Current Research in Bacteriology

Year: 2023 | Volume: 16 | Issue: 1 | Page No.: 17-26
DOI: 10.3923/crb.2023.17.26

Isolation and Characterization of Escherichia coli and Salmonella Bacteriophages from Poultry

Vasu Dev Hari Narayana Kola, Koigoora Srikanth , Shaik Muzammil Pasha, Yemgadda Sudhan Goutham and Chand Pasha

Abstract: Background and Objective: Escherichia coli and Salmonella species are major microbes that badly affect poultry. Various antibiotics are being used to control them and subsequently, antibiotic resistance is increased. Bacteriophages are better alternatives to control resistant E. coli and Salmonella species. Bacteriophages of choice are expected in the environment of their host bacteria. The present study aims to isolate bacteriophages of Escherichia coli and Salmonella species from poultry samples. Materials and Methods: Poultry litter samples were collected and isolated strains of E. coli and Salmonella species, were evaluated for their antibiotic resistance pattern and used to isolate the bacteriophages. Poultry litter aqueous suspension was filtered with 0.2 μ syringe filters and used as a phage source. Results: Isolated E. coli phage is specific to the isolated five strains of E. coli, having burst size of 120 plaque forming units per cell, tolerant to salt concentration 0.5-1.5%, temperature 37-40°C, pH 4-8 and found to be a tadpole shaped measuring a diameter of 647 nm and long non-contractile tail of 125 nm. Isolated Salmonella phage is specific to the isolated six strains of Salmonella, having burst size of 211 PFU per cell, tolerant to salt concentration 0.5-1.5%, temperature 37-40°C, pH 4-8 and found to be tadpole-shaped measuring icosahedral head of 60 nm in diameter and a tail of 32 nm in length. Conclusion: The isolated E. coli and Salmonella phages are specific to only E. coli and Salmonella, respectively. These phages can be used in poultry to control E. coli and Salmonella.

Keywords: Bacteriophage, Escherichia coli, Salmonella, antibiotic resistance and phage therapy

INTRODUCTION

Escherichia coli and Salmonella are the major microbes that are affecting the poultry industry adversely. Salmonella is a globally distributed food-borne pathogen. E. coli infections also cause severe losses to poultry appallingly. There is a considerable need for controlling the effect of these bacteria in the poultry industry. Bacteriophages are viruses that kill bacteria specifically. The use of E. coli and Salmonella bacteriophages as biocontrol agents has gained significant interest. The bacteriophages are abundant in the source of their specific bacteria. Phages have acquired the interest of researchers due to their host specificity, self-replication and abundance in the environment.

Increased demand for poultry products has forced the overuse of antibiotics and is causing resistant microbial infections. Interest has grown in phage therapy as an alternative treatment. The utilization of bacteriophage to kill resistant bacteria is a bright option for poultry to control diseases. Many bacterial phages are reported to control pathogenic E. coli and Salmonella in poultry. Escherichia coli and Salmonella are of main agents causing infections in poultry leading to huge losses to poultry and issues of public health¹. The E. coli infections lead to avian colibacillosis, respiratory diseases, bronchitis². Salmonella infections in poultry may cause pullorum disease (Salmonella pullorum), foul typhoid (Salmonella gallinarum), foul paratyphoid,etc³. To control the microbial load in commercial poultry, various antibiotics are being used and it is responsible for the increased emergence of antibiotic-resistant bacteria. Alternative strategies should be attempted to avoid the prevalence of microbial resistance^4-7. Phage therapy can be a potential alternative to antibiotic treatment and proper control of multi-drug resistant bacteria^8-11. Bacteriophages are potential therapeutic agents for bacterial diseases because they have high specificity and lysing of target bacteria¹². In the view of above, the present study was undertaken for the isolation of E. coli and Salmonella species from different poultry farms and rising bacteriophages against them. Subsequently, their utilization in poultry farms to control E. coli and Salmonella diseases.

