Production Of Bacteriophages Research Articles

A Critical Analysis of the Opportunities and Challenges of Phage Application in Seafood Quality Control.

Seafood is an important source of food and protein for humans. However, it is highly susceptible to microbial contamination, which has become a major challenge for the seafood processing industry. Bacteriophages are widely distributed in the environment and have been successfully used as biocontrol agents against pathogenic microorganisms in certain food processing applications. However, due to the influence of environmental factors and seafood matrices, using bacteriophages for commercial-scale biocontrol strategies still faces some challenges. This article briefly introduces the current processes used for the production and purification of bacteriophages, lists the latest findings on the application of phage-based biocontrol in seafood, summarizes the challenges faced at the current stage, and provides corresponding strategies for solving these issues.

A Recombinant RBD-Based Phage Vaccine Report: A Solution to the Prevention of New Diseases?

The safety, inherent immunogenicity, stability, and low-cost production of bacteriophages make them an ideal platform for vaccine development. Most vaccination strategies against COVID-19 have targeted the spike protein of SARS-CoV-2 to generate neutralizing antibodies. P1, a truncated RBD-derived spike protein, has been shown to induce virus-neutralizing antibodies in preclinical studies. In this study, we first investigated whether recombinant phages displaying P1 on the M13 major protein could immunize mice against COVID-19, and second, whether inoculation with 50 µg of purified P1 in addition to the recombinant phages would stimulate the immune systems of the animals. The results showed that the mice that received recombinant phages were immunized against the phage particles, but did not have anti-P1 IgG. In contrast, compared with the negative control, the group that received a combination of P1 protein and recombinant phage was immunized against the P1 protein. In both groups, CD4+ and CD8+ T cells appeared in the lung tissue. These results suggest that the number of antigens on the phage body plays a crucial role in stimulating the immune system against the bacteriophage, although it is immunogenic enough to function as a phage vaccine.

Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria

Bacteriophages are potent therapeutics against biohazardous bacteria, which rapidly develop multidrug resistance. However, routine administration of phage therapy is hampered by a lack of rapid production, safe bioengineering, and detailed characterization of phages. Thus, we demonstrate a comprehensive cell-free platform for personalized production, transient engineering, and proteomic characterization of a broad spectrum of phages. Using mass spectrometry, we validated hypothetical and non-structural proteins and could also monitor the protein expression during phage assembly. Notably, a few microliters of a one-pot reaction produced effective doses of phages against enteroaggregative Escherichia coli (EAEC), Yersinia pestis, and Klebsiella pneumoniae. By co-expressing suitable host factors, we could extend the range of cell-free production to phages targeting gram-positive bacteria. We further introduce a non-genomic phage engineering method, which adds functionalities for only one replication cycle. In summary, we expect this cell-free methodology to foster reverse and forward phage engineering and customized production of clinical-grade bacteriophages.

Participation of microbiologists Z.V. Ermolyeva and L.M. Yakobson in a scientific discussion about the fate of the production of Soviet cholera bacteriophages in 1967

Since the late 1920s in the USSR, along with many countries, there has been an interest in studying the phenomenon of bacteriophagy and its use in medicine. Bacteriophages are called bacterial viruses. Significant work on the study of bacteriophages and their use for medical purposes was carried out by Soviet microbiologists Zinaida Vissarionovna Ermolyeva and Lidiya Mikhailovna Yakobson. They paid especially great attention to the study of cholera bacteriophage, which during the Great Patriotic War helped prevent the cholera epidemic in frontline Stalingrad. In the 1940s due to the advent of the era of antibiotics with a wider range of applications, research interest in bacteriophages was waning. Nevertheless, in the prevention and treatment of a number of infectious diseases of bacterial origin, phage continued to be used in the following decades. In 1967, one of the countrys largest microbiologists sent an appeal to the Chief Sanitary Doctor of the USSR with a proposal to stop or reduce the production of cholera bacteriophage in the country. In this regard, a scientific discussion in which Z.V. Ermolyeva and L.M. Yakobson played an important role unfolded. Using the materials of the State Archive of the Russian Federation and the Russian State Archive of Economics, involving published scientific works on microbiology and medicine, an attempt is made to analyze this scientific discussion and establish its significance in the fate of the production of cholera bacteriophage in the USSR.

