Isolation and characterization of vB_SenS_Ib_psk2 bacteriophage against drug-resistant Salmonella enterica serovar Kentucky

Salmonella enterica serovar Kentucky is one of the food-borne zoonotic pathogens which is isolated in high frequency from poultry meat in the recent decades and is known for its multidrug resistance. The current study was aimed to isolate and characterize a bacteriophage against S. enterica serovar Kentucky isolate, 5925, which showed resistance to at least seven antibiotics and to study its efficiency to decontaminate S. Kentucky from chicken skin. The bacteriophage against S. enterica serovar Kentucky was isolated and was named vB_SenS_Ib_psk2 representing the place, source, and host. Electron microscopy revealed that the phage possesses isometric head and contractile tail, indicative of Siphoviridae family. Molecular detection of major capsid protein E gene yielded 511 bp, and NCBI blast analysis revealed that the phage belonged to the genus chivirus. The optimum temperature and pH for phage survival and multiplication were found to be − 20 to 42 °C and 6–10, respectively. One-step growth curve experiment of vB_SenS_Ib_psk2 revealed a latent period of 20 min and burst size of 253 phages/bacterial cell. The host susceptibility studies revealed that 83% of MDR isolates of S. enterica were susceptible to vB_SenS_Ib_psk2. Artificial spiking studies on chicken skin revealed that high multiplicity of infection (MOI) of phages of 106 pfu/mL is required for significant reduction (p ≤ 0.01) of bacterial concentration (0.14 ± 0.04) after 24-h incubation at 8 °C compared to group 1 (2.55 ± 0.89 cfu/mL).


Introduction
Nontyphoidal salmonellosis is one of the food-borne zoonotic illnesses affecting animal and human health.A variety of serovars, in particular, Typhimurium, Enteritidis, Kentucky, Infantis, Heidelburg, and Virchow, are involved in animal and human infections and food contamination.Invasive nontyphoidal salmonellosis is a condition in which bacteremia and mortality are reported in immunocompromised patients (Haselbeck et al. 2017).The recent studies have revealed the shift of serovars involved in iNTS from Tyhimurium and Enteritidis to Heidelburg, Infantis, Stanley, and Kentucky (Antunes et al. 2016).Salmonella enterica serovar Kentucky is one of the pathogens primarily associated with chicken and turkey meat (Le Hello et al. 2013).The recent concern about S.
Kentucky is the emergence of ciprofloxacin-resistant S. Kentucky ST 198 worldwide (Hawkey et al. 2019).The serovar further acquired antimicrobial-resistant genes by horizontal gene transfer and became resistant to cephalosporins and carbapenems (Fernández et al. 2018).Our recent studies revealed the emergence of resistance to fluoroquinolones and cephalosporins in NTS, especially S. Kentucky in India (Sharma et al. 2019;Inbaraj et al. 2022).The use of antibiotics in subtherapeutic concentration as growth promoters in poultry production is one of the major factors contributing to antimicrobial resistance development (Manyi-Loh et al. 2018).In case of Salmonella enterica, at least 50% of the multidrug-resistant isolates from poultry belong to the serovar Kentucky raising the risk of transmission of antimicrobial resistance genes to human food chain (Foley et al. 2011).Therefore, alternatives to antibiotics such as bacteriophages were used to control Salmonella enterica in various stages of poultry production chain (from farm to fork) (Hong et al. 2013;Kim et al. 2020).Bacteriophage-based biocontrol is one of the green approaches in which viruses against bacteria which possess narrow-spectrum or broad-spectrum host specificity targets only the pathogenic bacteria without altering the normal microflora of animal or food (Moye and Woolstonj 2018).There were previous studies depicting the bacteriophage-mediated control of various serovars of S. enterica by use as therapeutic, biocontrol, antibiofilm agents and as disinfectants that have been widely reviewed (Żbikowska and Dolka 2020; Moye and Woolstonj 2018;Endersen and Coffey 2020).Bacteriophages against S. Kentucky in poultry production and food safety are essential under Indian conditions as there was increase prevalence and AMR of that particular serovar (Sharma et al. 2019;Inbaraj et al. 2022).In the current study, isolation and characterization of bacteriophage active against drugresistant S. enterica serovar Kentucky and its efficiency in reducing the bacterial load in artificially contaminated chicken skin samples were observed.

