nations (Ghosh et al., 2019). Bacteriophages and their derivatives are among the most effective and potent methods for fighting multi-drug resistant (MDR) bacteria (Czaplewski et al., 2016). Soil, sewage and animal secretions are the most common sources of phages, allowing them to be isolated for therapeutic purposes. Phage therapy may be one of the most extensively used therapies for infections brought on by antibiotic-resistant bacteria in the current period of limited antibiotic supply (Górski, 2018).
           
    There is minimal information available on isolation, identification and antimicrobial activity of bacteriophages from India and no data are available from Mizoram. Accordingly, the present study was carried out to isolate, identify and molecularly characterize bacteriophages from sewage, evaluating their antimicrobial properties against multidrug-resistant (MDR) Escherichia coli isolated from pigs in Mizoram, India.

    MATERIALS AND METHODS

    Sewage samples (n=50) were collected in sterile containers from various places in Mizoram following the method described by Jothikumar et al., (2000) and carried to the laboratory maintaining a cold chain for processing. All the samples were inoculated in 5 mL nutrient broth (10×) with 1 mL of overnight grown E. coli (ATCC 43888), 400 µL of 0.1M CaCl2 and 0.1M MgCl2. The entire mixture was incubated aerobically at 37oC overnight in a shaking incubator at 150-200 rpm. Following incubation, the enriched broth was centrifuged at 10000 rpm for 15 minutes followed by collection of supernatant and sequential filtration utilizing 0.45 µm and 0.2 µm membrane filters. The filtrate was stored at 4oC for further use. A double agar overlay method (Adams, 1959) was performed to isolate the bacteriophage using hard agar at the bottom as the base and a mixture of phage particles and host cells (Escherichia coli ATCC 43888) in soft agar at the top. The phage isolates were characterized by turbidity reduction test in Escherichia coli (Shende et al., 2017) and spot assay (Adams, 1959) (Fig 1). Estimation of phage titer was done by enumerating the visible plaques in a double agar overlay plate (Fig 2) and PFU was used to measure the viable bacteriophage concentration using the following equation (Pelzek et al., 2013).

    PFU/ml = No. of visible plaques ×  1000 µl of phage stock plate × (dilution factor)

    Fig 1: Spot assay on Luria-bertani agar for demonstration of bacteriophages isolated from different sewage samples collected from various places of Aizawl.



    Fig 2: Plaque assay for demonstration of bacteriophages isolated from different sewage samples collected from various places of Aizawl.

     
    All the phage isolates were concentrated with 1M NaCl and PEG 8000 (10% w/v) and was kept overnight at 4oC to obtain the bacteriophage pellet, which was further dissolved in phage buffer (Anand et al., 2017). Phage DNA was extracted (Jothikumar et al., 2000), checked for purity and quantity by Nano-drop spectrophotometer and stored at -80oC. Restriction fragment length polymorphism (RFLP) was performed (Goodridge et al., 2003) for identification of bacteriophage isolates. Phage DNA was digested with EcoRI, BamHI, XhoI and HindIII enzymes following the guidelines of the manufacturer. Electrophoresis in a 1% agarose gel was used to separate the digested DNA fragments. The fragments were visualized in a UV transilluminator and documented using a gel documentation system. Fragment sizes were determined from co-migrating EcoRI and HindIII digests of lambda DNA (Fig 3). Morphological documentation bacteriophage isolates by Transmission Electron Microscopy (Fig 4) (Model No.: FEI TECHNAI G2 F20 X-Twin) to observe and document the phage morphology (Goodridge et al., 2003).

    Fig 3: Agarose gel electrophoresis of restriction endonuclease digestion of bacteriophages isolated from sewages of various places of Aizawl.



    Fig 4: (A) TEM observation of the phage virion revealed that the phage had an icosahedral capsid and a contractile tail, which comes under Myoviridae family. (B) Determined Myovirus with an icosahedral head and a contractile tails.


                   
    Total 192 porcine rectal swabs were collected from different places Mizoram and used for isolation and identification of E. coli. All the isolates were characterized phenotypically and genotypically (Mandakini et al., 2021; Phillips et al., 1995; Paton and Paton, 1998) and subjected to AST by disk diffusion assay on Mueller-Hinton agar (HiMedia, Mumbai) as per the guidelines of CLSI (2020) using 23 commercially available antimicrobial disks. Antibacterial property of bacteriophage isolates was observed by pour plate method in LB (Fig 5) (Kutter and Sulakvelidze, 2005). The titer of the phage was determined by plaque assay (Pelzek et al., 2013). 

    Fig 5: Demonstration of antibacterial property bacteriophages against the Escherichia coli isolated from animals.

    RESULTS AND DISCUSSION

    In the present study, 6 bacteriophages were isolated. Bacteriophages could not be recovered from soil and pig effluents and are isolated more frequently during the summer season before monsoon (Comeau et al., 2005), which might be due to the high concentration of bacterial substrate in the sewages or other effluents. During rainy seasons, the bacterial load decreases in the soil of effluents, which may reduce the propagation of bacteriophages. In our present study, all the bacteriophages were isolated during the summer months, which are in corroboration with the previous reports (Shende et al., 2017). All the bacteriophages were recovered using one bacterial host (E. coli, ATCC 43888). In previous reports, Bacillus cereus and Bacillus subtilis were found to be the best hosts for the growth of bacteriophages (Shende et al., 2017). Many reports indicated that either B. subtilis and/or E. coli can be used as host for bacteriophage isolation (Gillis and Mahillon, 2014). In comparison to E. coli, B. subtilis supports the growth of multiple morphotypes of phages (Shende et al., 2017). The B. subtilis phage appears to be the best option for isolation work because of its potent activity at both high and low pH and temperature. The size of the bacteria may be connected to lower E. coli phage recovery (Shukla and Hirpurkar, 2011).