The effect of two ribonucleases on the production of Shiga toxin and stx-bearing bacteriophages in Enterohaemorrhagic Escherichia coli

Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of intestinal pathogens responsible for a range of illnesses, including kidney failure and neurological compromise. EHEC produce critical virulence factors, Shiga toxin (Stx) 1 or 2, and the synthesis of Stx2 is associated with worse disease manifestations. Infected patients only receive supportive treatment because some conventional antibiotics enable toxin production. Shiga toxin 2 genes (stx2) are carried in λ-like bacteriophages (stx2-phages) inserted into the EHEC genome as prophages. Factors that cause DNA damage induce the lytic cycle of stx2-phages, leading to Stx2 production. The phage Q protein is critical for transcription antitermination of stx2 and phage lytic genes. This study reports that deficiency of two endoribonucleases (RNases), E and G, significantly delayed cell lysis and impaired production of both Stx2 and stx2-phages, unlike deficiency of either enzyme alone. Moreover, scarcity of both enzymes reduced the concentrations of Q and stx2 transcripts and slowed cell growth.

Strategy for mass production of lytic Staphylococcus aureus bacteriophage pSa-3: contribution of multiplicity of infection and response surface methodology

BackgroundAntibiotic-resistant bacteria have emerged as a serious problem; bacteriophages have, therefore, been proposed as a therapeutic alternative to antibiotics. Several authorities, such as pharmacopeia, FDA, have confirmed their safety, and some bacteriophages are commercially available worldwide. The demand for bacteriophages is expected to increase exponentially in the future; hence, there is an urgent need to mass-produce bacteriophages economically. Unlike the replication of non-lytic bacteriophages, lytic bacteriophages are replicated by lysing host bacteria, which leads to the termination of phage production; hence, strategies that can prolong the lysis of host bacteria in bacteria–bacteriophage co-cultures, are required.ResultsIn the current study, we manipulated the inoculum concentrations of Staphylococcus aureus and phage pSa-3 (multiplicity of infection, MOI), and their energy sources to delay the bactericidal effect while optimizing phage production. We examined an increasing range of bacterial inoculum concentration (2 × 108 to 2 × 109 CFU/mL) to decrease the lag phase, in combination with a decreasing range of phage inoculum (from MOI 0.01 to 0.00000001) to delay the lysis of the host. Bacterial concentration of 2 × 108 CFU/mL and phage MOI of 0.0001 showed the maximum final phage production rate (1.68 × 1010 plaque forming unit (PFU)/mL). With this combination of phage–bacteria inoculum, we selected glycerol, glycine, and calcium as carbon, nitrogen, and divalent ion sources, respectively, for phage production. After optimization using response surface methodology, the final concentration of the lytic Staphylococcus phage was 8.63 × 1010 ± 9.71 × 109 PFU/mL (5.13-fold increase).ConclusionsTherefore, Staphylococcus phage pSa-3 production can be maximized by increasing the bacterial inoculum and reducing the seeding phage MOI, and this combinatorial strategy could decrease the phage production time. Further, we suggest that response surface methodology has the potential for optimizing the mass production of lytic bacteriophages.

Personalized Production, Non-Genomic Engineering, and Time-Resolved Proteomics of Therapeutic Phages for Biohazardous and Multidrug-Resistant Bacteria

Bacteriophages are potent therapeutics against biohazardous bacteria, which rapidly develop multidrug resistance. However, routine administration of phage therapy is currently hampered by a lack of rapid phage production and efficient bioengineering and characterization capabilities. Thus, we have developed a comprehensive cell-free platform for time-efficient and personalized manufacturing, transient bioengineering, and proteomic characterization of a broad spectrum of phages. We validated 40 hypothetical proteins, including non-structural proteins, from T7 and CLB-P3 phages using high-resolution mass spectrometry and identified a new late-expressing gene cluster. Just a few microliters of the one-pot reaction produce effective doses of phages against enteroaggregative E. coli (EAEC) and potential biological warfare agents such as Yersinia pestis. By coexpressing suitable host factors, we could extend the range of cell-free phage production to include selective phages targeting gram-positive bacteria. We also introduce a transient, non-genomic phage engineering method, which adds functionalities for only one replication cycle. Finally, we showcase a pipeline for personalized phage therapy of a multidrug-resistant ESKAPE pathogen using a newly isolated and in-vitro produced phage against Klebsiella pneumoniae. We expect our cell-free methodology to enable accelerated reverse and forward phage engineering and the safe and customized production of clinical-grade therapeutic bacteriophages.

Single Mathematical Parameter for Evaluation of the Microorganisms' Growth as the Objective Function in the Optimization by the DOE Techniques.