Isolation, purification, and bulk production of bacteriophage against Salmonella enterica serovar Kentucky 5925
The sewage samples were collected from the pig farm located at ICAR-IVRI, Bareilly.The sample (40 ml) was poured into 4 ml of 10 × brain-heart infusion broth and mixed with 1 mL of overnight grown S. enterica serovar Kentucky (5925) culture followed by incubation at 37 °C for 48 h.The supernatant obtained after centrifugation at 8000 rpm for 10 min was filter-sterilized by 0.22 µm syringe filter (Axiva, India), and the bacteria-free filtrate (BFF) was obtained.Meanwhile, 200 µL of overnight grown culture of S. Kentucky (5925) was pipetted into 4 mL semisolid BHI broth, mixed gently, and was poured onto BHI plate.The BFF (5 µL) was spotted on to the plates, incubated overnight at 37 °C, and was observed for clear lytic zones.The phage which produced maximum clear zones was picked up and was further propagated using molten agar technique (Adams 1959) and incubated for 24 h at 37 °C.The plaques that appeared were harvested, filter sterilized (0.22 µm), and stored in SM buffer (100 mM NaCl, 10 mM MgSO 4 , 50 mM Tris-HCl, pH 7.5, and 0.01% (w/v) gelatin) at 4 °C.The plaque morphology is shown in Fig. 1(a).

Morphological characterization of Ib_psk2 by transmission electron microscopy
The phage suspension was treated with 5 M NaCl (2 mL) and PEG 8000 (2.2 g) at 4 °C overnight followed by centrifugation at 12,000 g for 30 min.The phage was further concentrated using chloroform extraction.The concentrated phage suspension (6 µL) was adsorbed to carbon-coated grid for 30 s.Then, the phage was negatively stained by 2% uranyl acetate for 30 s, and the excess stain was removed by washing in double distilled water.The phage was examined under JEM 1011 (JEOL, Japan) transmission electron microscope at different magnifications ranging from 80,000 to 150,000 X.

Thermal and pH stability studies
The bacteriophage of concentration 5 × 10 9 pfu/mL was subjected to wide temperature ranges, viz., − 20, 4, 21, 37, 42, 50, 60, 70, and 80 °C for a period of 1 h.Simultaneously, Ib_psk2 was exposed to pH ranging from 3 to 10 for 24 h.After treatment, the phage stability was observed using molten agar technique.The experiment was repeated thrice, and the mean ± standard error values were calculated.

One-step growth curve experiment
The bacterial overnight culture was mixed with phage suspension (MOI = 0.01) and was kept undisturbed for 10 min at room temperature.After 10 min, 100 µL of the resuspended pellet after centrifugation at 8500 × g was transferred to 10 mL BHI broth and incubated at 37 °C.Samples were withdrawn at 5,10,15,20,30,40,50, and 60 min, the supernatants were collected, and phage concentration was estimated using double agar overlay method.The method was repeated thrice, and the mean ± standard error values were calculated.

