Isolation and characterization of bacteriophages with lytic activity against multidrug-resistant non-typhoidal Salmonella from Nairobi City county, Kenya | BMC Infectious Diseases

Non-typhoidal Salmonella strains used in phage isolation

In this study, we utilized archived non-typhoidal Salmonella (NTS) isolates from previous research in Kenya [6], including a panel of four MDR Salmonella enterica serovars Typhimurium and Enteritidis (NCBI/Bioproject No. PRJEB19289 and Biosample accessions ERS4397787, ERS3403399, ERS3403411, and ERS4397849) with varying AMR patterns (Table 1). The strains were revived from a −80 °C freezer using tryptic soy agar (TSA) (Oxoid Ltd., Basingstoke, UK), and overnight colonies were sub-cultured into tryptic soy broth (TSB) (Oxoid Ltd., Basingstoke, UK), for overnight incubation. Antimicrobial susceptibility testing (AST) was repeated to confirm the MDR phenotype of the strains, using Kirby Bauer’s disc diffusion method following the Clinical Laboratory Standards Institute (CLSI) guidelines 2022 [38].

A 0.5 McFarland-equivalent suspension of the NTS bacterial strain was spread evenly on Mueller-Hinton Agar (MHA) (Oxoid Ltd., Basingstoke, UK), and allowed to air dry. Antibiotic disks were then placed on the bacterial lawn, and the plates were incubated overnight at 37 °C. The zones of inhibition were measured, and susceptibility was interpreted according to the 2022 CLSI guidelines. ESBL production was tested using a double-disc diffusion test, where cefotaxime/clavulanic acid (30/10 µg) and ceftazidime/clavulanic acid (30/10 µg) discs (BD, Franklin Lakes, NJ, USA), along with cefotaxime (30 µg) and ceftazidime (30 µg) discs without clavulanic acid (Oxoid Ltd., Basingstoke, UK), were used for AST as previously described [39, 40]. ESBL production was confirmed using the Phenotypic Confirmatory Disc Diffusion Test (PCDDT). An isolate was considered an ESBL producer if there was a > 5 mm difference in the zone of inhibition between cefotaxime with clavulanic acid and cefotaxime without clavulanic acid, or between ceftazidime with and without clavulanic acid. Conversely, isolates with a < 5 mm difference in the zones of inhibition were classified as non-ESBL producers. Escherichia coli (E. coli) NCTC 13,351 and E. coli ATCC 25,922 were the positive controls for ESBL and non-ESBL production, respectively.

The study used a panel of 13 antimicrobial agents from different classes, including penicillin (ampicillin (10 µg)), cephalosporins (ceftriaxone (30 µg), cefotaxime (30 µg), ceftazidime (30 µg), cefpodoxime (10 µg)), β-lactam-β-lactamase inhibitor (amoxicillin/clavulanic acid (20/10 µg)), quinolones (ciprofloxacin (5 µg)), nalidixic acid (30 µg)), aminoglycosides (gentamicin (10 µg), kanamycin (30 µg)), sulfonamides (trimethoprim-sulfamethoxazole (1.25/23.75 µg)), tetracyclines (tetracycline (30 µg)), and phenicol (chloramphenicol (30 µg)) all from Oxoid Ltd., Basingstoke, UK.

Collection of environmental samples

A one-time environmental sampling was conducted between April and October 2022 at seven locations in Nairobi City County, including open drains, rivers, and a dam. At each site, five samples were collected and pooled to form one composite sample, yielding in a total of seven composite samples, one from each location. The drains in Nairobi’s informal settlements are polluted with raw sewage and household waste, creating an ideal breeding ground for bacteriophages [41]. Four sampling points were from informal settlements, including Kamukunji (Majengo), Mukuru slums (River Ngong), Njiru River, and Kibera (Nairobi Dam), two points were selected from the Nairobi Wastewater Treatment Plant at Ruai (influent and effluent), and the other sampling point was at an open drain at Dagoretti Market (Supplementary Fig S1). The samples (100 mL) were collected in sterile Whirl-Pak bags (Whirl-Pak^® Sample Bag, Nasco, USA) and immediately transported in cool boxes to the laboratory at the Centre for Microbiology Research (CMR) in KEMRI for processing the same day after collection.

Phage isolation

We isolated phages as previously described [28, 42], with slight modification. The water samples were centrifuged at 10,000 × g for 10 min to decant the solid particles, and the supernatant was filtered through a 0.45 μm PES syringe filter (Scientific Laboratory Supplies Ltd, Nottingham, UK). To enrich phages, 10 mL of the filtrate was combined with 10 mL of TSB and 100 µl of exponentially growing MDR NTS strain (Table 1) subsequently added. The mixture was incubated overnight at 37 °C in an Eppendorf New Brunswick Innova 40 shaker incubator (Eppendorf SE, Hamburg, Germany) at 150 rpm to allow amplification of host-specific lytic phages.