The cultivation of bacteria sets a ground for studying biological processes in many scientific disciplines. The development of the bacterial population is commonly described with three factors that can be used to evaluate culture conditions. However, selecting only one of them for the optimization protocol is rather problematic and may lead to unintended errors. Therefore, we proposed a novel mathematical approach to obtain a single factor that could be used as the objective function to evaluate the whole growth dynamic and support the optimization of the biomass production process. The sigmoidal-shape curve, which is the commonly used function to plot the amount of biomass versus time, was the base for the mathematical analysis. The key process parameters, such as maximal specific growth rate and lag-phase duration were established with the use of mathematical coefficients of the model curve and combined to create the single growth parameter. Moreover, this parameter was used for the exemplary optimization of the cultivation conditions of Klebsiella pneumoniae that was cultured to be further used in the production of lytic bacteriophages. The proposed growth parameter was successfully validated and used to calculate the optimal process temperature of the selected bacterial strain. The obtained results indicated that the proposed mathematical approach could be effortlessly adapted for a precise evaluation of growth curves.

Production of Bacteriophages by Listeria Cells Entrapped in Organic Polymers.

Applications for bacteriophages as antimicrobial agents are increasing. The industrial use of these bacterial viruses requires the production of large amounts of suitable strictly lytic phages, particularly for food and agricultural applications. This work describes a new approach for phage production. Phages H387 (Siphoviridae) and A511 (Myoviridae) were propagated separately using Listeria ivanovii host cells immobilised in alginate beads. The same batch of alginate beads could be used for four successive and efficient phage productions. This technique enables the production of large volumes of high-titer phage lysates in continuous or semi-continuous (fed-batch) cultures.

Optimizing Propagation of Staphylococcus aureus Infecting Bacteriophage vB_SauM-phiIPLA-RODI on Staphylococcus xylosus Using Response Surface Methodology.

The use of bacteriophages for killing pathogenic bacteria is a feasible alternative to antibiotics and disinfectants. To obtain the large quantities of phages required for this application, large-scale production of bacteriophages must be optimized. This study aims to define conditions that maximize the phage yield of the virulent and polyvalent staphylococcal bacteriophage vB_SauM-phiIPLA-RODI in broth culture, using the food-grade species Staphylococcus xylosus as the host strain to reduce the risk of growing massive quantities of pathogenic bacteria and therefore, to ensure the safety of the final phage stock. The effect of four variables, namely initial bacterial concentration (5.66–8.40 log10 colony-forming unit (CFU)/mL), initial phage concentration (5–8 log10 plaque-forming unit (PFU)/mL), temperature (21–40 °C) and agitation (20–250 rpm), on phage yield (response) was studied by using response surface methodology (RSM). Successive experimental designs showed that agitation did not significantly impact phage yield, while temperature did have a significant effect, with 38 °C being the optimum for phage propagation. The results allowed the design of a model to describe phage yield as a function of the initial bacterial and phage concentrations at fixed agitation (135 rpm), and optimum temperature (38 °C). The maximum experimental phage yield obtained was 9.3 log10 PFU/mL, while that predicted by the model under the optimized conditions (7.07 log10 CFU/mL initial bacterial population and 6.00 log10 PFU/mL initial phage titer) was 9.25 ± 0.30 log10 PFU/mL, with the desirability of 0.96. This yield is comparable to that obtained when the phage was propagated on the original host, Staphylococcus aureus. Bacteriophage phiIPLA-RODI showed the same host range and very similar biofilm removal ability regardless of the staphylococcal species used for its propagation. The results presented in this study show the suitability of using a food-grade strain of S. xylosus for the propagation of S. aureus infecting phages and the application of RSM to define the optimal propagation conditions.

Bacteriophage Production in Bioreactors.

The optimal conditions for the production of virulent bacteriophages in bioreactors can vary greatly depending on the host-bacteriophage system used. We present a general method for the production of virulent bacteriophages in bioreactors that can be adapted to many host-bacteriophage systems and various operating conditions (reactor volume, medium composition, temperature, etc.). The procedures detail how to establish optimal initial infection conditions (infection load and initial multiplicity of infection (MOI)), prepare the host pre-culture and bioreactor, operate the bioreactor, and harvest the bacteriophage product. Batch operation is detailed but a short discussion addresses other modes of operation, namely two-stage continuous bioreactors and two-stage cycling bioreactors.