Isolation of phage genomic DNA
The phage suspension (10 mL) was filter sterilized using a syringe filter (0.22 µm) to remove residual bacteria, and further clearance by treatment with 0.1% chloroform was done.The chloroform residues were removed by centrifugation at 8000 rpm for 15 min.The phage particles were concentrated using 400 µL of 1 M ZnCl 2 for 5 min, and centrifugation was done at 10,000 rpm for 10 min at 4 °C.
The phage concentrate was suspended in 500-750 µL SM buffer.The phage suspension was treated with DNase I (1 U/ µL) and RNase A (10 mg/mL) for 1 h at 37 °C without shaking to remove bacterial nucleic acids.The enzyme reactions were stopped by treatment with 0.5 M EDTA (final concentration 20 mM) and boiling at 75 °C for 10 min (Jakočiūnė and Moodley 2018).The phage DNA was extracted by using DNeasy Blood & Tissue Kit (Qiagen, Germany) as per the manufacturers' instructions.

Molecular characterization of major capsid protein E of vB_SenS_Ib_psk2
The TEM findings morphologically revealed the family of the phage.Therefore, molecular characterization of phage was done by targeting the major capsid protein E of Ib_psk2 using forward (5′-TTC AGA CCC ACG GAT GGT TG-3′) and reverse (5′-AGA AAG CGG CTA CAA CAC GA-3′) primers (Phothaworn et al. 2019) (Eurofins Ltd., India).The PCR conditions involved an initial denaturation of 94 °C for 3 min followed by 35 cycles of initial heating at 94 °C for 60 s, annealing at 60 °C for 30 s, and 72 °C for 30 s followed by a final extension at 72 °C for 5 min.The PCR products were sequenced and edited using BIOEDIT software, and phylogenic tree was constructed using MegaX software by neighbor-joining method (Kumar et al. 2018), and the sequences were submitted to NCBI software.

Salmonella enterica isolates in vitro
The host range of vB_SenS_Ib_psk2 was assessed using 50 NTS isolates from farm animals, poultry, and environment, which were resistant to at least 3 or more antibiotics as mentioned in the previous study from our lab (Inbaraj et al. 2022).The purified phage suspension vB_SenS_Ib_psk2 (3 µL of 10 9 pfu/mL) was spotted on the lawn of each overnight-grown cultures (10 8 cfu/mL) and incubated at 37 °C as per modified bilayer plaque assay method (Kang et al. 2013).The experiment was repeated thrice, and the phage activity was labeled as + and − as per the zone of inhibition.

Bioassay on chicken skin
Chicken skin was procured from local meat shops and was cut into 2 × 2 cm, measuring 1 g.They were placed in individual sample boxes and frozen overnight at − 20 °C.The samples were thawed to 37 °C and further disinfected with UV irradiation inside Biosafety cabinet II (Haier) for 15 min.The overnight-grown S. Kentucky culture (10 6 cfu/ mL) of approximately 100 µL was applied to the skin surface to groups 1-3, whereas group 4 received treatment with approximately 10 4 cfu/mL bacterial concentration.The skin samples were kept undisturbed inside the cabinet for 30 min so that the bacteria get absorbed to the skin.After 30 min, distilled water treatment was applied to group 2, whereas groups 3 and 4 were treated with approximately 10 9 phages pfu/mL and were allowed to get adsorbed for 15 min, after which they were stored at 4 °C for 24 h.After 24 h, the samples were removed and washed with 9 mL of 0.1% BPW and serially diluted and plated in Xylose Lysine Tergitol4 (XLT4) agar plates in duplicates.The experiment was repeated thrice, and the mean ± standard error values were calculated.

Statistical analysis
The results were calculated as mean ± standard error and were analyzed using one-way ANOVA and Tukey's multiple range tests in GraphPad Prism software (version 9.3), and a P-value of ≤ 0.05 was considered significant.

Isolation, purification, and bulk production of bacteriophage against Salmonella enterica serovar Kentucky 5925
Initial testing of the bacteria-free filtrate from sample number 2 showed lysis of Salmonella Kentucky 5925.Therefore, further purification by molten agar technique was done, which revealed numerous circular, plagues which are clear in the center, indicative of virulent lytic phages.The plagues had a diameter of 0.5 to 1 mm.The phage was named as per the place of isolation in prefix (Izatnagar, pig shed), target host bacteria, and sample number in the suffix, namely, vB_ SenS_Ib_psk2.The phages were propagated using molten agar technique, harvested, filter-sterilized, and stored in 4 °C.The final concentration of the phages was found to be 6-7 × 10 14 pfu/mL.

