Phage isolation from environmental samples

1. Research Background

Antimicrobial resistance (AMR) has become a major threat to global public health. Infections caused by multidrug-resistant bacteria continue to spread in clinical settings, aquaculture, and food processing, and the development of traditional antibiotics is struggling to keep pace with the evolution of resistance. Bacteriophages, viruses that specifically infect bacteria, are the most numerous biological entities on Earth (estimated to be up to 10³¹ particles). They are widely present in sewage, soil, water bodies, feces, and various environmental substrates, forming a virtually inexhaustible natural antimicrobial resource.

However, the host spectrum of bacteriophages is typically extremely narrow, often precise down to the strain level. This means that it is almost impossible to "buy" a suitable bacteriophage from existing commercial products for a specific clinically resistant bacterium, foodborne pathogen, aquatic/livestock pathogen, or plant pathogen; instead, the target strain must be used as an indicator bacterium for targeted isolation from environmental samples. The success and efficiency of the isolation process directly determine the starting point for all subsequent downstream work, including bacteriophage therapy, biocontrol, and the development of diagnostic reagents.

Against this backdrop, Shutong Bio has launched a customized environmental phage isolation service targeting client-specified host bacteria. Leveraging our multi-source environmental sample collection network, standardized enrichment and screening system, and comprehensive characterization and quality control platform, we provide clients with a one-stop solution encompassing sample collection, enrichment and isolation, single-spot purification, biological characterization, whole-genome sequencing, and safety evaluation.

2. Sample Source and Biological Background

(1) Correlation between sample source and host distributionPhage abundance is highly correlated with the ecological distribution of its host bacteria, and the choice of sample source directly determines the isolation success rate. Municipal sewage and hospital sewage are the preferred sources of clinical pathogen phages; farm wastewater, manure, and bedding are suitable for livestock, poultry, and aquatic pathogens; soil and plant rhizosphere are suitable for isolating Bacillus, Pseudomonas, and plant pathogen phages; nearshore and freshwater samples are rich in Vibrio and Aeromonas phages; and food processing environment samples are suitable for the discovery of putrefactive bacteria and foodborne pathogen phages.

(2) Host-specific barrierThe structural differences in receptors on the surface of recipient bacteria (LPS, capsular polysaccharides, teichoic acid, pili, flagella, etc.), the presence of restriction modification systems (RM) and CRISPR/Cas immune systems, mean that the phage sensitivities of different strains of the same species may be completely different. Therefore, it is necessary to use the strain (or combination of strains) actually used by the customer as the indicator bacteria for screening.

(3) Differentiation between pyrolytic and solubilityPhages used for treatment, food preservation, and biocontrol must be strictly lytic (virulent) phages. Temperate phages may carry integrases, repressor proteins, and virulence/resistance genes, posing a risk of lysogen conversion and lateral gene transfer, and must be excluded during the isolation stage.

(4) Interference from the environmental matrixThe pH, ionic strength (especially Ca²⁺/Mg²⁺ concentration), organic matter load, suspended particles, and antibacterial residues of the sample can all significantly affect the phage adsorption efficiency and enrichment effect. Pretreatment and enrichment conditions need to be optimized for different matrix types.

[Image] Figure 1. Phage isolation and typical phage plaque morphology from environmental samples.

3. Mainstream Separation and Identification Strategies

3.1 Enrichment method (mainstream approach):

(1) Principle: After centrifugation and filtration through a 0.22 μm membrane to remove bacteria, the sample is co/cultured with the target host bacteria in the logarithmic growth phase and supplemented with concentrated nutrient medium, so that the low abundance of specific bacteriophages in the environment can be selectively amplified, and the detection rate is significantly improved after multiple rounds of enrichment.

(2) Advantages: High sensitivity, suitable for situations where the abundance of bacteriophages is extremely low or the host bacteria belong to rare groups, and is currently the most commonly used isolation route.

3.2 Direct Plating:

The Adams classic two-layer agar plate method was used, in which the sample filtrate and indicator bacteria were directly mixed and plated. This method preserves the diversity of the original community and avoids the competitive inhibition of rare phages by "dominant phages" during the enrichment process, making it suitable for projects that require the construction of a diverse phage library.

3.3 Continuous purification of single phagocytic plaques:

Uniform plaques are picked from the plate and purified and passaged for three or more consecutive times to ensure that a single phage strain with a pure genetic background is obtained, thus avoiding subsequent characterization bias caused by mixed strains.

3.4 Host spectrum and potency evaluation:

First, the bacterial strain group is rapidly screened using the spot test, and then the true proliferation capacity is quantitatively confirmed by the efficiency of plating (EOP). This can effectively eliminate false positives caused by high concentrations of phage lysin, such as "lysis from without".