Development of Biotechnology for Anthrax Bacteriophages Reproduction, and Comparative Analysis of Their Properties Stability and Diagnostic Value

Carried out has been complex comparative analysis of the production and diagnostic properties of such anthrax bacteriophages as Gamma A-26, Fah-RRIVV&M (Russian Research Institute of Veterinary Virology and Microbiology), R/D-Ph-6, BA-9, K RRIEV (Russian Research Institute of Experimental Veterinary), Saratov, and 186. It is revealed that all of the bacteriophages have the properties necessary for using them for preparation manufacturing purposes: they readily proliferate in liquid cultures of avirulent Bacillus anthracis strain - STI, and retain sufficient specific activity if stored at the optimum temperatures or, when exposed to short-term temperature excursions, within a period of two years. Most clearly expressed are the differences in diagnostic properties of the bacteriophages - specific activity and specificity. Put forward are the groups of bacteriophages that are the most relevant for identification or phage-typing of B. anthracis strains.

Phagomagnetic Separation and Electrochemical Magneto-Genosensing of Pathogenic Bacteria

This paper addresses the use of bacteriophages immobilized on magnetic particles for the biorecognition of the pathogenic bacteria, followed by electrochemical magneto-genosensing of the bacteria. The P22 bacteriophage specific to Salmonella (serotypes A, B, and D1) is used as a model. The bacteria are captured and preconcentrated by the bacteriophage-modified magnetic particles through the host interaction with high specificity and efficiency. DNA amplification of the captured bacteria is then performed by double-tagging polymerase chain reaction (PCR). Further detection of the double-tagged amplicon is achieved by electrochemical magneto-genosensing. The strategy is able to detect in 4 h as low as 3 CFU mL(-1) of Salmonella in Luria-Bertani (LB) media. This approach is compared with conventional culture methods and PCR-based assay, as well as with immunological screening assays for bacteria detection, highlighting the outstanding stability and cost-efficient and animal-free production of bacteriophages as biorecognition element in biosensing devices.

Two-stage, self-cycling process for the production of bacteriophages

BackgroundA two-stage, self-cycling process for the production of bacteriophages was developed. The first stage, containing only the uninfected host bacterium, was operated under self-cycling fermentation (SCF) conditions. This automated method, using the derivative of the carbon dioxide evolution rate (CER) as the control parameter, led to the synchronization of the host bacterium. The second stage, containing both the host and the phage, was operated using self-cycling infection (SCI) with CER and CER-derived data as the control parameters. When each infection cycle was terminated, phages were harvested and a new infection cycle was initiated by adding host cells from the SCF (first stage). This was augmented with fresh medium and the small amount of phages left from the previous cycle initiated the next infection cycle. Both stages were operated independently, except for this short period of time when the SCF harvest was added to the SCI to initiate the next cycle.ResultsIt was demonstrated that this mode of operation resulted in stable infection cycles if the growth of the host cells in the SCF was synchronized. The final phage titers obtained were reproducible among cycles and were as good as those obtained in batch productions performed under the same conditions (medium, temperature, initial multiplicity of infection, etc.). Moreover, phages obtained in different cycles showed no important difference in infectivity. Finally, it was shown that cell synchronization of the host cells in the first stage (SCF) not only maintained the volumetric productivity (phages per volume) but also led to higher specific productivity (phage per cell per hour) in the second stage (SCI).ConclusionsProduction of bacteriophage T4 in the semi-continuous, automated SCF/SCI system was efficient and reproducible from cycle to cycle. Synchronization of the host in the first stage prior to infection led to improvements in the specific productivity of phages in the second stage while maintaining the volumetric productivity. These results demonstrate the significant potential of this approach for both upstream and downstream process optimization.

Mastering of Technology of Production of Brucellosis Diagnostic Bacteriophages

Presented are technologies of production of brucellosis diagnostic bacteriophages developed on the basis of determination of optimal conditions of the phages reproduction. Stability of the finished product was evaluated using Shuhard control charts.

