Sfin-1 Bacteriophage: A Potent Weapon Against Drug-Resistant Shigella

Understanding Shigellosis: Causes and Treatment Challenges

What is Shigellosis?

Shigellosis is a significant public health threat in both developed and developing countries. It is an infectious disease caused by a group of bacteria known as Shigella. The genus Shigella includes four pathogenic serogroups: Shigella dysenteriae, Shigella flexneri, Shigella sonnei, and Shigella boydii. Shigellosis typically causes symptoms such as diarrhea, fever, and stomach cramps.

How is Shigellosis Contracted?

Individuals can contract shigellosis by ingesting contaminated food or water, or through direct contact with the stool of an infected person. This highlights the ease of transmission and the importance of hygiene.

Current Treatments and Emerging Resistance

Antibiotic therapy, using drugs like ciprofloxacin and azithromycin, is the current mainstay of treatment for shigellosis. However, there is a growing concern due to the emergence of multidrug-resistant strains of Shigella. This resistance poses a significant challenge to effective treatment.

The Promise of Bacteriophage Sfin-1

Lytic bacteriophages, which destroy antibiotic-resistant Shigella species, show great potential in this context. Therefore, their identification and detailed characterization are crucial. The bacteriophage Sfin-1 demonstrates potent lytic activity against multidrug-resistant isolates of Shigella flexneri, Shigella dysenteriae, and Shigella sonnei.

Bacteriophage Sfin-1 Isolation and Enrichment

Collection Site and Enrichment Process

A water sample containing bacteriophages was collected from the Ganga River in West Bengal, India. To enrich the concentration of bacteriophages, the water sample was mixed with S. flexneri 2a strains and 10% (w/v) peptone, then incubated at 37°C for 24 hours.

Sequence Alignment Methodology

Bacteriophage sequences were subsequently aligned using ClustalW in MEGA7 with default parameters to facilitate genetic analysis.

Morphology of Bacteriophage Sfin-1

Key Structural Features (Figure 1)

Phage Sfin-1 shows potent lytic activity against multidrug-resistant isolates. Morphologically, the phage has an isometric head and a non-contractile tail, to which a basal tuft is attached.

Bacterial Viability and Multiplicity of Infection (MOI)

Impact of Sfin-1 on Host Cell Lysis (Figure 2)

Host cell lysis caused by phage Sfin-1 demonstrated a direct relationship between the multiplicity of infection (MOI) and cell death. Higher MOIs lead to destabilization of the outer membrane, subsequently causing cell lysis.

MOI and Cell Death Correlation

Phage Sfin-1 was tested at MOIs of 0.001, 0.01, and 0.1. Bacterial cell viability significantly decreased upon infection with MOIs of 0.1, 0.01, and 0.001, indicating effective lytic activity even at low phage concentrations.

Thermal and pH Stability of Sfin-1

Heat Stability Profile (Figure 3)

The activity of phage Sfin-1 remained moderately stable when warmed at 37°C or 50°C for 5 minutes. However, activity decreased significantly to 0.1–0.01% when incubated at 60°C or 70°C for 5 minutes. When heated at 80°C or 90°C for 5 minutes, only 0.001% activity was retained.

• Most phages remained active after 60 minutes of incubation at 37°C or 50°C.
• Only 0.1% and 0.01% of phages remained active after 60 minutes at 60°C and 70°C, respectively.
• Phage activity remarkably decreased at 80°C or 90°C after the same incubation period.

These results suggest that phage Sfin-1 exhibits moderate thermal stability to heat stress at both 37°C and 50°C.

pH Stability Characteristics

Highest activity was observed after 1 hour of incubation at pH 7.0 and 37°C, while activity reduction was noted at other pH levels. Approximately 42.7% and 10.8% recovery of infectious phage Sfin-1 was observed at pH 5.0 and 12.0, respectively. This suggests that while extreme and lower pH levels affect phage stability, a significant fraction of Sfin-1 remains active, indicating good pH stability across a wide range.

Sfin-1 Latent Period and Burst Size

Replication Kinetics (Figure 4)

Burst size was determined as a ratio of the average bacteriophage particles produced after the burst and the average number of phage particles absorbed. The replication kinetics of Sfin-1 varied depending on the host strain:

• S. flexneri 2a and S. dysenteriae 1: Latent period of approximately 5 minutes, with an average burst size of 27–28 PFU/cell.
• S. sonnei: Latent period of 10 minutes, with an average burst size estimated at 146 PFU/cell.

Implications for Phage Therapy

A larger burst size is generally more advantageous for therapeutic applications, as it indicates a higher yield of new phage particles per infected cell, enhancing the phage's ability to clear bacterial infections.

Sfin-1 Genome: Head and Tail Component Genes

Genetic Basis of Viral Structure

Sequence-based prediction of the phage Sfin-1 genome identified that upstream and downstream gene clusters are involved in viral head morphogenesis and tail component formation.

Homology to Phage Mu F-like Proteins

Specifically, CDS3, CDS4, and CDS5, which are likely to produce phage capsid and scaffold proteins, belong to the Phage Mu F-like protein family. Members of this family are known to be required for viral head morphogenesis.

Genes Involved in Sfin-1 Cell Lysis

Mechanism of Lysis and Outer Membrane Interaction

The genes involved in cell lysis are associated with the bacterial outer membrane, particularly its LPS components. The outer membrane of Gram-negative bacteria contributes to their structural integrity and protects them from chemical attack. Phage-mediated lysis involves mechanisms that destabilize this crucial outer membrane, leading to bacterial cell death.

Sfin-1 Host Cell Receptor

Lipopolysaccharide (LPS) as the Primary Receptor (Figure 9)

The adsorption of phage Sfin-1 to its host is mediated by the outer membrane lipopolysaccharide (LPS) structure. When LPS serves as the phage receptor, it often leads to strain-specific adsorption, meaning the phage may only infect certain bacterial strains based on their LPS composition.

Key Conclusions and Therapeutic Potential of Sfin-1

Summary of Findings

This study concludes the identification and characterization of a thermostable and wide-range pH tolerant Siphoviridae phage, Sfin-1, which effectively infects and lyses important antibiotic-resistant Shigella species. Notably, this is the first reported phage capable of infecting both S. flexneri and S. dysenteriae.

Evidence Supporting Sfin-1's Efficacy

The research provides comprehensive evidence, including:

• Complete physical characterizations.
• Detailed sequence analysis and genome annotation of phage Sfin-1.
• Identification of phage structural proteins through LC-MS/MS analysis.
• Phylogenetic analysis indicating Sfin-1 belongs to the T1-like bacteriophage group, suggesting packaging via the headful packaging method.
• Further analysis of the phage Sfin-1 cell wall receptor revealed that it recognizes the LPS O-antigen as its primary receptor for adsorption.

Future Therapeutic Applications

This study strongly suggests that Sfin-1 phage holds significant promise for therapeutic application against multidrug-resistant shigellosis, offering a potential alternative to conventional antibiotic treatments.

Personal Reflections and Key Learnings

Insights from the Sfin-1 Research

From this paper, two key insights were gained:

1. The activity of phages is highly dependent on its pH and thermal stability, influencing whether the phage remains active or inactive. This highlights the importance of environmental factors in phage efficacy.
2. The bacterium Shigella and the disease shigellosis have significant public health relevance due to its ease of spread and prevalence in human populations, underscoring the urgent need for effective treatments like phage therapy.