3.5 Morphological and genomic identification:

Transmission electron microscopy (TEM) with negative staining was used to observe the morphology and structural parameters of bacteriophages; whole genome sequencing combined with bioinformatics annotation was used to systematically screen integrase, lysogenization module, virulence factor (VFDB) and drug resistance gene (CARD/ResFinder), and classification was completed according to the latest classification framework of the International Committee on Taxonomy of Viruses (ICTV) (Note: the family/level classification based on morphology such as Siphoviridae/Myoviridae/Podoviridae was abolished in 2022 and is now uniformly classified into the new classification system under the class Caudoviricetes).

4. Core Application Areas

（1）Phage therapy and personalized medicine: Targeted screening of highly efficient lytic phages for clinically drug/resistant bacterial isolates, supporting compassionate use, personalized cocktail formulations, and early resource reserves for phage drug pipelines.

（2）Food safety and biological preservation: Isolate bacteriophages targeting foodborne pathogens such as Salmonella, Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli O157:H7 for use in the development of processing environment disinfection and biological preservatives.

（3）Biological control in agriculture and aquacultureTo obtain bacteriophages targeting Vibrio aquaticus, Aeromonas hydrophila, pathogenic Escherichia coli in livestock and poultry, and plant pathogens (such as Ralstonia solanacearum and Ralstonia solanacearum) as alternatives to antibiotic reduction.

（4）Source tracing and control of bacteriophage contamination in industrial fermentationThe goal is to isolate bacteriophages from contaminated fermenters and workshop environments, identify the source of contamination and the type of bacteriophage, and provide a basis for the selection of anti-phage strains and process disinfection solutions.

（5）Phage Resource Library and Component MiningTo build phage resource libraries for research institutions and enterprises, and to discover high-value functional elements such as lyases, depolymerases, and receptor-binding proteins.

（6）Detection and Diagnostic DevelopmentUsing highly specific bacteriophages obtained through isolation as a framework, we develop rapid detection tools such as report bacteriophages and bacteriophage biosensors.

5. Project Process and Validation

We provide a one/stop service from protocol design to delivery of bacterial/viral strains, ensuring that every delivered phage has a clear origin, well-defined characteristics, and complete data.

（1）Protocol Design and Host Bacterial IdentificationClearly define the target strain, application scenario, and acceptance criteria; customers can provide their own strains or choose host strains from our in-stock strain platform.

（2）Sample collection and preprocessingBased on the ecological niche of the target bacteria, a sampling scheme was designed to complete the collection, centrifugation, filtration and preservation of samples from multiple locations.

（3）Enrichment and primary screeningAfter multiple rounds of enrichment, positive results were determined using the double-layer plate method and the droplet method, and the morphology and initial titer of the phage plaques were recorded.

（4）Single-spot purification and amplificationAfter three consecutive rounds of single-spot purification, liquid amplification was performed to obtain high-titer phage stock solution.

（5）Biological characteristic characterizationThe study aimed to determine the host spectrum and EOP, one-step growth curve (latency and lysis rate), optimal multiple of infection (MOI), temperature and pH stability, and UV and disinfectant tolerance.

（6）Purification and Quality ControlPurification is achieved using PEG precipitation, cesium chloride density gradient centrifugation, or chromatography, and endotoxin removal and quantitative detection are provided to meet the needs of animal experiments and therapeutic applications.

（7）Identification and PreservationComplete TEM morphological identification, whole genome sequencing and safety bioinformatics analysis, and provide glycerol preservation or freeze-drying preservation services.

[Illustration] Figure 2: Project Flowchart

6. Introduction to Bacteriophage Isolation Services

6.1 Core services include:

（1）Customized phage isolationUsing customer-specified strains as targets, we isolate lytic bacteriophages from environmental samples from multiple sources, with acceptance standards set at "N bacteriophages delivered per host bacterium".

（2）High-efficiency amplification and purificationWe offer a range of purification solutions from laboratory to therapeutic levels, including endotoxin removal and testing reports.

（3）Biological characteristic characterizationThe complete set of characterization data includes host spectrum, EOP, one-step growth curve, and stability.

（4）Whole genome sequencing and safety analysisComplete genome assembly and annotation, with a focus on identifying lysogenization modules, virulence genes, and drug resistance genes, and providing safety evaluation opinions.

（5）Cocktail Pairing DesignBased on host spectrum complementarity, receptor differences, and resistance evolution patterns, we designed phage combinations with broader coverage and lower resistance rates.

（6）bacteriophage preservation and freeze-dryingProvides long-term preservation services and stability data.

6.2 Technical Advantages:

（1）Host bacterial resource matchingWe have our own in-stock microbial strain platform and standard strain library, which allows customers to quickly start projects even when they do not have host microorganisms.

（2）Multi-source sample networkIt covers a wide range of sample sources, including municipal and hospital sewage, aquaculture wastewater, soil, water bodies, and food processing environments, significantly improving the separation success rate.