An optimized procedure for efficient phage display of antibody fragments with a low folding efficiency

We recently developed an efficient bacterial expression system for phagemid-coded antigen-binding fragments of antibody (Fabs) without the use of a helper bacteriophage. This system is characterized by an unusually long cultivation at a low temperature and gentle induction of Fab expression without the addition of the inducer isopropyl-β- D-thiogalactopyranoside (IPTG). This method allows for a high yield production of Fabs fused with phage gene III coat protein, even when the protein is defective in its folding ability. With this cultivation procedure, we aimed here at improving the production and selection efficiency of filamentous bacteriophages displaying functional Fabs on their surface (Fab-phages) that have high affinity but low folding ability. The Fab components of the Fab-phages used were clonally related but differed in their affinity and folding ability. The production of the functional Fab-phages was quantitatively evaluated under various culture conditions. With conventional phage particle preparation, the production of functional Fab-phages was significantly biased according to the folding ability of the displayed Fabs, and affinity-based biopanning was therefore unsuccessful. In contrast, with the present procedure employing cultivation at 25 °C for 16 h without IPTG induction, functional Fab-phages were produced without any such dependence on folding ability. With this optimized library, affinity-based biopanning was successful. Especially noteworthy, bead-based biopanning accurately discriminated between high affinity Fab-phages and Fab-phages with low or middling affinity. In obtaining Fab-phages with high affinity but low folding ability, these optimized procedures for both cultivation and selection were essential.

Presence of activatable Shiga toxin genotype (stx(2d)) in Shiga toxigenic Escherichia coli from livestock sources.

Stx2d is a recently described Shiga toxin whose cytotoxicity is activated 10- to 1000-fold by the elastase present in mouse or human intestinal mucus. We examined Shiga toxigenic Escherichia coli (STEC) strains isolated from food and livestock sources for the presence of activatable stx(2d). The stx(2) operons of STEC were first analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis and categorized as stx(2), stx(2c vha), stx(2c vhb), or stx(2d EH250). Subsequently, the stx(2c vha) and stx(2c vhb) operons were screened for the absence of a PstI site in the stx(2A) subunit gene, a restriction site polymorphism which is a predictive indicator for the stx(2d) (activatable) genotype. Twelve STEC isolates carrying putative stx(2d) operons were identified, and nucleotide sequencing was used to confirm the identification of these operons as stx(2d). The complete nucleotide sequences of seven representative stx(2d) operons were determined. Shiga toxin expression in stx(2d) isolates was confirmed by immunoblotting. stx(2d) isolates were induced for the production of bacteriophages carrying stx. Two isolates were able to produce bacteriophages phi1662a and phi1720a carrying the stx(2d) operons. RFLP analysis of bacteriophage genomic DNA revealed that phi1662a and phi1720a were highly related to each other; however, the DNA sequences of these two stx(2d) operons were distinct. The STEC strains carrying these operons were isolated from retail ground beef. Surveillance for STEC strains expressing activatable Stx2d Shiga toxin among clinical cases may indicate the significance of this toxin subtype to human health.

Frequency, size and distribution of bacteriophages in different marine bacterial morphotypes

The frequencies of cells containing mature phages, the burst sizes, the phage head sizes and the distribution of phages inside cells of different bacterial morphotypes were investigated in the northern Adriatic Sea. Coccoid bacteria more frequently (2 .5%) contained mature phages than rodshaped bacteria (1.2%) and spirillae (1.4 %). Including an estimation of non-v~sible infection we found that up to 27 % of rods were infected with viruses, up to 79% of cocci and up to 100% of spirillae. The highest overall infection frequency of the entire bacterial community was 30 %. The percentage of rods with mature phages was significantly correlated to increasing rod densities. It is suggested that a threshold density of about 2 X 10' rods ml-' exists that is necessary for infection with phages. No threshold densities could be determined for cocci and spirillae. Burst sizes varied strongly between different host morphotypes. The burst sizes of rods increased significantly with the frequency of rods containing mature phages, probably as a result of superinfection of bacteria with phages. The volume of the host cells seemed to influence the number of phages produced per cell. Most of the phages within rods and all phages within spirillae were smaller than 60 nm, whereas the majority of phages within cocci were larger than 60 nm. Analyses of the distribution of phages inside the cells showed that phages were frequently concentrated in 2 defined areas at the 2 opposite ends of both rods and spirillae. Our results from an in situ study suggest that the production of bacteriophages is strongly influenced by the structure of the bacterial community, i.e. by the relative abundances of the various morphotypes.

Temperate Bacteriophages of Bacillus amylolquefaciens

SUMMARY: We examined the production of temperate bacteriophages by several strains of Bacillus amyloliquefaciens. Each of the seven strains we induced with mitomycin C produced at least one temperate phage capable of infecting strains of Bacillus subtilis; some of the phages could also infect other B. amyloliquefaciens strains. It is probable that some of the bacteria also produce bacteriocins.

Anomalous induction of mycobacteriophages mediated by mitomycin C.

Mitomycin C has been found to stimulate the production of long-tailed defective bacteriophages and poly tails in thick cell wall mycobacterial mutants.