WITHDRAWN: Isolation and characterization of vB_SenS_Ib_psk2 bacteriophage against drug resistant Salmonella enterica serovar Kentucky

doi:10.21203/rs.3.rs-1900211/v1

Salmonella enterica serovar Kentucky is one of the food-borne zoonotic pathogens known for multidrug resistance. The current study was aimed at isolating and characterization of bacteriophages against the pathogen. The bacteriophage against S. enterica serovar Kentucky was isolated and was named vB_SenS_Ib_psk2, representing the virus family, place, source, and host. Electron microscopy revealed that the phage possesses an isometric nonenveloped head and a noncontractile tail, indicative of the Siphoviridae family. Molecular detection of the major capsid protein E gene yielded 511 bp and NCBI blast analysis revealed that the phage belonged to the genus chivirus. Temperature and pH were found to be optimal at 20 to 42° C and 6–10, respectively for phage survival and multiplication. A one-step growth curve experiment revealed a latent period of 20 min and a burst size of 253 phages/cell. 83% of MDR isolates of S. enterica were susceptible to vB_SenS_Ib_psk2. Studies in chicken skin revealed that an MOI of 10⁶ is required for significant reduction of the bacteria.

Bacteriophage

Salmonella

Antimicrobial

Resistance

Biocontrol

Chicken

Nontyphoidal salmonellosis (NTS) is one of the foodborne zoonotic illnesses affecting food safety and animal and human health. A variety of serovars, in particular, Typhimurium, Enteritidis, Kentucky, Infantis, Heidelburg, and Virchow are involved in animal, human infections, and food contamination. Invasive nontyphoidal salmonellosis (iNTS) is a condition in which bacteremia and mortality are reported in immunocompromised patients (Haselbeck et al. 2017). Recent studies revealed the shift of serovars involved in iNTS from Typhimurium 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 and contaminated food (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 become resistant to cephalosporins and carbapenems (Fernández et al. 2018). Recent studies in India revealed the emergence of resistance to fluoroquinolones and cephalosporins in NTS, especially, S. Kentucky (Sharma et al. 2019; Inbaraj et al. 2022). The use of antibiotics in sub-therapeutic doses as growth promoters in poultry production is one of the major factors contributing to antimicrobial resistance (AMR) development (Manyi-Loh et al. 2018). In the case of foodborne bacteria such as Salmonella enterica, at least 50% of the multidrug-resistant isolates from poultry belong to the serovar Kentucky raising the risk of transmission of AMR genes to the human food chain (Foley et al. 2011). Therefore, an alternative to antibiotics such as bacteriophages were used to control Salmonella enterica at various stages of the poultry production chain (from farm to fork). Bacteriophages-based biocontrol is one such green technology-based approach in which viruses against bacteria are used to control bacterial growth. They possess host specificity targeting only the pathogenic bacteria without altering the normal microflora of animals or food. Previous studies depicting bacteriophage-mediated control of various serovars of S. enterica have been widely reviewed (Żbikowska et al. 2020; Moye et al. 2018; Endersen and Coffey 2020). In the current study, isolation, and characterization of bacteriophage against drug-resistant S. enterica serovar Kentucky and its efficiency in artificially contaminated chicken skin samples were performed.

Bacteria

Salmonella enterica serovar Kentucky (No 5925) was characterised by culture in Hektoen enteric plates, biochemical tests, and invA gene PCR. Antimicrobial susceptibility testing revealed that the bacteria was resistant to seven antibiotics, namely Am, AMC, CIP, Tet, LVX, NX, and S (Inbaraj et al., 2022).