MATERIALS AND METHODS

Sample collection: Samples containing E. coli, Salmonella and their bacteriophages are poultry litter and drainage samples collected from 25 poultry farms of Telangana and Andhra Pradesh, India collected during April and May, 2022.

Isolation and identification of E. coli and Salmonella: Pure E. coli and Salmonella strains were isolated from collected poultry samples. The samples were suspended in sterile normal saline and spread on MacConkey agar and Salmonella Shigella agar, Himedia, India incubated at 37°C for 24 hrs. Growth on specific media, microscopic morphology, and biochemical tests such as indole test, methyl red test, voges proskauer test, citrate test, TSIA (triple sugar iron agar) test, urease test, dulcitol fermentation test and lysine decarboxylase test were determined¹³.

Antibiotic susceptibility test: Antibiotic susceptibility of E. coli and Salmonella was determined by kirby-bauer disc-diffusion method¹⁴. Eight different antibiotics were used in these tests ampicillin, norfloxacin, tetracycline, ciprofloxacin, kanamycin, amikacin, streptomycin and amoxicillin obtained from Merck India.

Isolation of the bacteriophages
Bacteriophage enrichment: Bacteriophage enrichment was done by taking 4 mL of 0.2 μ filtered sample water suspension (phage source), 1mL of 10x luria broth and 1 mL of exponential growing bacteria and incubating at 37°C for 24 hrs. Then the suspension was centrifuged at 15000 rpm for 5 min and filtered through a 0.2 μ syringe filter. The filtrate was mixed with pure culture and overlaid using double agar layered-based plaque assay method¹⁵.

Detection of bacteriophages/ plaque assay: In a sterile Eppendorf, 0.2 μ syringe filtered 100 μL of bacteriophage source and 100 μL of exponential bacterial culture were added and incubated at 37°C for 15 min, then it was mixed with 5 mL low melting agar (0.8%) and poured onto a nutrient agar plate. Allowed the low melting agar to solidify for 30 min at room temperature and then plates were incubated inverted at 37°C for 24 hrs.

Purification of phage: Using a sterile scalpel, an isolated plaque was picked from the overlayed nutrient agar plate and suspended with 500 μL of phage buffer and diluted. A dilution was mixed with exponential bacterial culture, incubated and underwent double agar layered-based plaque assay¹⁵. Individual plaque obtained in this method is selected.

Host inactivation studies: Pure E. coli and Salmonella were inoculated separately into two flasks containing nutrient broth and incubated at 37°C for 24 hrs. Then the two flasks were infected with 0.2 μ filtered phages and incubated at 37°C with gentle shaking. The sample was collected from both flask every 1 hr, till 8 hrs consecutively. The hourly samples of both flasks were spread on the nutrient agar plates, respectively for the viability of host cells. The numbers of colonies in the hourly samples were counted by using the colony counter multilab India. The time required to kill 90% of initial cells was measured.

Burst size determination: An isolated plaque was picked into a sterile Eppendorf containing 500 μL of phage buffer and then it was added to 500 μL of bacterial culture in an Eppendorf and 100 μL of the mixture was undergone double-layered agar-based plaque assay¹⁵.

Salt, heat and pH tolerance: Salt tolerance was determined by treating the phage filtrate in successive tubes containing nutrient broth with additional 0.5, 1.5, 2.5, 3.5 and 4.5% salt, incubated at 37°C for 1 hr. Heat tolerance was determined by treating the phage samples at 37, 40, 50, 60, 75 and 85°C for 1 hr. The pH tolerance was determined by treating phage filtrate in consecutive tubes containing nutrient broth of 1 to 10 pH ranges, incubated at 37°C for 1 hr. This bacteriophage filtrate treated with different salt, heat and pH concentrations was overlayed using double layered gar-based plaque assay method¹⁵.

Purification of phages
Phage purification with centrifugation: Phage lysate was made cell-free by centrifuging at 5000 rpm for 10 min and the clear lysate was again centrifuged at 15000 rpm for 5 hrs to precipitate phages. The pellet was suspended in the phage buffer.