    Based on the virulence gene profile, 59/163 pathogenic E. coli isolates that were isolated and identified from pigs. Previously from this laboratory, 2291 E. coli from 790 fecal samples from pigs in India’s Northeast region was reported (Mandakini et al., 2015). Similarly, Lalzampuia et al., (2013) also identified 102 E. coli from 53 Mizoram pig fecal samples. Forty two E. coli from 19 chickens that died from severe diarrhoea were also reported by Dutta et al., (2011). 

    The type of samples, the media employed, the researchers’ laboratory procedures, the host’s treatment status and other factors can affect the rate of E. coli isolation from fecal samples. There is considerable variation in the number of E. coli colonies obtained from a same sample. In the present study, four distinct pathotypes of E. coli including STEC (n=32), EPEC (n=6), EHEC (n=8) and ETEC (n=13) were recorded. Earlier, from the same laboratory, various pathotypes of E. coli were also reported (Das et al., 2021; Das et al., 2024; Kylla et al., 2017). The pathogenic E. coli isolated from livestock and poultry are always associated with public health significance due to their proximity to humans. As the human dwellings are very close to the pig housings and share a common source of water, transmission of such organisms between pigs to humans is possible (Puii et al., 2019).
           
    Majority of E. coli isolates were multi-drug resistant (MDR) and exhibited resistance to at least three classes of antibiotic. Among the 59 pathogenic E. coli isolates, imipenem was recorded as the most sensitive (88.44%). Maximum resistance was recorded against amoxicillin (91.53%) followed by ciprofloxacin (86.44%) and cefpodoxime (81.36%). Previously, Kylla et al., (2017) also reported a similar type of observation in E. coli isolates from pigs in 4 NE states of India. Carballo et al., (2013) observed that the most gram-negative bacteria antimicrobial resistances were to tetracycline (66.7%), ampicillin (52.4%), cephalothin (46.0%), chloramphenicol (44.4%), nalidixic acid (39.7%) and trimethoprim-sulphamethoxazole (33.3%).
           
    The bacteriophage lytic activity was tested against 59 pathogenic E. coli isolates, of which 28.81% were positive. The agar overlay and spot test methods revealed that E. coli phage had clear spots or plaques against 17 E. coli isolates. One of the critical factors influencing phage antimicrobial activity is host range, which is likely to be influenced by phage tail morphology. The phage isolates were effective against multiple E. coli pathotypes, whereas majority of the phage isolates showed the lytic spectrum against STEC pathotypes. The efficacy of bacteriophage isolates against different E. coli pathotypes was variable. The P-5 isolate could not exhibit any bacteriolytic activity against any of the E. coli pathotypes, which may be due to the non-availability of any specific surface attachment molecules on the E. coli isolates. Manohar et al., (2019) characterized Escherichia phage myPSH2311 and reported broad host range activity belonging to 6 different pathotypes including EAEC, EHEC, EIEC, EPEC, ETEC and UPEC besides few unknown pathotypes, which are in corroboration of our report, where the bacteriophages exhibited lytic activity for multiple pathotypes of E. coli. In the present study, isolated phages showed lytic activity against the five isolates of EHEC, which is also in corroboration of the report of Viazis et al., (2011), who reported 7/8 phages as highly effective against 16 EHEC strains.
                   
    Although the high specificity of the bacteriophages is their greatest strength, it also poses a challenge to their widespread use. Accurate targeting is ensured by the great specificity. Therefore, one crucial step in achieving the application is suitably expanding and altering the phage’s host range. Genetic engineering has been shown to alter the host spectrum of phages, but only for a small number of phages that elucidate the mechanism of receptor-binding protein binding on the host (Yoichi et al., 2005). Bacteriophage replication is essentially host genus-specific however; members of the family Enterobacteriaceae are so closely related that polyvalent phages are common, particularly in the E. coli-Shigella-Klebsiella group. There are only a few phages that have broad host specificity across bacterial genera. Accordingly, the observation in the present study might be considered as one of the highly specific broad-spectrum bacteriophages recovered from sewage. Further studies will be required to characterize the phage isolates and to determine their host range before recommending them as potential antibacterial agents against major MDR bacterial pathogens.

    CONCLUSION

    Therefore, it may be concluded that the bacteriophages isolated and identified from sewages of Mizoram exhibited antimicrobial activities against field isolates of multidrug-resistant pathogenic E. coli. Bacteriophages could be potential candidates in the future as alternatives to conventional antimicrobial agents to contain the menace of AMR in the environment.

    ACKNOWLEDGEMENT

    The authors are thankful to the DBT sponsored project on “DBT-NER Institutional Level Biotech Hubs at College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, Mizoram (Phase-II)” (BT/NER/143/SP44310/2021) for financial support and the Dean, CVScandAH, Central Agricultural University, Aizawl, Mizoram - 796015, India for providing research facility during this study.
     
    Author contributions
     
    Tarakchi M. Sangma: laboratory activities, sample collection; Tapan Kumar Dutta: Conceptualization, manuscript preparation; Parimal Rouchoudhury C: Data analysis; Prasant Kumar Subudhi: Manuscript editing; Indranil Samanta: Manuscript editing; Samiran Bandyopadhayay: Manuscript editing.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of the affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish or preparation of the manuscript.

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