Isolation, purification, and bulk production of bacteriophage against Salmonella enterica serovar Kentucky 5925

The sewage samples were collected from the pig/porcine shed at ICAR-IVRI, Bareilly. The sample (40 ml) was poured into 4 ml of 10X Brain Heart Infusion broth and mixed with 1 ml of overnight grown S. enterica serovar Kentucky (5925) culture, followed by incubation at 37ºC for 48 hrs. The supernatant obtained after centrifugation at 8000 rpm was sterilised by passing through a syringe filter (0.22 µm, Axiva, India) and the bacteria-free filtrate (BFF) was obtained. Meanwhile, 200 µl of the overnight grown culture of S. Kentucky (5925) was mixed with 4 mL of semisolid BHI broth gently and poured onto the BHI plate. The BFF (5µL) were spotted on the plates, incubated overnight at 37º C, and observed for clear lytic zones. The phage which produced the maximum clear zones was picked up and further propagated using the molten agar technique (Adams, 1959) and incubated for 24 hrs at 37ºC. The plaques appeared were harvested, filter sterilized (0.22µm) and stored in SM buffer (100mM NaCl, 10mM MgSO₄, 50mM Tris-HCl, pH 7.5 and 0.01% (w/v) gelatin) at 4ºC. The plaque morphology was shown in Figure 1 (a).

Morphological characterization of Ib_psk2 by transmission electron microscopy

The phage suspension was treated with 5M NaCl (2 ml) and PEG 6000 (2.2 g) at 4ºC overnight, followed by centrifugation at 12000 g for 30 min. The phage was further concentrated by chloroform extraction. The concentrated phage suspension (6 µL) was adsorbed to the carbon-coated grid for 30 s. The phage was negatively stained by 2% uranyl acetate for 30 s and the excess stain was removed by washing with double distilled water. The phage was examined with the JEM 1011 (JEOL, Japan) Transmission Electron Microscope at different magnifications ranging from 80000-150000X magnification as shown in Fig. 1 (b).

Thermal and pH stability studies

The bacteriophage of concentration 5× 10⁹was subjected to a wide temperature range, viz., -20, 4, 21, 37, 42, 50, 60, 70, and 80º C for a period of 1 hr. Simultaneously, Ib_psk2 was exposed to pH ranging from 3-10 for 24 hrs. After treatment, the phage stability was observed using the molten agar technique. The results were plotted as graphs, as shown in Fig. 2 (a, b).

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, centrifugation at 8500× g for 10 min was done and the 100 µl of the resuspended pellet was transferred to 10 ml of BHI broth and incubated at 37º C. Samples were withdrawn at 5, 10, 15, 20, 30, 40, 50, 60 min and the phage concentration was estimated using the double agar overlay method. The growth frequency was plotted as a graph, as shown in Fig. 3.

Isolation of phage genomic DNA

The phage suspension (10 ml) was filtered to remove residual bacteria, and further clearance by treatment with 0.1% chloroform was achieved. Then centrifugation at 8000 rpm for 15 min was done to get rid of chloroform. Phage concentration was done by treatment with 400 µl of 1M ZnCl₂ 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), RNase A (10 mg/mL) for 1 h at 37 ºC, without shaking, to remove bacterial nucleic acids. The enzyme reactions were stopped by treatment with 0.5M EDTA (final concentration 20 mM) and boiling at 75ºC for 10 min (Jakočiūnė and Moodley, 2018). The phage DNA was extracted by using the DNeasy Blood & Tissue Kit (Qiagen, Germany) as per the manufacturers’ instructions.

Molecular characterization of major capsid protein E of vB_SenS_Ib_psk2

Based on the TEM findings, the family to which the phage belongs was revealed. Therefore, molecular characterization of phage by available published primers targeting the phage family gene viz. major capsid protein E of Ib_psk2 using forward (5’-TTCAGACCCACGGATGGTTG-3’) and reverse (5’-AGAAAGCGGCTACAACACGA-3’) primers (Phothaworn et al. 2019). The PCR products were sequenced and edited using BIOEDIT software. A phylogenetic tree was constructed using MegaX software by the neighbor-joining method (Kumar et al. 2018) and the sequences were submitted to NCBI software. Figure 4 depicts a phylogenetic analysis of the major capsid protein E gene of vB_SenS_Ib_psk2.