Chloroform: The phage lysate was centrifuged at 5000 rpm for 10 min and the cleared phage lysate was taken into phage buffer and treated with 15% chloroform. As the chloroform sediments, the top layer was taken and centrifuged at 5000 rpm for 10 min and the supernatant was filtered through a 0.2 micron syringe filter.

Poly ethylene glycol: By centrifugation of 15000 rpm for 5 min, the cells were removed and the supernatant was collected. The PEG 8000 was added to the supernatant solution to make a 2% concentration and stirred at 4°C overnight to precipitate the bacteriophages. Then the solution was centrifuged at 15000 rpm for 10 min, bacteriophages were collected as pellets and suspended in phage buffer and dialyzed.

Transmission electron microscopy of phages: One drop of the purified phage suspension was placed on a copper grid with carbon-coated Formvar film for 10 min at room temperature. As 4% aqueous phosphotungstic acid was used for staining at pH 7. The sample was air-dried overnight and examined with a Zeiss TEM 900 electron microscope, Carl Zeiss AG it was operated at 50 kV. The phage particles were visualized using the Image SP software V2.5 SYSPROG under the guidance of TRS, Duncelbuch, Moorenweis, Germany and a CCD (charge-coupled device) camera Horiba instruments, Piscataway, New Jersey, United States.

Determination of host range: The host range of obtained phages was determined by E. coli, Salmonella and Campylobacter. As 1 mL of pure E. coli, Salmonella and Campylobacter were spread on nutrient agar plates, respectively. While 50 μL of phages was sprayed on the nutrient agar plate with pure E. coli/Salmonella/Campylobacter culture. These plates were incubated at 37°C for 24 hrs. Then plates were observed for plaques.

Bacteriophage efficacy studies: The plaque formation ability of phages on each bacterial strain or the effectiveness of phage on each bacterial strain was determined by 100 μL of phage and 100 μL of pure isolates of different strains, respectively, mixed with low melting agar overlayed on a nutrient agar plate and incubated at 37°C for 24 hrs. The number of plaques was counted. The highest efficacy was considered in that bacterial strain, where the highest number of plaques was produced.

Statistical analysis: Experiments were repeated three times in triplicates (n = 9) and the average values were provided in the results.

RESULTS

Isolation and identification of poultry E. coli and Salmonella: Pure E. coli was isolated from poultry samples, collected from various parts of Telangana and Andhra Pradesh. The E. coli was identified by growth on MacConkey agar, microscopy and biochemical characteristics and the results were presented in Table 1.

Five E. coli strains were isolated from different poultry samples. They were identified as E. coli based on pink colonies on MacConkey agar and transparent colonies on Salmonella Shigella agar.

They were gram-negative, rod-shaped and 1-3×0.4-0.7 μm in size. In the biochemical tests, it was indole positive, methyl red positive, vogues prausker test negative and citrate test negative.

Pure Salmonella was isolated from poultry samples, collected from various parts of Telangana and Andhra Pradesh. Salmonella was identified by growth on Salmonella Shigella agar, microscopy and biochemical characteristics and the results were presented in Table 2.

Five Salmonella strains were isolated from different poultry samples. They were identified as Salmonella based on Transparent colonies on MacConkey agar, black colonies on Salmonella Shigella agar. They were gram-negative, Straight rods, 0.7-1.5×2-5 micrometers.

Biochemical tests: It was urease negative, dulcitol fermentation test negative, lysine decarboxylase test positive and in Triple Sugar Iron Agar test specific to Salmonella by the formation of red slant, yellow butt and H₂S production.

Antibiotic susceptibility test: Results showed that a high rate of resistance was against ampicillin, amoxicillin and tetracycline, followed by amikacin, norfloxacin, streptomycin, ciprofloxacin, kanamycin. Resistance was observed for all E. coli and Salmonella strains.

