Salmonella clinical isolates and antimicrobial susceptibility testing

Among thirty-one Salmonella isolates applied for the isolation of bacteriophages, twenty-six isolates were previously identified and susceptibility profiled in our Lab [15]. S. Paratyphi A, S. Paratyphi B, S. Paratyphi C clinical isolates, and a standard strain S. Typhimurium ATCC 14,028, were supplied by the Bacterial Bank of Animal Health Research Institute (AHRI, Egypt). Moreover, S. Typhi clinical isolate was kindly provided by the Central laboratories of the Ministry of Health and Health Insurance, Egypt (Table 1). Bacterial isolates were serologically identified at AHRI according to the White-Kauffman-Le Minor scheme, as reported by the Word Health Organization (WHO) Collaborating Centre for Reference and Research on Salmonella (WHOCC-Salm) [16] and they were also assessed for their antibiotic susceptibility pattern according to CLSI guidelines using the Kirby-Bauer disk diffusion [17]. MDR was determined as previously described [12].

Table 1 Salmonella serovars applied in bacteriophage isolationIsolation of Salmonella-specific bacteriophages

The bacterial host employed in phage isolation was S. Typhimurium ATCC 14,028, the isolate was cultured overnight in Tryptic Soy Broth (TSB), and the bacterial count was obtained by dilution in TSB to match the turbidity of 0.5 McFarland standard (108 colony forming unit (CFU)/mL). Samples employed for bacteriophage isolation were collected from different poultry markets in Cairo and Giza governorates as raw chicken rinse samples. Samples were filtered using filter paper to remove suspended particulates [18]. A fresh rinse of raw chicken (5 mL) was incubated with 5 mL of a mixture of the previously prepared bacterial isolate in addition to 50 mL of double-strength TSB (supplemented with 10 mM CaCl2 and 1 M MgSO4). The mix was incubated overnight at 37 °C (200 rpm), followed by centrifugation at 2817 x g for 20 min using 1010 Centrifuge, Centurion Scientific ltd, UK. The supernatant was shaken with chloroform (1% v/v) for 30 min at room temperature to kill the bacterial cells followed by getting rid of the bacterial cell remnants by centrifugation at 2817 x g for 10 min. The obtained phage lysate was kept at 4 °C [19].

Screening for the phage lytic activity against MDR Salmonella

The phage lysate was screened for its lytic activity against MDR Salmonella serovars using a spot test [19]. The plaque assay was carried out to settle the titer of the isolated phage(s) in their initial lysate using the standard double agar overlay (DAO) method [20, 21]. Ten-fold serial dilution of the phage lysate in saline-magnesium (SM) buffer: NaCl, 5.8 g, 100 mM. MgSO4•7H2O, 2 g, 8 mM. Tris-Cl (1 M, pH 7.5), 50 ml, 50 mM. H2O, to 1 L. (Biodiagnostics ®, Cairo, Egypt) was done. Each phage dilution was mixed with an equal volume of the bacterial host followed by adding this mixture to a 3 mL soft overlay composed of double-strength TSB and agar at a concentration of 0.75 g/100 mL [22]. Each mixture was then poured and evenly distributed over a layer of TSA previously prepared. The plates were left to completely solidify undisturbed and were incubated in an upright position at 28 °C overnight. The plaques were counted and the phage titer was calculated as previously reported [23].

Purification of the isolated bacteriophages

To ensure the purity of the suspended phage lysate, a sterile spatula was used to pick out a single well-defined plaque and suspend it in SM buffer. It was then left for 2 h and then the obtained suspension was incubated overnight with fresh bacterial host, centrifuged, treated with chloroform, and kept at 4 °C [19].

Phages propagation

Phage cocktail propagation was performed by repeating the previously described method for phage isolation three times using aliquots of the phage lysate as an alternative to the chicken rinse samples [19, 24].

Characterization of the isolated bacteriophages showing lytic activity against MDR Salmonella isolates

Characterization was assessed using a spot test which showed either a positive (clear spot) or negative result (no spot) indicating that the phage was either active or inactive after being challenged using different Salmonella isolates and also at different pH values, temperatures, or organic solvents.

Host range

The host range of the isolated phage cocktail was evaluated using a spot test against 31 MDR Salmonella isolates [25]. For each tested bacterial isolate, a plate with a TSA base layer was overlaid with 3 mL of double-strength TSB agar inoculated with the tested MDR Salmonella isolate. Then, each plate was spotted with 15 µL of the phage lysate, overnight incubated followed by visual examination. The phage lytic effect was detected by the presence of an inhibition zone in the area of the applied spot surrounded by a well-grown confluent sheet of bacterial growth.