Host range determination studies of nontyphoidal Salmonella enterica isolates in-vitro

The host range of vB_SenS_Ib_psk2 was assessed using 50 NTS isolates from farm animals, poultry, and the 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 10⁹ pfu/ml) was spotted on the 70 µl (10⁸ cfu/ml) overnight grown cultures and incubated at 37º C as per the modified bilayer plaque assay method (Kang et al., 2013). As per the zone of inhibition, the phage activity was labelled as + and -, respectively.

Bioassay on chicken skin

The 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. About 100 µl of overnight S. Kentucky culture (10⁶cfu/ml) was applied to the skin surface of groups 1–3 and 10⁴cfu/ml to group 4 and allowed to get absorbed for 30 min. One ml of distilled water was applied to group 2, 10^-9phages were applied to the treatment groups 3, 4 and allowed to get adsorbed for 15 min, after which they were stored at 4 ̊ C for 24 h. After 24 hrs, the samples were removed and washed with 9 ml of 0.1% BPW and serially diluted and plated on XLT4 agar in duplicates.

Statistical analysis

Each experiment was repeated thrice and the results were calculated as mean ± standard error and analysed 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

On preliminary analysis, the bacteria-free filtrate (sample number 2) showed lysis of Salmonella Kentucky 5925. Therefore, further examination by the molten agar technique revealed numerous circular plagues that are clear in the center, indicative of virulent lytic phages. The plagues had a diameter of 0.5 to 1 mm. The phages were propagated using the molten agar technique, harvested, filter sterilized, and stored at 4°C. The final concentration of the phages was found to be 6-7×10¹⁴ 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 a long, non-contractile tail (237.52±46.88nm), characteristic of the Siphoviridae family. Therefore, the phage was named vB_SenS_Ib_psk2 based on TEM findings.

Thermal and pH stability

When vB_SenS_Ib_psk2 was exposed to different temperatures ranging from-20 °C to 42°C, the phage concentration remained to be 426±50.07×10⁷, which then significantly decreased (p ≤ 0.0001) to 80 ±10 ×10⁷ pfu/ml during incubation at 50°C. Phage survival was not observed when incubated at 60, 70, and 80°C. The phage could not survive and multiply at the extreme pH of 3. At pH 4 and pH 5, the concentration of phage (27±19.42, 293.33 ± 37.86×10⁷) was significantly reduced (p 0.0001, p 0.01). The phage concentration remained to be 657.33±109.22 ×10⁷without any significant change in pH ranging from 6-10.

One-step growth curve

The burst size of vB_SenS_Ib_psk2 was 253 phages/cell and the latency period was 20 min.

Molecular characterization of vB_SenS_Ib_psk2's major capsid protein E

The genomic DNA of vB_SenS_Ib_psk2 was isolated and its concentration was checked by nanodrop and was found to be 19ng/µl. A 511 bp product was obtained from PCR amplification of the major capsid protein E gene. The sequenced PCR products were analysed by NCBI blast, which revealed that vB_SenS_Ib_psk2 is a chivirus, belonging to the siphoviridae family under the order Caudovirales. Alignment and editing of the sequences were done by using BIOEDIT software and submitted to NCBI (Accession number: OL603975). Figure 4 depicts a phylogenetic analysis of the major capsid protein E gene of vB_SenS_Ib_psk2.

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. Most of the isolates were of poultry origin and belonged to the serovar Kentucky. Apart from Kentucky, serovars such as Virchow, Paratyphi B, Infantis, Typhimurium, etc. were also found to be susceptible. The source, serovar, year of isolation, antimicrobial susceptibility, and phage susceptibility were tabulated in Table 1.