（3）Strict criteria for determining virulent bacteriophagesThe dual confirmation of phenotype (no lysogen formation, no turbid plaques) and genome (no integrase/repressor/drug resistance genes) helps to avoid biosafety risks at the source.

（4）Platform IntegrationThe isolated phages can be seamlessly integrated into our phage gene editing, phage library, bacterial prephage prediction, and genetically modified microbial safety assessment platform, forming a complete "isolation-modification-evaluation" chain.

（5）Full deliveryWe provide phage stock solution, preservation tubes, and complete experimental data reports. The data format can be customized according to the client's application or publication requirements.

7. Case Introduction

Case Study: Personalized Phage Cocktail Therapy for Disseminated Pan-Drug-Resistant Acinetobacter baumannii Infection

Project content: Using extensively drug-resistant Acinetobacter baumannii isolated from patients as indicator bacteria, personalized phage cocktails were selectively isolated from environmental samples and administered via intravenous and percutaneous catheter routes, successfully treating critically ill patients who had failed conventional anti-infective therapy.

(1) Research Background

A 68-year-old male patient contracted extensively drug-resistant Acinetobacter baumannii during overseas travel, which led to necrotizing pancreatitis and infection of abdominal pseudocysts. Multiple antibiotic treatments failed, and the patient went into septic shock and coma. With conventional anti-infective methods exhausted, the treatment team initiated personalized phage therapy under the FDA's emergency investigational new drug (eIND) framework in the United States.

(2) Scheme Design

Using a clinical strain of Acinetobacter baumannii isolated from the patient as the sole indicator bacterium, two research institutions (Texas A&M University's Phage Technology Center and the U.S. Naval Medical Research Center) conducted parallel enrichment and screening of phages from environmental samples such as sewage, each obtaining phages with lytic activity against the clinical strain, and constructing two phage cocktails. After amplification, purification, and endotoxin removal, one group of phages was injected directly into the peritoneal abscess cavity via a percutaneous catheter, while the other group was administered systemically via intravenous route. Changes in the patient's strain's phage sensitivity were continuously monitored throughout the treatment process.

(3) Experimental conclusions

Several days after the initiation of intravenous administration, the patient regained consciousness, and infection markers and overall condition continued to improve, ultimately achieving clinical cure. During treatment, a bacterial variant resistant to the initial cocktail was observed. The research team then isolated a new phage with lytic activity against this variant from environmental samples and incorporated it into the treatment regimen, re-establishing the antibacterial effect. No serious phage-related adverse reactions were observed throughout the entire treatment course.

(4) Significance of application

This case is the first reported example in the United States of successful phage therapy for systemic drug-resistant bacterial infections via intravenous administration, establishing a personalized phage therapy paradigm of "targeting the patient's bacterial strain → targeted isolation from environmental samples → cocktail formulation → resistance monitoring and dynamic supplementation." It clearly demonstrates that rapid, reliable, and repeatable environmental phage isolation capabilities are both the prerequisite and the bottleneck for phage therapy and biocontrol to move from concept to application, and are precisely the core value of our isolation services.

8. References

[1] O'Neill J. (2016). Tackling Drug-Resistant Infections Globally: Final Report and Recommendations. Review on Antimicrobial Resistance.

[2] Van Twest R, Kropinski AM. (2009). Bacteriophage enrichment from water and soil. Methods in Molecular Biology, 501, 15-21.

[3] Kropinski AM, Mazzocco A, Waddell TE, Lingohr E, Johnson RP. (2009). Enumeration of bacteriophages by double agar overlay plaque assay. Methods in Molecular Biology, 501, 69-76.

[4] Hyman P, Abedon ST. (2009). Practical methods for determining phage growth parameters. Methods in Molecular Biology, 501, 175-202.

[5] Mirzaei MK, Nilsson AS. (2015). Isolation of phages for phage therapy: a comparison of spot tests and efficiency of plating analyses for determination of host range and efficacy. PLoS ONE, 10(3), e0118557.

[6] Gill JJ, Hyman P. (2010). Phage choice, isolation, and preparation for phage therapy. Current Pharmaceutical Biotechnology, 11(1), 2-14.

[7] Bonilla N, et al. (2016). Phage on tap – a quick and efficient protocol for the preparation of bacteriophage laboratory stocks. PeerJ, 4, e2261.

[8] Ackermann HW. (2009). Basic phage electron microscopy. Methods in Molecular Biology, 501, 113-126.

[9] Pirnay JP, et al. (2015). Quality and safety requirements for sustainable phage therapy products. Pharmaceutical Research, 32(7), 2173-2179.

[10] Turner D, et al. (2023). Abolishment of morphology-based taxa and change to binomial species names: 2022 taxonomy update of the ICTV bacterial viruses subcommittee. Archives of Virology, 168(2), 74.

[11] Schooley RT, et al. (2017). Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection. Antimicrobial Agents and Chemotherapy, 61(10), e00954-17.

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