Screening for phage by spot assay

We followed the previously described spot assay protocol [28, 42] for phage screening. The enriched culture was centrifuged at 10,000 × g for 10 min and filtered through 0.45 μm filters. To prepare the bacterial lawn, 100 µL of an overnight culture of the respective NTS host bacteria (Table 1.) was added to tryptic soy broth (TSB) containing 0.7% agar at 45 °C and poured onto tryptic soy agar (TSA) (Oxoid Ltd., Basingstoke, UK) to form a lawn. The lawn was allowed to cool, and 10 µl of the enriched filtrate was spotted and incubated overnight at 37 °C. Clear zones (plaques) indicated the presence of phages.

Phage purification

To purify the phage, we followed a procedure described by Kazibwe et al. [42]., where the filtrate containing phage, after spot assay, was serially diluted (ten-folds) using sterile saline magnesium (SM) buffer (100 mM sodium chloride, 10 mM magnesium sulfate, 50 mM Tris-HCl, pH 7.5 and 0.01% weight by volume gelatin). The spot assay was repeated by spotting 2 µl of each dilution on a lawn of bacteria on a TSA plate labeled with dilutions ranging from 10⁻¹ to 10⁻⁸. The plates were allowed to air dry and incubated overnight at 37 °C. The plaque assay was then performed by mixing 100 µl of bacteria strain with 100 µl of phage from the highest dilution in 5mL of 0.7% soft agar. The soft agar was spread on TSA, allowed to gel at room temperature, and incubated overnight at 37 °C. The plaques were examined based on morphology, and the distinct single plaques were picked using a sterile pipette tip for further propagation.

Purification was conducted in five rounds of plaque assays, picking individual plaques each round. After purification, phages with uniform and distinct plaques were harvested in 1 mL sterile SM buffer at room temperature for 30 min before centrifuging at 4000 × g for 5 min. The phages were filtered through 0.22 μm PES syringe filters (Scientific Laboratory Supplies Ltd, Nottingham, UK) and stored at 4 °C as working stock, with small aliquots in 20% glycerol stored at −80 °C for further analysis. All purified phages were named according to the system described by Adriaenssens and Rodney Brister [43]. The phage name starts with the word Salmonella, followed by the word phage, and then a unique identifier, which is a serial number that begins with the prefix KE (Kenya). For instance, the first purified phage was named Salmonella phage vB_SenST11_KE01. The third part of the name vB_SenST11_KE01 denotes ‘virus of Bacteria’, infecting Salmonella Enteritidis Sequence Type 11 and then the unique identifier. In the subsequent processes during phage characterization, the unique identifier has been used in phage labeling (e.g. KE01).

Phage titer determination

The concentration of the phages was determined following the agar overlay method [42], with serial dilutions (10⁻¹ to 10⁻⁸) of the purified phages prepared using SM buffer. A 100 µl of respective NTS host bacteria was inoculated into 5 mL soft agar and poured on a gridded TSA plate. We performed a spot test for all dilution factors to determine the highest dilution that showed lysing. Subsequently, we conducted plaque-forming assay using the highest dilution, counted the number of plaques formed, and expressed phage titer in plaque-forming units per milliliter (PFUs/mL) as follows;

(:text{P}text{h}text{a}text{g}text{e}:text{t}text{i}text{t}text{e}text{r}:(text{P}text{F}text{U}text{s}/text{m}text{L})=frac{text{N}text{u}text{m}text{b}text{e}text{r}text{s}:text{o}text{f}:text{p}text{l}text{a}text{q}text{u}text{e}text{s}:text{p}text{e}text{r}:text{p}text{l}text{a}text{t}text{e}}{text{V}text{o}text{l}text{u}text{m}text{e}:text{p}text{l}text{a}text{t}text{e}text{d}:text{i}text{n}:text{m}text{l}text{s}:times::text{d}text{i}text{l}text{u}text{t}text{i}text{o}text{n}:text{f}text{a}text{c}text{t}text{o}text{r}:}) [44].