Bioassay on chicken skin

Phage application did not affect bacterial concentration in groups 2 and 3. However, after 24 hours of incubation at 8 °C, group 4 showed a significant decrease in bacterial concentration (p 0.01). The concentration of bacteria and reduction after phage treatment are given in Table 2.

Food safety is an important public health issue of major concern as foodborne illness affects 600 million people worldwide, claiming 420,000 lives (WHO, 2020). Campylobacteriosis and nontyphoidal salmonellosis are the major foodborne infections acquired through the consumption of poultry meat (Z˙bikowska et al. 2020). Nontyphoidal salmonellosis affects humans, especially the immunocompromised ones. The infection is transmitted through the faeco-oral route and food, especially of animal origin, acts as a 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 research revealed that the shift from serovars Pullorum and Gallinarum to Enteritidis and Typhimurium happened over the past decades. In recent years, serovars such as Heidelberg and Kentucky have been frequently isolated from poultry and cause iNTS infections in humans (Foley et al. 2011). In addition, the emergence of multidrug-resistant S. enterica serovar Kentucky has added the additional risk of foodborne illness with multidrug-resistant bacteria. Even though such a shift in serovars has been well documented in developed countries, recent studies in South Asian regions have also revealed an increase in the prevalence of MDR serovar Kentucky (Barua et al., 2012; Fagbamila et al., 2017; Sharma et al., 2019; Inbaraj et al., 2022). The use of the same class of antibiotics in human medicine and the livestock sector is a major contributor to the development of antimicrobial resistance. Therefore, there is a necessity to search for alternatives that can be targeted against MDR bacteria in livestock production and food safety. Hence, a bacteriophage against MDR Salmonella enterica serovar Kentucky was isolated and characterized. The bulk production yielded a concentration of 10¹⁴. 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, characterised by long, non-contractile tails and a nonenveloped head (Jurczak-Kurek et al. 2016) which was further confirmed by PCR. NCBI nucleotide blast of the sequences revealed that the phage belonged to a chivirus of the siphoviridae family. According to Phothaworn et al. (2019), chi-like viruses were isolated from 50% of samples from Thai poultry farms, and these viruses adsorb to bacteria flagella and LPS. The stability studies of vB_SenS_Ib_psk2 revealed that though the phage can tolerate lower temperatures, its concentration reduced significantly at 50°C after which, the phage could not survive. Similar observations were 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 6–10 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 10–21 min (Litt et al. 2018). The burst size was found to be 253 phages/infected cells which was slightly above the average burst size of 50–100 phages/infected cells of other siphoviridae phages. The extended latent period may also be the reason for the larger burst size in addition to the type of media used and the growth rate of the 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, rough, and some untypeable serovars. The host specificity of phages is due to their adsorption capacity on 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, hence being called a 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 a special affinity toward chicken meat; hence, it is frequently isolated from poultry meat (Salehi et al. 2016). The experiment revealed that a significant reduction in bacterial population from chicken skin was obtained from a high MOI (10⁶) of vB_SenS_Ib_psk2 compared to a low MoI. A high MOI is 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 the bacterial count, though washing the chicken carcass was believed to remove the bacteria. Even though the experiment showed a significant reduction in bacterial population after high MOI treatment, the use of phage cocktails which recognise different bacterial receptors for adsorption is advisable to prevent phage resistance to highly evolving serovars 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 the lysogenic life cycle (Phothaworn et al. 2019). Therefore, whole genome sequencing and analysis of phages is further warranted in the selection of phages for therapy and use as biocontrol agents.

The serovar prevalence and antimicrobial resistant pattern of NTS isolates change over every decade. In recent decades, 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 replace the chemical treatment of food products with green technology and can contribute to food safety.