Longevity test

Aliquots of the previously prepared bacteriophage lysate were maintained at 4, 37, and − 80 °C. The phages were examined for their lytic activity at 1, 2, 3, 4, 5, 6, 7, 15, 30, 60, and 90-day intervals using spot test [19].

Thermal stability

Aliquots of the phage lysates were incubated at different temperature ranges between 30 and 65 °C (5-degree intervals) for 1 h followed by an examination of their lytic activity using spot test. The thermal inactivation point was considered as the temperature at which a complete loss of the phage lytic activity was recorded [25].

pH stability

The phage lysate cocktail was mixed with an equal volume of TSB at different pH ranges between 1 and 13. The prepared suspensions were left for 1 h at room temperature then the phage lytic activities were observed using spot test [26].

Sensitivity to organic solvents

The phage lysates were treated with different concentrations of ethanol, isopropyl alcohol, and chloroform (10, 30, 50, and 100% v/v), incubated at room temperature for 1 h, and evaluated for their lytic activity using spot test according to the studies conducted by Abd-Allah et al. and Oduor et al. [19, 27].

Morphology of the isolated bacteriophages

Morphological characterization of the isolated phages against MDR Salmonella was performed using a transmission electron microscope (TEM) via preparation of a high-titer phage lysate previously prepared using 2 to 3 successive propagations. Purification was performed by centrifugation for 25 min at 7826 x g twice, followed by syringe filtration (0.22 μm). Samples were sent to NanoTech for Photo Electronics Co. Giza, Egypt, for characterization by TEM. They were prepared following the procedure described by Kalatzis et al. [28] and examined using a transmission electron microscope (JEOL_JEM_2100 Electron Microscope Siemens & Halske, Germany) [25, 28].

Molecular analysis of the isolated bacteriophagesExtraction of nucleic acid and genomic sequencing

The genomic DNA of the purified phage lysate was extracted using QIAamp® DNA Minikit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Ensuring the quality and quantity of the extracted DNA was carried out as recommended by the specifications outlined in the kit’s user guide.

Genomic sequencing of phage genome

The phage lysate was sequenced in an Illumina MiSeq instrument (Illumina, La Jolla, CA, USA), at Children Cancer Hospital 57,357, Cairo, Egypt, and the library was set up by the Nextera XT DNA Library preparation kit (San Diego, CA, USA). The obtained contigs were assembled using the StadenPackage software v2. Further confirmation of certain gaps has been performed using the Oxford Nanopore Sequencing [29] which was carried out at HITS Solutions, Co, (https://www.hitssolutions.com/), Cairo, Egypt.

Library preparation and Oxford nanopore sequencing

Preparation of the genomic library was carried out using a Rapid Barcoding Kit (SQK-RBK004; Oxford Science Park, OX4 4DQ, UK) according to the manufacturer’s protocol. The quality of the Fastq reads was assessed using Fastqc. Subsequently, reads that were of low quality or insufficient length were removed using the NanoFilt tool https://github.com/wdecoster/nanofilt (accessed on October, 2023) [30]. Adapter sequences present in the reads were then eliminated using Porechop_ABI https://github.com/bonsai-team/Porechop_ABI (accessed on October, 2023) [31]. The filtered reads were then subjected to Denovo assembly and polishing using Flye https://github.com/fenderglass/Flye (accessed on October, 2023) and Medaka https://github.com/nanoporetech/medaka (accessed in October, 2023), respectively. The final consensus sequence was analyzed using PATRIC BRC [32] https://www.bv-brc.org/ (accessed on 23 October 2023) and analyzed using RAST algorithm [33]. The final assembled consensus sequence of each phage genome was annotated [34] and submitted to the NCBI GenBank database under the accession code, OR757455 and OR757456. The BLAST Ring Image Generator (BRIG) tool v0.95 (https://sourceforge.net/projects/brig/ (accessed on 25 October 2023) was employed to create the circular image [35].

Microencapsulation of Salmonella phages cocktail using freeze drying

Freeze drying was applied as a method for microencapsulation of the isolated phages using Whey protein isolate (WPI) protein (Sigma Aldrich, Saint Louis, MO, USA) which was prepared as previously reported [36]. Trehalose dihydrate (Advent Chembio Pvt Ltd, India) was immediately incorporated into the whey protein solution at a ratio of 1:3 according to the optimized formulation as demonstrated by Petsong et al. [37] to achieve 10% (w/v) total solid. Phages lysate (109 PFU/mL) was added to the mixture to obtain 10% (v/v). The mixture was held at -80˚C for 12 h. The frozen mixture was dried at -50˚C using a laboratory scale freeze-dryer (CoolSafe 55, ScanLaf A/S, Lynge, Denmark) under vacuum (Welch, 8912 Vacuum pump, Gardner Denver Thomas, Inc, Welch Vacuum Technology Niles, IL, USA) at approximately 30 pounds per square inch (psi) for 48 h. Dry lyophilized powder was obtained (10 ± 0.05 g) and employed for the assessment of the phage titer [37].