The E. coli and Salmonella are highly susceptible to ciprofloxacin, kanamycin and streptomycin, whereas they are highly resistant to ampicillin, amoxicillin and tetracycline (Table 3).

Bacteriophage enrichment: Phage enrichment filtrate contained numerous phages and formed plaques of varying sizes specific to E. coli and Salmonella strains.

Detection of bacteriophages/plaque assay: In the plaque assay, after incubation, bacteriophage plaque formation was determined and plaques were counted as plaque-forming units (PFU). Salmonella phages were small and round as in Fig. 1. The E. coli phages were large and oval as in Fig. 2.

Phage purification: The plaque was purified and used in the plaque assay method which produced plaques specific to E. coli and Salmonella on nutrient agar plates. The single and isolated plaque was selected for pure phage.

Host inactivation studies: The number of colonies in hourly samples was counted using the colony counter. The viable cell count was more till 1 hr, from the 2nd hr, the number of viable cells started decreasing in descending order. As 90% of E. coli cells were inactivated in 5 hrs whereas in Salmonella it took 3 hrs only.

Burst size determination: Plaques were observed on the nutrient agar plates. The plaque with the largest burst size was the bacteriophage with higher effectivity.

Among them, for E. coli, lambda phage produced a burst size of 120 PFU per cell and for Salmonella, SAL-PG phage produced a burst size of 211 PFU per cell.

Salt tolerance: Phage filtrate was subjected to various salt concentrations of 0.5 to 4.5% (Fig. 3). At salt concentrations of 2.5 to 4.5%, the phages were decreased by 20, 30 and 50%, respectively. The phage filtrate was identical to the control at 0.5 and 1.5%, indicating the ability of phages to survive in a narrow salt concentration range of 0.5 to 1.5%.

Temperature tolerance: Phage filtrate was subjected to various temperatures: 37, 40, 50, 60, 75 and 85°C (Fig. 4). There was no decrease in phages till 40°C. At 50 and 60°C phages were reduced because the number of plaques was decreased consecutively in decreasing order. Phages were not present at 75 and 85°C indicating the non-survival of phages at 75 and 85°C treated for 1hr.

pH tolerance: Phage filtrate was subjected to various pH ranges from 1 to 10 (Fig. 5). The phages were reduced 60, 40 and 30% at 1, 2 and 3 pH, respectively.

From pH range 4 to 8, the phage filtrate was identical to the control, indicating the ability of phages to survive in a broad range of pH. The phages were reduced by 20 and 50% at 9 and 10 pH, respectively.

Transmission electron microscopy of phages: The E. coli phage-phages were like lambda phages in morphological appearance, having an icosahedral head with a diameter of 647 nm and long non-contractile tails of 125 nm as in Fig. 6.

The Salmonella phage-phage was SAL-PG phage, with an icosahedral head of 60 nm in diameter and a tail of 32 nm in length as in Fig. 7.

Host ranges: Host ranges were determined using E. coli, Salmonella and Campylobacter. The SAL-PG bacteriophage infected Salmonella isolates only and Lambda phages infected E. coli isolates only. The bacteriophages didn’t infect Campylobacter.

Bacteriophage efficacy studies: Plaque formation efficiency of lambda phages on five E. coli strains and plaque formation efficiency of SAL-PG on five Salmonella strains was obtained. The E. coli phage has infected and lysed all five E. coli strains, Salmonella phage has killed four of five Salmonella strains isolated from poultry samples.

Bacteriophage isolation and purification: The E. coli bacteriophages were isolated from poultry samples, phages were like lambda phages in morphological appearance, having an icosahedral head with a diameter of 647 nm and a long non-contractile tail of 125 nm based on TEM. Salmonella bacteriophages were isolated from poultry samples.

Among them, SAL-PG bacteriophage formed round and big plaques in Salmonella species. SAL-PG was selected for Salmonella species and lambda phages were selected for E. coli species based on host range and clear plaques.