NTS Nontyphoidal Salmonella

iNTS invasive Nontyphoidal Salmonella

MOI Multiplicity of Infection

TEM Transmission Electron Microscopy

AM Ampicillin

AmC Amoxicillin—clavulanic acid

CIP Ciprofloxacin

LVX Levofloxacin

NA Nalidixic acid

SXT Trimethoprim/Sulphamethoxazole

Tet Tetracycline

S Streptomycin

Author contributions

SI was involved in phage isolation, experimental design, and phage studies. PT and AV were involved in Salmonella isolates serotyping, maintenance, and molecular studies. RKA was involved in chicken skin experimental studies. Overall supervision was done by PC and V.K.C All authors reviewed the manuscript.

Funding

Not applicable

Compliance with ethical standards

Ethical Approval

The article does not contain any studies with human participant or animals

Adams MH (1959) Bacteriophages. Interscience Publishers Inc, New York
Antunes P, Mourão J, Campos J, Peixe L (2016) Salmonellosis: the role of poultry meat. Clin Microbiol Infect 22(2):110–121
Augustine J, Bhat SG (2015) Biocontrol of Salmonella Enteritidis in spiked chicken cuts by lytic bacteriophages ΦSP-1 and ΦSP-3. J Basic Microbiol 55(4):500–503
Bao H, Zhang H, Wang R (2011) Isolation and characterization of bacteriophages of Salmonella enterica serovar Pullorum. Poult Sci 90(10):2370–2377
Barua H, Biswas PK, Olsen KE, Christensen JP (2012) Prevalence and characterization of motile Salmonella in commercial layer poultry farms in Bangladesh. PLoS ONE 7:e35914
Endersen L, Coffey A (2020) The use of bacteriophages for food safety. Curr Opin Food Sci 36:1–8
Fagbamila IO, Barco L, Mancin M, Kwaga J, Ngulukun SS, Zavagnin P et al (2017) Salmonella serovars and their distribution in Nigerian commercial chicken layer farms. PLoS ONE 12:e0173097
Fernández J, Guerra B, Rodicio MR (2018) Resistance to carbapenems in non typhoidal Salmonella enterica Serovars from Humans, Animals and Food. Vet Sci 5(2):40
Foley SL, Nayak R, Hanning IB, Johnson TJ, Han J, Ricke SC (2011) Population dynamics of Salmonella enterica serotypes in commercial egg and poultry production. Appl Environ Microbiol 77(13):4273–4279
Haselbeck AH, Panzner U, Im J, Baker S, Meyer CG, Marks F (2017) Current perspectives on invasive nontyphoidal Salmonella disease. Curr Opin Infect Dis 30(5):498–503
Hawkey J, Le Hello S, Doublet B, Granier SA, Hendriksen RS, Fricke WF, Ceyssens PJ, Gomart C, Billman-Jacobe H, Holt KE, Weill FX (2019) Global phylogenomics of multidrug-resistant Salmonella enterica serotype Kentucky ST198. Microb Genom 5(7):e000269
Inbaraj S, Agrawal RK, Thomas P, Mohan C, Agarwal RK, Verma MR, Chaudhuri P (2022) Antimicrobial resistance in Indian isolates of non typhoidal Salmonella of livestock, poultry and environmental origin from 1990 to 2017. Comp Immunol Microbiol Infect Dis 80:101719
Jakočiūnė D, Moodley A (2018) A Rapid Bacteriophage DNA Extraction Method. Methods Protoc 1(3):27
