Bacteriophages, ubiquitous and diverse on Earth, infect and replicate within bacterial hosts, playing a crucial role in microbial ecosystems. Despite their importance, their presence may disrupt industrial processes. We have developed a method using bacterial lipopolysaccharides to eliminate bacteriophages from Salmonella cultures.
Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and interactions. These organisms are viruses that infect and replicate within bacterial hosts. Phages are ubiquitous on Earth, highly diverse, and very abundant. While bacteriophages have valuable roles in different environments and are a key area of research in microbiology and ecology, their presence can be undesirable in certain industrial processes or products. Considering the abundance and ubiquity of bacteriophages on Earth, the design of procedures for the removal of bacteriophages from bacterial cultures is crucial in diverse laboratory and industrial applications to preserve the integrity of the cultures and ensure accurate experimental results or product quality. Here, we have fine-tuned a protocol to eliminate the bacteriophages from infected Salmonella enterica cultures, using a strategy based on the use of lipopolysaccharides (LPS) located in the outer membrane of Gram-negative bacteria. Bacterial LPS plays an important role in host recognition by phages, and we make use of this property to design an effective procedure for the removal of phages, which use LPS as a receptor, in Salmonella bacterial cultures.
Microbial populations are faced with multiple challenges in natural environments, and an especially severe threat is the potential of infection by bacteriophages, the viruses that infect bacteria1. These viruses are widespread on the planet, exhibiting great diversity and abundance2,3,4,5. Although bacteriophages are diverse in size, morphology, and genomic organization, all share the same structure: a DNA or RNA genome enveloped by a capsid formed by phage-encoded proteins6. Bacteria have evolved a diverse array of defense mechanisms against them7. A key aspect of bacteriophage infection, which is relevant for characterization as well as for detection, is the receptor binding domains present on tail fibers. Bacteriophages have proteins on their surface called receptor-binding proteins or tail fibers for recognizing and binding to specific receptor sites on the surface of the bacterial cell. In the case of Gram-negative bacteria, the recognition of surface structures, such as lipopolysaccharides (LPS), outer membrane proteins, pili, and/or flagella, are involved in phage-bacteria interaction8. This interaction between bacteriophages and bacteria is highly specific and depends mainly on their ability to attach to host surfaces. The O-antigen of lipopolysaccharide is a commonly used receptor9.
The investigation of bacteriophage-bacteria interactions is not only fascinating from a biological standpoint but also has practical applications in areas such as phage therapy and biotechnology. While bacteriophages have valuable roles in various contexts, for example, altering microbial populations10, their presence can be undesirable in certain industrial processes. In pharmaceuticals, biotechnology, and food production, the presence of bacteriophages can impact the quality and safety of the final products, making their removal essential to meet quality standards. In bioprocessing and biomanufacturing, where bacterial cultures are used to produce various compounds (e.g., proteins, enzymes, or antibiotics), the presence of bacteriophages can lead to the disruption of production processes due to their ability to balance the bacterial population in every shared environment. Phages can occasionally turn the industrial microbiologist's professional life into a nightmare11. The design of effective procedures to remove phages is critical to ensure consistent and reliable production, enhancing process efficiency. Apart from these industrial aspects, in a research laboratory setting, where precision and reproducibility are crucial, the elimination of bacteriophages is essential for obtaining accurate and reliable results. Furthermore, the removal of phages can also be used to simulate varying environments to test different hypotheses12. Removing phages can also be very useful in the research environment since many phage-based studies, such as the enumeration of bacteria following phage application, would benefit from a step to remove phages in order to produce much more reliable viable counts.
Phage removal based on colony isolation would take several days to ensure that colonies are phage-free, whereas the procedure described here allows the generation of phage-free cultures in hours. This protocol permits us to follow the evolution of bacterial cultures without stopping them from isolating colonies. In this sense, it is possible to simulate fluctuating environments (presence and/or absence of phages) to test different hypotheses. Furthermore, this protocol permits the qualitative and quantitative analysis of the presence of phages in a bacterial culture.
In summary, designing cost-effective procedures for the removal of bacteriophages is crucial for maintaining product quality, safety, and process efficiency in various industries and for advances in basic and applied research. Here, we describe a highly effective protocol based on the use of LPS for the removal of bacteriophages, which use LPS as a receptor, from infected Salmonella cultures, which is both time-efficient and requires minimal equipment.
Diverse strategies are employed by bacteriophages to recognize and infect bacterial hosts. Different molecular structures on the surface of bacteria can act as phage receptors: protein, polysaccharide, lipopolysaccharides (LPS), and carbohydrate moieties20. In Gram-negative bacteria, LPS is a common receptor for phages. In addition, other receptors are outer membrane proteins, pili, and flagella21.