Morphological characterization of Ib_psk2 by electron microscopy
Transmission electron microscopy findings revealed that Ib_psk2 possessed isometric capsids (63.61 ± 4.93 nm) and long, non-contractile tail (237.52 ± 46.88 nm), characteristic of Siphoviridae family.Therefore, the phage was named vB_SenS_Ib_psk2 based on TEM findings.The plaque and phage morphology of Ib_psk2 is depicted in Fig. 1(a) and (b), respectively.

Thermal and pH stability
When vB_SenS_Ib_psk2 was exposed to different temperatures ranging from − 20 to 42 °C, the phage concentration was found to be 426 ± 50.07 × 10 7 pfu/mL, which then significantly decreased (p ≤ 0.0001) to 80 ± 10 × 10 7 pfu/mL during incubation at 50 °C.Phage survival was not observed when incubated at 60, 70, and 80 °C.Ib_psk2 could not survive and multiply at the extreme pH of 3. Phage concentration (27 ± 19.42, 293.33 ± 37.86 × 10 7 pfu/mL) was significantly reduced (p ≤ 0.0001 and p ≤ 0.01) at pH 4 and 5, respectively.The phage concentration remained to be 657.33 ± 109.22 × 10 7 pfu/mL without any significant change at pH ranging from 6 to 10.The graphical representation of temperature and pH stability of vB_SenS_Ib_psk2 is depicted in Fig. 2(a) and (b), respectively.

One-step growth curve
After 10 min of adsorption time, the phage counts were 293 ± 24.0 pfu/mL at 5 min, followed by 366.7 ± 15.9 pfu/ mL at 10 min, 466.7 ± 33.3 pfu/mL at 20 min, followed by Fig. 2 Stability tests of phage vB_SenS_Ib_psk2under: a temperature ranging from − 20 to 50 °C and the phage concentration was expressed as (mean ± standard error) pfu/ml (plaque-forming units/ml).The phage concentration reduced significantly (p ≤ 0.0001) at 50 ℃.b pH stability of phage vB_ SenS_Ib_psk2 ranging from 3 to 10.The phage concentration was expressed as (mean ± standard error) pfu/mL.Significant reduction in phage concentration (p ≤ 0.0001) was observed at pH 4 a significant increase of 719.3 ± 12.24 pfu/mL at 30 min, which further decreased to 655.7 ± 18.66 and 644.7 ± 74.24 pfu/mL at 40 and 50 min, respectively.The burst size of vB_SenS_Ib_psk2 was 253 phages/cell.The latency period is found to be approximately 20 min and is graphically depicted in Fig. 3.

Molecular characterization of major capsid protein E of vB_SenS_Ib_psk2
The genomic DNA of vB_SenS_Ib_psk2 was isolated, its concentration was checked by an ND-1000 spectrophotometer (Nano-Drop Technologies, USA) and was found to be 19 ng/µL, and the major capsid protein E gene-based PCR amplification yielded a 511-bp length gene product.The sequenced PCR products were analyzed by NCBI blast, which revealed that vB_SenS_Ib_psk2 is a chivirus belonging to Siphoviridae family under the order Caudovirales.The alignment and editing of the sequences were done using BIOEDIT software and were submitted to NCBI (accession number: OL603975.1).Phylogenetic analysis of major capsid protein E gene of vB_SenS_Ib_psk2 is depicted in Fig. 4.