Jurczak-Kurek A, Gąsior T, Nejman-Faleńczyk B, Bloch S, Dydecka A, Topka G, Necel A, Jakubowska-Deredas M, Narajczyk M, Richert M, Mieszkowska A (2016) Biodiversity of bacteriophages: morphological and biological properties of a large group of phages isolated from urban sewage. Sci Rep 6(1):1–17
Kang HW, Kim JW, Jung TS, Woo GJ (2013) wksl3, a New biocontrol agent for Salmonella enterica serovars Enteritidis and Typhimurium in foods: characterization, application, sequence analysis, and oral acute toxicity study. Appl Environ Microbiol 79(6):1956–1968
Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol Biol Evol 35:1547–1549
Le Hello S, Bekhit A, Granier SA, Barua H, Beutlich J, Zając M, Münch S, Sintchenko V, Bouchrif B et al (2013) The global establishment of a highly-fluoroquinolone resistant Salmonella enterica serotype Kentucky ST198 strain. Front Microbiol 4:395
Litt PK, Saha J, Jaroni D (2018) Characterization of bacteriophages targeting non-O157 Shiga Toxigenic Escherichia coli. J Food Prot 81(5):785–794
Manyi-Loh C, Mamphweli S, Meyer E, Okoh A (2018) Antibiotic use in agriculture and its consequential resistance in environmental sources: potential public health implications. Molecules 23(4):795
Moye ZD, Woolston J, Sulakvelidze A (2018) Bacteriophage applications for food production and processing. Viruses 10(4):205
Nilsson AS (2014) Phage therapy—constraints and possibilities. Ups J Med Sci 119(2):192–198
Phothaworn P, Dunne M, Supokaivanich R, Ong C, Lim J, Taharnklaew R, Vesaratchavest M, Khumthong R, Pringsulaka O, Ajawatanawong P, Klumpp J, Brown N, Imam M, Clokie MRJ, Galyov EE, Korbsrisate S (2019) Characterization of Flagellotropic, Chi-Like Salmonella Phages Isolated from Thai Poultry Farms. Viruses 11(6):520
Salehi S, Howe K, Brooks J, Lawrence ML, Bailey RH, Karsi A (2016) Identification of Salmonella enterica serovar Kentucky genes involved in attachment to chicken skin. BMC Microbiol 16(1):168
Sharma J, Kumar D, Hussain S, Pathak A, Shukla M, Prasanna Kumar V, Anisha PN, Rautela R, Upadhyay AK, Singh SP (2019) Prevalence, antimicrobial resistance and virulence genes characterization of nontyphoidal Salmonella isolated from retail chicken meat shops in Northern India. Food Control 102:104–111
Sun WJ, Liu CF, Yu L, Cui FJ, Zhou Q, Yu SL, Sun L (2012) A novel bacteriophage KSL-1 of 2-Keto-gluconic acid producer Pseudomonas fluorescens K1005: isolation, characterization and its remedial action. BMC Microbiol 12:127
WHO: Food safety fact sheet (2020) Retrieved from https://www.who.int/news-room/fact-sheets/detail/food-safety
Yang M, Liang Y, Huang S, Zhang J, Wang J, Chen H, Ye Y, Gao X, Wu Q, Tan Z (2020) Isolation and Characterization of the Novel Phages vB_VpS_BA3 and vB_VpS_CA8 for Lysing Vibrio parahaemolyticus. Front Microbiol 11:259
Żbikowska K, Michalczuk M, Dolka B (2020) The use of bacteriophages in the poultry industry. Anim (Basel) 10(5):872