Phage host range determination

We determined the phage host range by spot test as described by Esmael et al. [28], using 12 Salmonella strains – MDR S. Typhimurium, ESBL-producing S. Typhimurium, MDR S. Enteritidis, ciprofloxacin-resistant S. Enteritidis, recently isolated S. Typhimurium and S. Enteritidis (from an ongoing study (2022)), ATCC 13,076 S. Enteritidis, NCTC 3048 S. Typhimurium, S. Arizonae, S. Dublin, S. Heidelberg, and S. Typhi. A bacterial lawn was prepared on TSA by spreading 100 µL of each tested strain. Once the lawn had air-dried, 2 µL of each individual phage stock was spotted onto the surface and allowed to air dry. The plates were then incubated overnight at 37 °C. The study considered spot tests showing a clear zone as susceptibility and the absence of a clear zone or plaque as resistance of a bacterial strain to the tested phages, respectively. Although the term “broad host range” is commonly used to describe phages capable of lysing multiple strains or species, there is currently no universally accepted numerical threshold for defining this based solely on intra-species lytic activity [45, 46]. Here, we considered phages that lysed more than 80% of the study bacterial strains as having a broad host range.

Determining the efficiency of plating (EOP)

We evaluated the efficiency of plating (EOP) of phages that showed > 80% host range following Kotter’s protocol with slight modifications [47]. Phage titer was determined using all the susceptible bacteria strains and the titer obtained from its isolation host, with the EOP calculated as follows;

We interpreted the EOP ratio as follows: ≥ 0.5 as high production efficiency, ≥ 0.1 to < 0.5 as medium production efficiency, 0.001 to 0.1 as low production efficiency, and ≤ 0.001 as inefficient [48,49,50].

Determination of thermal and pH stability

Phages with > 80% host range were analyzed further for thermal and pH stability, as described by Bao et al. [51]. Phage lysates stored at 4 °C were used as the reference titer to establish the baseline for thermal stability testing. Aliquots of each phage were then incubated at various temperatures, including − 80 °C, −20 °C, 4 °C, 20 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, and 80 °C. These temperature ranges covered freezing and fridge temperatures, the phage storage conditions [52], and room temperatures between 20 °C and 30 °C in Kenya for the better part of the year [53]. It also included body temperatures, critical in phage application for therapy ranging from 30 °C to 40 °C [54], with high temperatures of 50 °C to 80 °C studied to inform phage packaging during phage therapy production [55].

A volume of 50 µl phage was aliquoted in PCR tubes and incubated for 60 min in respective temperatures in a freezer or thermal cycler. After incubation, we held the phages at room temperature for 30 min and determined their titer. For pH stability testing, SM buffer was adjusted to pH values of 1, 3, 7, 9, 11, and 13 by adding 1 M NaOH or 1 M HCl drop by drop until the desired pH was reached, as measured with a pH meter (Thermo Scientific, Roskilde, Denmark). Phages were then incubated in the adjusted SM buffers at 37 °C for 60 min, and titers were subsequently evaluated. Phages incubated at pH 7 served as the control to assess stability across the pH range. All experiments for phage titer followed the double-layer agar plate method using 0.7% soft agar and TSA.

Effects of phages on NTS biofilms

The effectiveness of the selected phages on biofilms formed by their respective NTS host strains (MR2829, MCC1462, and MB1102) was quantitatively determined as previously described [28, 56]. Single colonies of the NTS host strain (Table 1.) were cultured in TSB at 37 °C, 200 rpm for 24 h. Following incubation, the bacterial culture was diluted 1:100 in fresh TSB. Then, 100 µl of the diluted NTS strain culture was aliquoted into 96-well microplates, in triplicates for two sets, and incubated at 30 °C for 72 h. The TSB was carefully replaced every 24 h to avoid disturbing the biofilm layer by gently aspirating the media and replenishing it with fresh TSB. For negative control, three wells contained TSB media with no bacteria. We treated one set of the wells containing bacteria with their respective phages and the second set with PBS and incubated at 37 °C for 24 h. After incubation, we washed the wells five times to remove planktonic cells and then air-dried them. We added 98% methanol into wells for 10 min, discarded methanol, and air-dried the plates before staining wells with 1% crystal violet for 45 min and eluting with 33% acetic acid, and Microplate Reader (ELx808 Bio Tek Instruments, Winooski, USA) used to read the optical densities (OD) of the wells at 630 nm wavelength.

Phage genomic DNA extraction and sequencing

Phages were propagated using host bacteria strains to achieve titers exceeding 1 × 10¹⁰ plaque-forming units per milliliter (PFUs/mL) as described by Jakočiūnė & Moodley [57]. Before phage DNA extraction, we treated 1 mL of the phage suspension with 2.5 U/ml of DNase I (Thermo Fisher Scientific, USA) and 0.07 mg/ml of RNase A (Thermo Fisher Scientific, USA) to degrade bacterial DNA and RNA respectively, and utilized the Phage DNA Isolation Kit (Norgen Biotek Corp., Thorold, ON, Canada) following the manufacturer’s instructions [58]. The quality and quantity of the extracted DNA were assessed using the Nanodrop One spectrophotometer and the Invitrogen™ Qubit™ 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). DNA library preparation was performed using Nextera XT Library protocol (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The genomes were sequenced using the Illumina NextSeq 2000 sequencing platform using 2 × 150 bp paired-end reads.