Host range determination studies of nontyphoidal Salmonella enterica isolates in vitro
Out of the 50 MDR Salmonella enterica isolates tested, 43 (84%) were found to be susceptible to phages by spot test.Majority of the susceptible isolates were of poultry origin and belonged to the serovars Kentucky, Virchow, Paratyphi B, Infantis, and Typhimurium.The source, serovar, year of Fig. 3 One-step growth curve experiment and the phage concentration was expressed as (mean ± standard error) phages/cell.The burst size and latency period of vB_SenS_Ib_psk2 were found to be 253 phages/cell and 20 min, respectively Fig. 4 Phylogenetic analysis of major capsid protein E gene of vB_SenS_Ib_psk2 using MegaX software.The bacteriophage, vB_SenS_Ib_psk2 was found to be phylogenetically close to chi-viruses isolated from poultry farms of Thailand

Bioassay on chicken skin
Phage application reduced bacterial concentration in group 2 (distilled water treatment) and group 3 (S.Kentucky 10 5 cfu/ mL + phage concentration 10 9 pfu/mL).However, group 4 showed significant reduction (p ≤ 0.01) in bacterial concentration (0.14 ± 0.04) after incubation at 8 °C for 24 h in comparison with group 1 (non-treated control group spiked with S. Kentucky).The concentration of bacteria and reduction after phage treatment are given in Table 2.