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

Serial No	Year of isolation	Serovar	Source	Antimicrobial susceptibility	Phage susceptibility
1	2017	Kentucky	Poultry	CIP NA LVX	+
2	2017	Kentucky	Poultry	Tet S NA LVX	+
3	2017	Kentucky	Poultry	AM Tet S CIP NA	+
4	2016	Kentucky	Poultry	Tet S CIP NA LVX	+
5	2016	Kentucky	Poultry	AM Tet S CIP NA LVX	+
6	2016	Kentucky	Poultry	AM Tet CIP LVX NA AmC	+
7	2017	Kentucky	Poultry	AM Tet AmC CIP LVX NA	+
8	2017	Kentucky	Poultry	AM Tet CIP LVX NA AmC	+
9	2017	Kentucky	Poultry	Tet SXT CIP NA LVX	-
10	2015	Untypeable	Environment	AM S CIP	+
11	2016	Kentucky	Poultry	AM C Tet SXT S NA CIP LVX	+
12	2017	Kentucky	Poultry	AM Tet CIP LVX NA AmC	+
13	2017	Kentucky	Poultry	AM S Tet SXT NA	+
14	2016	Kentucky	Poultry	AM Tet S CIP NA LVX AmC	+
15	2017	Kentucky	Poultry	AM Tet S CIP NA LVX AmC	-
16	1995	Paratyphi B	Poultry	S Tet NA	+
17	2017	Typhimurium	Poultry	Tet LVX NA	+
18	2017	Kentucky	Poultry	AM Tet CIP NA	+
19	2017	Virchow	Pig	AM C Tet AmC LVX	+
20	2002	Sandiego	Pig	AM SXT Tet NA	+
21	2016	Rough	Pig	AM S CIP Tet SXT LVX NA AmC	+
22	2015	Infantis	Environment	Tet SXT NA	-
23	2010	Typhimurium	Poultry	AM S SXT	-
24	2016	Kentucky	Poultry	AM Tet SXT S CIP NA LVX	_
25	2016	Kentucky	Poultry	AM Tet S CIP NX LVX	+
26	2002	Untypeable	Goat	AM SXT Tet NA	-
27	2017	Kentucky	Poultry	AM Tet S CIP NA LVX AmC	+
28	2016	Kentucky	Poultry	AM Tet SXT S CIP NA LVX AmC	+
29	2017	Kentucky	Poultry	AM Tet S CIP NA LVX	+
30	2016	Kentucky	Poultry	AM S CIP NA LVX AmC	+
31	2002	Untypeable	Calf	AM SXT Tet NA	+
32	2015	Infantis	Environment	Tet SXT S NA	+
33	2015	Takorodi	Environment	Tet SXT NA	+
34	2002	Rough	Goat	AM SXT Tet NA	+
35	2002	Lagos	Environment	AM S SXT Tet NA	+
36	2017	Kentucky	Poultry	AM S Tet SXT CIP NA LVX AmC	-
37	2013	Enteritidis	Poultry	C S Tet AmC NA	-
38	2017	Virchow	Pig	AM C CIP Tet SXT NA	+
39	2005	Haifa	Poultry	AM S SXT Tet	+
40	2015	Typhimurium	Poultry	Tet SXT S NA	+
41	2002	Untypeable	Pig	AM SXT Tet NA	+
42	2004	Virchow	Poultry	AM Tet SXT NA	+
43	2016	Typhimurium	Pig	Am S SXT CIP NA LVX AmC	+
44	2015	Untypeable	Environment	Am S CIP Tet LVX NA AmC	+
45	2015	Typhimurium	Poultry	AM S CIP Tet AmC LVX NA	+
46	2015	Untypeable	Environment	Tet SXT NA	+
47	2016	Kentucky	Poultry	AM Tet S NA LVX	+
48	2002	Untypeable	Poultry	AM SXT Tet NA	+
49	2017	Typhimurium	Poultry	SXT LVX NA	+
50	2017	Virchow	Pig	AM C S Tet SXT NA	+

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

Treatment groups	Group 1 Positive control	Group 2 Distilled water treated	Group 3 Kentucky (10⁵cfu/ml)+ phage (10⁹)pfu/ml	Group 4 Kentucky(10³ cfu/ml)+ phage (10⁹ pfu/ml)
Parameters	Group 1 Positive control	Group 2 Distilled water treated	Group 3 Kentucky (10⁵cfu/ml)+ phage (10⁹)pfu/ml	Group 4 Kentucky(10³ cfu/ml)+ phage (10⁹ pfu/ml)
Concentration (log₁₀ cfu/g)	2.55±0.89	2.21±0.56	1.89±0.41	0.14±0.04
Reduction in bacterial concentration (log₁₀ cfu/g)	-	0.34	0.66	(*)

*P≤ 0.01

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

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Editorial Note

Abstract

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Introduction

Materials And Methods

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Tables

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