The specific interaction between bacteriophages and bacteria based on the recognition of LPS8 has been exploited in this work for the development of a highly efficient protocol for the elimination of bacteriophages in infected bacterial cultures (Figure 1 and Figure 2). Our protocol does not favor the selection of phage-resistant cells; it only eliminates bacteriophages (Figure 6). Both phage-susceptible and phage-resistant cells remain in bacterial culture after performing this protocol of phage removal.
The traditional standard practice when a phage infection occurs is to attempt to eliminate all the contaminated material, followed by cleaning and sterilization22. The decontamination procedure involves exposing the bacterial culture to stressful conditions, such as high temperatures, in order to partially or completely eliminate the bacterial cells. As described in representative results, the crucial step in this protocol is the incubation of phages-infected bacterial cultures with commercial LPS, a non-harmful substance for bacterial cultures. This helps to preserve the viability of bacterial cultures and offers significant advantages for industrial applications in fermenters and bioreactors.
The incubation time in this protocol is 2 h to ensure sufficient time for phage lysis of bacterial cells. If different bacterial strains and bacteriophages are to be used, this parameter should be considered and defined by the user. In this case, an assay similar to that described in Figure 4 should be performed prior to the experiment.
Interestingly, the efficacy of this cleaning protocol could also be analyzed by employing an assay that monitors the phage content of a given sample. In this sense, epigenetic biosensors are a novel tool for bacteriophage detection23. A well-known phage biosensor able to detect coliphages, which use LPS as a receptor, is the opvAB::gfp system13,18,23,24. This phage biosensor detects an increase in the OpvABON subpopulation in the presence of phages that use O-antigen as a receptor. In this sense, we could use an opvAB::gfp fusion to monitor LPS-binding phages in various steps of this protocol and/or diverse media and conditions. These approaches could be valuable in determining the timing and locations at which an effective protocol may be necessary.
While LPS recognition is common, phages can also utilize a variety of other surface receptors on bacterial cells for attachment and infection. Here, we have used the Gram-negative Salmonella as a representative enterobacteria and the bacteriophage 9NA that uses LPS as a receptor and genome ejection trigger. Other enterobacteria phages (e.g., Escherichia coli T5) bind loosely to LPS and require an outer membrane protein for genome injection. The protocol described is applicable for bacteriophages that recognize and need the O-antigen of LPS for successful infection, such as 9NA, Det7, and P2213,25,26,27. Accordingly, the successful implementation of this protocol for the phage decontamination of bacterial cultures involves determining whether the source of the phage infection requires recognizing LPS in the host.
In conclusion, and despite the potential limitations of the protocol, our representative results clearly demonstrate that this method is a powerful tool to clean bacterial Salmonella cultures of bacteriophages that use LPS as a receptor and genome ejection trigger.
The authors have nothing to disclose.
We thank Dr. Carmen R. Beuzón and Rocío Carvajal-Holguera for helpful discussions and suggestions. This work was supported by the grant PID2020-116995RB-I00 funded by MICIU/AEI/ 10.13039/5011100011033 and the VI Plan Propio de Investigación y Transferencia from the Universidad de Sevilla.
20 mL syringe | BD Discardit II | 300296 | No special requirements |
50 mL conical tubes | Avantor | 525-0610 | No special requirements |
90 x 14 mm Petri dishes | Deltalab | 200209 | No special requirements |
Agar | Sigma-Aldrich | A1296 | No special requirements |
Bacteriophage lysate | Minimal concentration: 109 PFU/mL | ||
Centrifuge | Eppendorf | No special requirements | |
Chloroform | Panreac | 131252 | No special requirements |
Citric acid · H2O | Merck | 1.00247 | |
Colony counter | No special requirements | ||
Evans Blue | Sigma-Aldrich | E-2129 | |
Flasks | No special requirements | ||
Fluorescein sodium salt | Sigma-Aldrich | F-6377 | |
Forceps | No special requirements | ||
Glass tubes | No special requirements | ||
Glass tubes for lysate | No special requirements | ||
Glucose | Sigma-Aldrich | G7021 | |
K2HPO4 | Merck | 1.05104.1000 | |
K2HPO4 anhydrous | Merck | 1.05104 | |
Lipopolysaccharide from Salmonella enterica serotype Typhimurium | Sigma-Aldrich | L6511-25 mg | Dissolved in sterile water |
Membrane 0.45 µm | MF-Millipore | HAWP02500 | No special requirements |
MgSO4 · 7 H2O | Merck | 1.05886 | |
NaCl | Sigma-Aldrich | S9888 | No special requirements |
NaNH4HPO4 · 4 H2O | Sigma-Aldrich | S9506 | |
Peptone | iNtRON | Ba2001 | No special requirements |
Syringe Filter 0.22 µm | Millex | SLGSR33SB | No special requirements |
Toothpicks | No special requirements | ||
Tryptone | Panreac | 403682.1210 | No special requirements |
Vacuum pump | Thermo Scientific | No special requirements | |
Yeast extract | iNtRON | 48045 | No special requirements |
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