In vitro antimicrobial activity of microencapsulated phages

Freeze-dried phage powder (1 g) was resuspended in SM buffer (10 mL) and incubated for 1 h at room temperature while shaking (220 rpm) (ThermoStableTM IS-30 model, DAIHAN Scientific, Korea). Plaque-forming units were determined to evaluate the ability of the phage cocktail to retain its lytic activity post-encapsulation. The plaque forming units were evaluated against the three selected MDR bacterial hosts. The encapsulation efficiency (EE) was estimated using the identified phage titer originally applied for phage encapsulation (TP) compared to the phage titer recovered from the dried powder (RP), using the following formula: EE (%) = RP/TP × 100 [37].

In vivo antimicrobial potential of microencapsulated phagesLaboratory animals and experimental design

Sixty-five male white albino mice aged 6 weeks and weighing between 100 and 125 g were used as animal models throughout the experiment. Animals were kept in open cages, and given antibiotics-free food as previously reported [38]. Animals were maintained on an alternate 12 h light-dark cycle, with a constant temperature of 25 °C controlled by air conditioning at the Animal House Facility (Faculty of Pharmacy, Ahram Canadian University, Giza, Egypt). Dealing with animals was carried out according to the regulations of the animal care and use committee of the Faculty of Pharmacy, Ain Shams University (ACUC-FP-ASU) as recommended by the National Regulations on Animal Welfare and the Institutional Animal Ethical Committee. Animals were categorized into thirteen groups (5 mice / each) as previously described [38]. Fecal samples from 5 mice belonging to different groups were obtained before the beginning of the experiment and checked for the absence of Salmonella infection. Treatment was done by either the free phage lysate cocktail, the formulated microencapsulated phage cocktail, or by using the vehicle only (10% whey protein and trehalose in ratio 3:1 dissolved in sterile distilled water containing 10% TSB) as a control (Table 2). Each mouse was infected orally with a single dose (0.2 mL) of Salmonella suspension of the selected serovar (1.5 × 108 CFU/mL). On the third day post infection, infected mice were treated with either the free form of the phage cocktail or the microencapsulated phage daily for 9 days (1 × 108 PFU/mL, 0.2 mL/dose). On the 12th day, mice were weighed prior to slaughtering, and a part of the liver of each mouse in each group was aseptically removed and homogenized for determination of the bacterial viable count [38]. All internal mice organs were preserved in 10% formalin for further histopathological investigations.

Table 2 Categorization of animals and the description of each groupHistopathology

For the histopathological analysis, the liver, spleen, intestine, and mesenteric lymph nodes (MLN) were fixed in 10% neutral-buffer formalin [39]. The samples were obtained from each of the dissected organs, washed with tap water, and dehydrated for 30 min using diluted ethanol. The samples were incubated in a 1:1 mixture of ethanol and xylene for 30 min, washed twice using xylene for 1 h, and moved to xylene and paraffin mixture for 30 min. Transverse Sect. (5 mm) were dewaxed at 60 °C, immersed in xylene for 1 h, rehydrated using a series of ethanol for 2 min, and washed using tap water. Sections were stained using hematoxylin and eosin, mounted using Entellan embedding agent, and examined [40] for histopathological abnormalities at the Pathology laboratory (AHRI, Giza, Egypt) in a blind manner.

Bacterial viable count

Dissected livers were weighed and homogenized in SM buffer using a hand-held tissue grinder. Ten-fold serial dilutions of homogenized liver were prepared and plated onto Salmonella-Shigella agar plates and incubated at 37 °C for 24 h. Bacterial counts were measured per one gram of the homogenized tissue.

Statistical analysis

Data were analyzed by one-way analysis of variance (ANOVA) using Graph pad Instat-3 software (Graph Pad Software Inc., USA), based on the normality of the data that was assessed using the Kolmogorov-Smirnov and Shapiro-Wilk tests (Table S1, Figures S1 and S2) and the homogeneity of variances for log bacterial count that was tested using Levene’s test (Tables S3). Results were considered significant at P values less than 0.001 (Table S3) and were presented as means ± standard deviation.