Discussion
Food safety is an important public health issue of major concern as food-borne illness affects 600 million people approximately worldwide, claiming 420,000 lives (WHO 2020).Campylobacteriosis and nontyphoidal salmonellosis are the major foodborne infections acquired through consumption of poultry meat (Żbikowska and Dolka 2020).Non-typhoidal salmonellosis affects human beings especially the immunocompromised individuals and animals.The infection is transmitted through feco-oral route and food, especially of animal origin that acts as major carrier of the organism.The prevalence of various serovars of NTS varies over time and is influenced by biosecurity measures, surveillance, vaccination, competitive exclusion, etc.Previous studies revealed that the shift from serovars Pullorum and Gallinarum to Enteritidis and Typhimurium happened over the past decades.In the recent years, serovars such as Heidelberg and Kentucky were frequently isolated from poultry and iNTS infections in humans (Foley et al. 2011).In addition, the emergence of multidrug-resistant S. enterica serovar Kentucky also has added the additional risk of food-borne illness with multidrug-resistant bacteria.
Even though such shift in serovars was well documented in developed countries, recent studies in South Asian countries also revealed the increase in prevalence of MDR serovar Kentucky (Barua et al. 2012;Fagbamila et al. 2017;Sharma et al. 2019;Inbaraj et al. 2022).Use of same class antibiotics in human medicine and livestock sector is one of the major contributing factors of antimicrobial resistance development.
Therefore, there arises a necessity to search for alternatives which can be targeted against MDR bacteria in livestock production and food safety.Hence, bacteriophage against MDR Salmonella enterica serovar Kentucky 5925 was isolated and characterized.The isolate was selected as the target host as it was found to be multidrug resistant to at least 7 antibiotics, namely, ampicillin, tetracycline, streptomycin, ciprofloxacin, nalidixic acid, levofloxacin, and amoxicillinclavulanic acid, as revealed in our previous study (Inbaraj et al. 2022).The bulk production yielded a concentration of 10 14 pfu/mL.The major advantage of bacteriophage is that its production can be scalable under laboratory conditions.Transmission electron microscopy studies revealed that the phage belonged to the family Siphoviridae characterized by long, non-contractile tails (Jurczak-Kurek et al. 2016), which was further confirmed by PCR.NCBI nucleotide blast of the sequences revealed that the phage belonged to chivirus genus of the Siphoviridae family.Phothaworn et al. (2019) reported that chi-like viruses were isolated from 50% samples from poultry farms of Thailand and that these viruses utilize bacteria flagella and LPS for adsorption.The stability studies of vB_SenS_Ib_psk2 revealed that though the phage can tolerate lower temperature, its concentration reduced significantly at 50 °C, after which phage could not survive.Similar observation was reported on Siphoviridae phages against Vibrio parahaemolyticus (Yang et al. 2020).Acidic pH 3-5 was found to be lethal to vB_SenS_Ib_psk2.The alkaline pH ranging from 6 to 10 was found to be optimum for survival and multiplication of vB_SenS_Ib_psk2.The phage was isolated from pig shed drainage which may contain the alkaline intestinal contents and hence the sewage pH.Alkalinity-tolerant phages can be used as disinfectants along with alkaline sanitizers in poultry meat processing plants (Litt et al. 2018).The latent period of vB_SenS_Ib_ psk2 was found to be 20 min, which was earlier reported as between 10 and 21 min (Litt et al. 2018).The burst size was found to be 253 phage/infected bacterial cell, which was slightly above the average burst size of 50-100 phage/ infected cell of Siphoviridae phages.The extended latent period may also be the reason for larger burst size in addition to type of media used and growth rate of bacterial host (Bao et al. 2011;Litt et al. 2018;Sun et al. 2012).In vitro host specificity tests revealed that vB_SenS_Ib_psk2 was effective against various multidrug resistant isolates belonging to Kentucky, Paratyphi B, Virchow, Infantis, and rough and some untypeable serovars.The host specificity of phages is due to its adsorption capacity to bacterial receptors.When the phage uses common antigens expressed across all serovars/strains/related species as receptors, then it can able to lyse maximum bacterial strains/species and hence called as broad-spectrum phage (Nilsson 2014).The biocontrol efficiency of vB_SenS_Ib_psk2 was evaluated in chicken skin samples artificially spiked with S. Kentucky 5925.Salmonella enterica serovar Kentucky is the major organism affecting post slaughter processing of poultry meat.The flagellar genes of this organism possess special affinity toward chicken meat, hence frequently isolated from poultry meat (Salehi et al. 2016).The experiment revealed that significant reduction in bacterial population from chicken skin was obtained from high MOI (10 6 ) of vB_SenS_Ib_psk2 compared to low MoI.A high MOI is actually required as the bacterial multiplication might be reduced under refrigeration temperature and due to single phage usage (Augustine and Bhat 2015).Treatment with distilled water did not show any significant reduction in bacterial count, though washing of the chicken carcass was believed to remove the bacteria.Even though the experiment showed significant reduction in bacterial population after high MOI treatment, use of phage cocktails which recognize different bacterial receptors for adsorption is advisable to prevent phage resistance to highly evolving serovar such as Kentucky.PCR sequencing and NCBI blast analysis revealed that the phage belonged to the genus chivirus.Whole-genome sequencing of chi-like viruses revealed that they possess an integrase gene characteristic of lysogenic life cycle (Phothaworn et al. 2019).Therefore, whole-genome sequencing and analysis of phages are further warranted in selection of phages for therapeutic and biocontrol purposes.

Conclusion
The serovar prevalence and antimicrobial resistant pattern of NTS isolates change over every decade.In the recent decade, S. Kentucky has emerged as one of the most prevalent and antimicrobial-resistant serovars in poultry meat.Therefore, phage isolation and characterization against S. Kentucky were done in the current study.The bacteria possess receptors to get attached to chicken skin.Phage treatment of chicken skin artificially spiked with S. Kentucky was attempted, which has yielded promising results.Therefore, bacteriophages can be one of the green alternatives and can contribute to food safety.

Fig. 1
Fig. 1 Morphology of phage vB_SenS_Ib_psk2: a plaque morphology revealing clear and round plaques of 0.5 to 1 mm and b transmission electron microscopy revealed icosahedral head and non-contractile tail, indicative of Siphoviridae family (bar: 200 nm)

Table 1
Salmonella enterica isolates, their serotypes, source and year of isolation, antimicrobial, and phage susceptibility

Table 2
Biocontrol studies of phage vB_SenS_Ib_psk2 in chicken skin artificially spiked with Salmonella enterica serovar Kentucky