DISCUSSION

Isolated E. coli phage is specific to the isolated five strains of E. coli, having a burst size of 120 PFU per cell, tolerant to salt concentrations 0.5-1.5%, temperatures 37, 40°C, pH 4-8 and found to be a tadpole-shaped, measuring a diameter of 647 nm and long non-contractile tail of 125 nm. Isolated Salmonella phage is specific to the isolated five strains of Salmonella, having a burst size of 211 PFU per cell, tolerant to salt concentrations of 0.5-1.5%, temperatures of 37-40°C, pH 4-8 and being found to be tadpole-shaped, measuring an icosahedral head of 60 nm in diameter and a tail of 32 nm in length. The present phages raised against E. coli and Salmonella are specific to E. coli and Salmonella respectively, not infecting other bacteria, hence can be a good source for phage therapy as reported by Nilsson¹⁶. The E. coli phage has infected and lysed all five E. coli strains, Salmonella phage has killed four of five Salmonella strains isolated from poultry samples. Bacterial infections in commercial poultry are challenging^17-19. Uncontrolled usage of antibiotics in poultry is leading to the development of antibiotic-resistant micro flora^20,21. Bacteriophages offer great potential as an alternative to antibiotics in poultry²².

Bacteriophages effectively kill resistant bacteria to reduce the prevalence of antibiotic resistance^23-26. The E. coli and Salmonella are predominant microbial pathogens of commercial poultry²⁷. As bacteriophages and their host bacteria will be present in the same environment, poultry samples were used to isolate E. coli and Salmonella strains and also to isolate the bacteriophages against this bacteria²⁸. E. coli and Salmonella species were isolated from poultry samples as reported by some researchers^28,29. The E. coli strains are known for the high mortality of chickens and found to be reduced by phages^28,30. Salmonellosis poses a health threat to farmers and consumers and bacteriophages were found to reduce salmonellosis³¹. The supplementation of E. coli and Salmonella phages increased the Lactobacillus concentration confirming the improved gut ecosystem³². Phage specificity for target and lysis of bacteria should be high to prevent non-specific bacterial targeting³². As Nillson¹⁶ has reported 20% infectivity in E. coli phages and 50% infectivity in Salmonella phages. Both the phages are having high infectivity and phage inactivation rates. The 90% of E. coli cells were inactivated in 5 hrs, whereas, in Salmonella it took 3 hrs only. The inactivation rate was reported 4 and 8 hrs for E. coli and Salmonella, respectively³³. The bacteriophages were able to survive in a wide range of pH (4 -8), resistant at 40°C for 1 hr and tolerating up to 1.5% salt as also reported by a researcher¹.

Bacteriophages for specific bacteria can be isolated in the bacterial habitat and can be used against the specific bacteria. The present isolated bacteriophages are specific to the host isolated and hence can be used to control E. coli and Salmonella spp.

CONCLUSION

Isolated E. coli phage is specific to the isolated five strains of E. coli, having burst size of 120 plaque forming units per cell, tolerant to salt concentration 0.5-1.5%, temperature 37-40°C, pH 4-8 and found to be a tadpole-shaped measuring a diameter of 647 nm and long non-contractile tail of 125 nm. Isolated Salmonella phage is specific to the isolated six strains of Salmonella, having burst size of 211 PFU per cell, tolerant to salt concentration 0.5-1.5%, temperature 37-40°C, pH 4-8 and found to be tadpole-shaped measuring icosahedral head of 60nm in diameter and a tail of 32 nm in length. Bacteriophages against E. coli and Salmonella are highly specific, lytic, tolerant to broad environment conditions and kill specific bacteria in a short duration.

SIGNIFICANCE STATEMENT

As poultry is rich in E. coli and Salmonella spp. prepared phages can be used in poultry applications. Cocktail of E. coli and Salmonella bacteriophages may be effective against poultry E. coli and Salmonella.

REFERENCES