Genome annotation and comparative genome analysis

Bioinformatics analyses were as described by Shen and Millard [59]. We assessed the quality of sequence raw reads using FastQC v0.12.1, with adapters, overrepresented sequences, and poor-quality bases trimmed off using Fastp v0.20.1. Seqtk version 1.4-r122 was used for read subsampling to attain 50-100x genome coverage. Genome assembly was performed using Shovill v1.1.0 with default settings, applying SPAdes as the assembler. Genome completeness was checked using checkv version 1.0.3. Phage genome annotation was performed using Pharokka v1.7.1, which uses PHANOTATE as the default gene caller and integrates tRNAscan-SE to predict tRNA genes. The linear genome map was constructed using Proksee (https://proksee.ca/, accessed on 30 June 2025). PhageLead online tool (https://phageleads.dk/, accessed on 06 June 2024) used to screen for lysogeny, AMR, and virulence genes to assess the suitability of the phages for therapeutic use. Additionally, phages lifestyle was determined using PhaTYP2, a lifecycle prediction tool in PhaBOX2 (https://phage.ee.cityu.edu.hk/ accessed on 01 May 2025). Presence of AMR genes was further assessed using Resistance Gene Identifier (https://card.mcmaster.ca/analyze/rgi accessed on 01 May 2025) as well as abricate version 1.0.1 (https://github.com/tseemann/abricate accessed on 01 May 2025). The allergenic potential of phage proteins was evaluated using AllerCatPro 2.0 (https://allercatpro.bii.a-star.edu.sg/ accessed on 28 April 2025), which predicts allergenicity based on sequence similarity, structural features, and epitope matching [60].

To assess the genetic relatedness of the phages, a phylogenetic tree was constructed using the Molecular Evolutionary Genetics Analysis (MEGA11) program. Multiple sequence alignment of the major head protein nucleotide sequences was performed with the ClustalW algorithm using default settings. The phylogenetic tree was generated using the neighbor-joining method with 1000 bootstrap replicates [61]. An online tool, the Virus Intergenomic Distance Calculator (VIRIDIC) (https://rhea.icbm.uni-oldenburg.de/viridic, accessed on 10 June 2024), was used to assess the phage’s inter-genomic similarities, Clinker [62] was used to compare phages proteins, while the Virus Classification and Tree Building Online Resource (VICTOR) (https://ggdc.dsmz.de/victor.php, accessed on 10 June 2024) was used to assess the relatedness of our study phages with those reported in other studies using whole genome sequences. Roary was employed to analyze and compare the presence or absence of various genes across different phage genomes, as described by Page et al. [63].

Morphological characterization of phages

The morphology of the study phages was characterized using Transmission Electron Microscopy (TEM). Phages were propagated to achieve high titers exceeding 1 × 10¹⁰ PFU/mL and inactivated using paraformaldehyde following the protocol described by Möller et al. [64]. Briefly, 500 µL of phage suspension was mixed with 55 µL of a concentrated paraformaldehyde solution to achieve a final concentration of 2% in 0.05 M HEPES buffer (pH 7.2). The mixture was incubated for 30 min at 25 °C followed by an additional 30 min at 37 °C with shaking. Samples were then shipped at room temperature to the Advanced Light and Electron Microscopy Unit (ZBS 4) at the Robert Koch Institute, Berlin, for negative-staining TEM analysis.

For sample preparation, 10 µL of the inactivated phage suspension was applied to a pre-treated electron microscopy grid (coated with Alcian blue) and incubated at room temperature for 10 min as described by Laue, M [65]. The grid was washed three times distilled water, stained with 0.5% uranyl acetate for a short incubation, and dried using filter paper. Phage particles were visualized using a Tecnai Spirit transmission electron microscope (Thermo Fisher) operated at 120 kV. Images were captured using a side-mounted CMOS camera (Phurona, EMSIS) at a resolution of 4100 × 3000 pixels.

Data analysis

We conducted all the experiments in triplicates and the data entry was done on Microsoft Excel. Phage titer for each experiment was converted to log₁₀ PFUs/mL. The effect of phage on biofilms was assessed by comparing the optical density readings of bacterial wells treated with PBS to those treated with phage. Statistical significance was determined using GraphPad Prism 8.0.2 (GraphPad Software, Inc., San Diego, CA, USA) by performing Mann-Whitney U test with confidence intervals set at 95% and a statistical significance at p < 0.05.